Some genetic and biochemical aspects of myoclonus.

Can a gene defect be responsible for the occurrence in an individual, at a particular age, of such a muscle twitch followed by relaxation called: "myoclonus" and defined as sudden, brief, shock-like movements? Genetic defects could indeed determine a subsequent cascade of molecular events (caused by abnormal encoded proteins) that would produce new aberrant cellular relationships in a particular area of the CNS leading to re-built "myoclonogenic" neuronal networks. This can be illustrated reviewing some inherited neurological entities that are characterized by a predominant myoclonic picture and among which a clear gene defect has been identified. In the second part of this chapter, we will also propose a new point of view on how some structural genes could, under certain conditions, when altered, produced idiopathic generalized epilepsy with myoclonic jerks, taking juvenile myoclonic epilepsy (JME) and the myoclonin (EFHC-1) gene as examples.

Seasonal variation in androgen-metabolizing enzymes in the diencephalon and telencephalon of the male European starling (Sturnus vulgaris).

In seasonally breeding songbirds, seasonal fluctuations occur in serum testosterone (T) concentrations and reproductive behaviours. Many T-dependent behaviours are regulated by the activity of androgenic and oestrogenic metabolites within specific brain regions. Male European starlings breed in spring when circulating T concentrations peak. T and its metabolites act within portions of the diencephalon to regulate the pituitary-gonadal axis and to activate courtship and copulation. Song in male starlings is critical for mate attraction during the breeding season and is regulated by steroid-sensitive nuclei in the telencephalon and diencephalon. Outside the breeding season, T is undetectable, however, males continue to sing at high levels. This suggests that singing outside of the breeding season might not be T-dependent as it appears to be in the spring. Alternatively, singing when T is low might continue to be regulated by T due to increased sensitivity of the brain to the action of the steroid. This increased sensitivity could be mediated by changes in intracellular T metabolism leading to increased production of active or decreased production of inactive metabolites. To explore the relationship between T-metabolism and reproductive behaviour, we analysed seasonal changes in the activity of four brain T-metabolizing enzymes: aromatase, 17beta-hydroxysteroid dehydrogenase (17beta-HSDH), 5alpha-reductase (all three convert T into active metabolites) and 5beta-reductase (converts T into an inactive metabolite) in the diencephalon and telencephalon. In the anterior and posterior diencephalon, the highest aromatase was observed in spring when this region is critical for courtship and copulation. In the telencephalon, aromatase was highest and 5beta-reductase was lowest throughout the winter months well prior to the reproductive season and these enzymes presumably maximize T-activity within this region. Although these data do not indicate whether the metabolic changes occur specifically within song nuclei, these findings are compatible with the idea that singing in male starlings outside the breeding season may be regulated by steroids despite the presence of low serum T concentrations. Overall, seasonal changes in T-metabolizing enzymes appear to play a significant role in seasonal changes in behaviour and reproductive physiology.

Distribution of DARPP-32 immunoreactive structures in the quail brain: anatomical relationship with dopamine and aromatase.

We recently demonstrated that dopamine (DA) as well as different DA receptor agonists and antagonists are able to decrease within a few minutes the aromatase activity (AA) measured in vitro in homogenates or in explants of the quail preoptic area - hypothalamus. In addition, DA also appears to regulate AA, in vivo presumably by modifying enzyme synthesis. The cellular mechanisms and the anatomical substrate that mediate these controls of AA by DA are poorly understood. Tyrosine hydroxylase-immunoreactive (TH-ir) fibers and punctate structures have been previously observed in close vicinity of aromatase-immunoreactive (ARO-ir) cells in the quail medial preoptic nucleus (POM) and bed nucleus striae terminalis (BST) but these fibers could reflect a noradrenergic innervation. We also do not know whether aromatase cells are dopaminoceptive. The main goal of the present study was therefore to bring more information on the anatomical relationships between aromatase expressing neurons and the dopaminergic system in the quail brain. The visualization by immunocytochemistry of DA and of the D1 receptor associated protein DARPP-32 was used to address these questions. DA-ir fibers were observed in the quail forebrain and overlapped extensively with nuclei that contain high densities of ARO-ir cells such as the POM and BST. This confirms that the previously reported TH-ir innervation of ARO-ir cells is, at least in part, of dopaminergic nature. DARPP-32-immunoreactive cells were found in periventricular position throughout the hypothalamus. DARPP-32-ir cells were also observed in telencephalic and mesencephalic areas (hyperstriatum accessorium, paleostriatum, nucleus intercollicularis, optic tectum). DARPP-32-ir fibers were widespread in tel-, di-, and mes-encephalic areas. The highest densities of immunoreactive fibers were detected in the lobus parolfactorius, paleostriatum augmentatum and substantia nigra/area ventralis of Tsai. In double-labeled sections, appositions between DARPP-32 fibers and ARO-ir cells were present in the dorsolateral POM and BST but DARPP-32 immunoreactivity was not detected in the ARO-ir perikarya (no colocalization). These data confirm the presence of a dopaminoceptive structures within the main cell clusters of ARO-ir cells in the quail brain but provide no evidence that these ARO-ir cells are themselves dopaminoceptive. Because DARPP-32 is not present in all types of cells expressing DA receptors, the presence of DA receptors that would not be associated with DARPP-32 in ARO-ir cells still remains to be investigated

Immunocytochemical localization of ionotropic glutamate receptors subunits in the adult quail forebrain.

The excitatory amino acid glutamate is implicated in the central control of many neuroendocrine and behavioral processes. The ionotropic glutamate receptors are usually divided into the N-methyl-D-aspartate (NMDA) and non-NMDA (kainate and AMPA) subtypes. Subunits of these receptors have been cloned in a few mammalian species. Information available in birds is more limited. In quail, we recently demonstrated that glutamate agonists (kainate, AMPA, and NMDA) rapidly (within minutes) and reversibly decrease in vitro aromatase activity like several other manipulations affecting intracellular HCa(2+) pools. Aromatase catalyzes the conversion of androgens into estrogens which is a limiting step in the control by testosterone of many behavioral and physiologic processes. Therefore, glutamate could control estrogen production in the brain, but the anatomic substrate supporting this effect is poorly understood. In quail, aromatase is mainly localized in the preoptic-hypothalamic-limbic system. We visualized here the distribution of the major ionotropic glutamate receptors in quail by immunocytochemical methods by using commercial primary antibodies raised against rat glutamate receptor 1 and receptors 2-3 (GluR1, GluR2/3: AMPA subtype, Chemicon, CA), rat glutamate receptors 5-7 (GluR5-7: kainate subtype, Pharmingen, CA), and rat NMDA receptors (NMDAR1, Pharmingen, CA). Dense and specific signals were obtained with all antibodies. The four types of receptors are broadly distributed in the brain, and, in particular, immunoreactive cells are identified within the major aromatase cell groups located in the medial preoptic nucleus, ventromedial hypothalamus, nucleus striae terminalis, and nucleus taeniae. Dense specific populations of glutamate receptor-immunoreactive cells are also present with a receptor subtype-specific distribution in broad areas of the telencephalon. The distribution of glutamate receptors, therefore, is consistent with the idea that these receptors could be located at the surface of aromatase-containing cells and mediate the rapid regulation of aromatase activity in a direct manner.

Localization and controls of aromatase in the quail spinal cord.

In adult male and female Japanese quail, aromatase-immunoreactive cells were identified in the spinal dorsal horns from the upper cervical segments to the lower caudal area. These immunoreactive cells are located mostly in laminae I-III, with additional sparse cells being present in the medial part of lamina V and, at the cervical level exclusively, in lamina X around the central canal. Radioenzyme assays based on the measurement of tritiated water release confirmed the presence of substantial levels of aromatase activity throughout the rostrocaudal extent of the spinal cord. Contrary to what is observed in the brain, this enzyme activity and the number of aromatase-immunoreactive cells in five representative segments of the spinal cord are not different in sexually mature males or females and are not influenced in males by castration with or without testosterone treatment. The aromatase activity and the numbers of aromatase-immunoreactive cells per section are higher at the brachial and thoracic levels than in the cervical and lumbar segments. These experiments demonstrate for the first time the presence of local estrogen production in the spinal cord of a higher vertebrate. This production was localized in the sensory fields of the dorsal horn, where estrogen receptors have been identified previously in several avian and mammalian species, suggesting an implication of aromatase in the modulation of sensory (particularly nociceptive) processes.

Distribution of aromatase activity in the brain and peripheral tissues of passerine and nonpasserine avian species.

Many behavioral effects of testosterone on hypothalamic and limbic brain areas are mediated by the action, at the cellular level, of estrogens derived from local testosterone aromatization. Aromatase activity and cells containing the aromatase protein and mRNA have accordingly been identified in the brain areas involved in the control of behavior. The presence of an unusually high level of aromatase activity has been detected in the telencephalon of one songbird species, the zebra finch (Taeniopygia guttata), and it is suspected that this high telencephalic aromatase may be a specific feature of songbirds but this idea is supported only by few experimental data. The distribution of aromatase activity in the brain of zebra finches and of one nonsongbird species, the Japanese quail (Coturnix japonica), was compared with the distribution of aromatase activity in the brain of four species of free-living European songbirds, the chaffinch (Fringilla coelebs, Fringillidae), willow warbler (Phylloscopus trochilus, Sylviidae), great tit (Parus major, Paridae), and pied flycatcher (Ficedula hypoleuca, Muscicapidae). High levels of enzyme activity were observed in the diencephalon of all species. The high levels of aromatase activity that had been observed in the zebra finch telencephalon and were thought to be typical of songbirds were also present in the four wild oscine species but not in quail. None of these songbird species had, however, a telencephalic aromatase activity as high as that in the zebra finch, which may represent an extreme as far as the activity of this enzyme in the telencephalon is concerned. Measurable levels of aromatase activity were also detected in all songbird species in the liver and in the three other brain areas that were assayed, the optic lobes, cerebellum, and brain stem, with the exception of the cerebellum in willow warblers and quail, but no detectable activity was observed in the testes, muscle, and adrenals of all species. Additional studies will be needed to identify the functional significance of estrogen synthesis in areas that are not classically known to be implicated in the control of reproduction. Within a given species, the birds that had the highest plasma testosterone levels also displayed the highest levels of diencephalic aromatase activity and the interspecies differences in the two variables were positively related. This raises the possibility that the absolute level of diencephalic aromatase represents a species-specific characteristic under the control of plasma testosterone levels. There was, in contrast, no correlation between the aromatase activity in the telencephalon and the plasma testosterone levels but the enzyme activity was correlated with the plasma levels of luteinizing hormone. These data bring additional support to the idea that the diencephalic and telencephalic aromatases are controlled by independent mechanisms.

Effects of sexual interactions with a male on fos-like immunoreactivity in the female quail brain.

Sexual interactions can cause changes in plasma hormone levels and activate immediate early genes within the mammalian brain. There are marked anatomical differences between the regions activated that relate directly to the sexual specific behaviour and neuroendocrinology of each sex. The aim of this study was to determine if such a sexual dimorphism exists in birds by examining the brain regions stimulated in adult virgin female Japanese quail (Coturnix japonica) during sexual behaviour and comparing this to previously reported data concerning males. Female quail were allowed to freely interact with adult males and both female and male sexual behaviour was recorded. Contrary to previous findings in male quail, no significant induction of Fos-like immunoreactive (FLI) cells was observed following sexual interactions in the preoptic area of females; this area is fundamentally involved in the control of male-type copulatory behaviour. Sexual interactions significantly induced FLI cells in the hyperstriatum ventrale, the part of the archistriatum just lateral to the anterior commissure, and the nucleus intercollicularis. Moreover, prominent activation was detected throughout most of the ventromedial nucleus of the hypothalamus, a region reported to be rich in oestrogen receptors. FLI induction was not a consequence of sexual behaviour induced changes in luteinizing hormone (LH) as plasma LH levels were unaltered. Instead, brain activation must be a consequence of copulation-associated somatosensory inputs or direct stimuli originating from the male. Male quail, like the majority of other birds, lack an intromittant organ (penis) so that the somatosensory inputs to the female are rather different from those in mammals; the precise nature of these inputs is yet to be determined.

Steroid sensitive sites in the avian brain: does the distribution of the estrogen receptor alpha and beta types provide insight into their function?

Studies in avian species have often been useful in elucidating basic concepts relevant to the regulation of reproductive behaviors by sex steroid hormones. Once a link between a steroid hormone and a behavioral response has been established, one can use the localization of steroid hormone receptors in the brain to facilitate the identification of neural circuits that control behavior. The recent identification of a second type of estrogen receptor called estrogen receptor beta or ERbeta has raised new issues about the action of steroid hormones in the brain. A hypothesis has been proposed by Kuiper et al. [1998] based on studies in mammalian species suggesting that ERalpha (the name given to the ER that was previously described) is important for reproduction while ERbeta is more important for non-reproductive functions. In this paper we apply this hypothesis more generally by examining possible functions of ERbeta in avian species. We have initiated studies of the ERbeta in the brain of two avian species, the Japanese quail (Coturnix japonica) and the European starling (Sturnus vulgaris). ERbeta was cloned in both species and the mRNA for this receptor type was localized in the brain employing in situ hybridization histochemistry methods. In both species ERbeta was found to be diffusely present in telencephalic areas consistent with a role for this receptor subtype in cognitive functions. However, ERbeta mRNA was also found in many brain areas that are traditionally thought to be important in the regulation of reproductive functions such as the preoptic region, the bed nucleus of the stria terminalis and the nucleus taeniae. Of the two receptor types, only mRNA for ERalpha was observed in the telencephalic vocal control nucleus HVc of male starlings. Steroid receptors in this nucleus are thought to be an example of an evolutionary specialization that has evolved to coordinate the production of courtship vocalizations with other aspects of reproduction. The lack of ERbeta mRNA expression in HVc is consistent with the hypothesis that ERalpha is preferentially involved in reproductive behaviors while ERbeta is involved in the steroid regulation of other neural functions. However, the widespread occurrence of ERbeta in other nuclei involved in reproductive function suggests that one must be cautious about the general applicability of the above hypothesis until more is known about ERbeta function in these other nuclei.

Estrogen receptor-beta in quail: cloning, tissue expression and neuroanatomical distribution.

A partial estrogen receptor-beta (ERbeta) cDNA had been previously cloned and sequenced in Japanese quail. The 3'- and 5'-rapid amplification of cDNA ends techniques were used here to identify a cDNA sequence of the quail ERbeta that contains a complete open reading frame. For the first time in an avian species, this cDNA sequence and the corresponding amino acid sequence are described. They are compared with the known ERbeta sequences previously described in mammals and with the ERalpha sequences identified in a selection of mammalian and avian species. The analysis by Northern blotting of the ERbeta mRNA expression in the brain and kidneys revealed the presence of several transcripts. The presence of ERbeta identified by reverse transcriptase-polymerase chain reaction demonstrated a widespread distribution quite different from the distribution of ERalpha. The complete neuroanatomical distribution of ERbeta mRNA as determined by in situ hybridization with 35S- and 33P-labeled oligoprobes is also presented. Transcripts are present in many nuclei implicated in the control of reproduction such as the medial preoptic nucleus, the nucleus striae terminalis, and the nucleus taeniae, the avian homologue of the amygdala. These data demonstrate the presence of ERbeta in a nonmammalian species and indicate that the (neuro)-anatomical distribution of this receptor type has been conserved in these two classes of vertebrates. The role of this receptor in the control of reproduction and other physiological processes should now be investigated.

Mating-induced Fos and aromatase are not co-localized in the preoptic area.

Male sexual behavior is determined by the interaction of endocrine and environmental stimuli originating from the female, yet it is unknown how and where these stimuli are integrated within the brain. Activation of copulatory behavior by testosterone is limited by its central aromatization into an estrogen in the preoptic area. We investigated whether mating-induced neuronal activation as identified by the expression of the immediate early gene Fos occurs in aromatase-immunoreactive (ARO-ir) cells of the male quail preoptic area. Fos-immunoreactive (ir) cells were observed within and lateral to these ARO-ir cells groups but few ARO-ir cells contained Fos-ir indicating that mating-related stimuli do not directly affect estrogen-synthesizing cells.

Regional distribution and control of tyrosine hydroxylase activity in the quail brain.

Tyrosine hydroxylase (TH) activity, the rate-limiting step in the synthesis of catecholamines, was quantified in the preoptic area-hypothalamus of adult male Japanese quail by a new assay measuring the tritiated water production from 3,5-[3H]-L-tyrosine. Maximal levels of activity were observed at a 20-25 microM concentration of substrate, with more than 50% inhibition of the activity being recorded at a 100 microM concentration. TH activity was linear as a function of the incubation time during the first 20 min and maximal at a pH of 6.0. TH was heterogeneously distributed in the quail brain with highest levels of activity being found (in decreasing order) in the mesencephalon, diencephalon, and telencephalon. Given the large size of the telencephalon, this is the brain area that contains, as a whole, the highest level of enzyme activity. TH inhibitors that have been well-characterized in mammals, such as 3-iodo-L-tyrosine and L-alpha-methyl-p-tyrosine (AMPT) completely inhibited the enzyme activity at a 100 microM concentration. In mammals, the accumulation of catecholamines exerts a negative feedback control on TH activity. Similar controls were observed in the quail brain. Two inhibitors of the DOPA decarboxylase that should lead to accumulation of DOPA depressed TH activity by 60% or more, and the inhibitor of the dopamine beta-hydroxylase, fusaric acid that should cause an accumulation of dopamine, suppressed 90% of the TH activity. The addition of exogenous DOPA, dopamine, or norepinephrine to the brain homogenates also strongly inhibited TH activity, independently confirming the feedback effects of the enzyme products on the enzyme activity. These data demonstrate that TH activity in the quail brain is heterogeneously distributed and acutely regulated, as it is in mammals, by the accumulation of its products and of the derived catecholamines.

Partial cloning and distribution of estrogen receptor beta in the avian brain.

A partial estrogen receptor beta (ER-beta) cDNA was isolated from testicular quail RNA by RT-PCR with degenerate primers specific to the rat ER-beta sequence. A high expression of ER-beta was demonstrated by RT-PCR in the telencephalon, diencephalon, pituitary, testis and kidneys of male quail but little or no expression was detected in the cerebellum, pectoral muscle and adrenal gland. In situ hybridization with a 35S-labelled oligoprobe in sections through the preoptic area-rostral hypothalamus identified high expression in the medial preoptic nucleus, bed nucleus striae terminalis and nucleus taeniae. These data demonstrate the presence of an ER-beta in brain areas implicated in the control of reproduction in a non-mammalian species.

Neuroanatomical distribution and variations across the reproductive cycle of aromatase activity and aromatase-immunoreactive cells in the pied flycatcher (Ficedula hypoleuca).

The anatomical distribution and seasonal variations in aromatase activity and in the number of aromatase-immunoreactive cells were studied in the brain of free-living male pied flycatchers (Ficedula hypoleuca). A high aromatase activity was detected in the telencephalon and diencephalon but low to negligible levels were present in the optic lobes, cerebellum, and brain stem. In the diencephalon, most aromatase-immunoreactive cells were confined to three nuclei implicated in the control of reproductive behaviors: the medial preoptic nucleus, the nucleus of the stria terminalis, and the ventromedial nucleus of the hypothalamus. In the telencephalon, the immunopositive cells were clustered in the medial part of the neostriatum and in the hippocampus as previously described in another songbird species, the zebra finch. No immunoreactive cells could be observed in the song control nuclei. A marked drop in aromatase activity was detected in the anterior and posterior diencephalon in the early summer when the behavior of the birds had switched from defending a territory to helping the female in feeding the nestlings. This enzymatic change is presumably controlled by the drop in plasma testosterone levels observed at that stage of the reproductive cycle. No change in enzyme activity, however, was seen at that time in other brain areas. The number of aromatase-immunoreactive cells also decreased at that time in the caudal part of the medial preoptic nucleus but not in the ventromedial nucleus of the hypothalamus (an increase was even observed), suggesting that differential mechanisms control the enzyme concentration and enzyme activity in the hypothalamus. Taken together, these data suggest that changes in diencephalic aromatase activity contribute to the control of seasonal variations in reproductive behavior of male pied flycatchers but the role of the telencephalic aromatase in the control of behavior remains unclear at present.

Distribution of androgen receptor-immunoreactive cells in the quail forebrain and their relationship with aromatase immunoreactivity.

The distribution of androgen receptor-like immunoreactive (AR-ir) cells in the quail brain was analyzed by immunocytochemistry with the use of the affinity-purified antibody PG-21-19A raised against a synthetic peptide representing the first 21 N-terminal amino acids of the rat and human AR. This antibody is known to bind to the receptor in the absence as well as in the presence of endogenous ligands, and it was therefore expected that a more complete and accurate characterization of AR-ir cells would be obtained in comparison with previous studies using an antibody that preferentially recognizes the occupied receptor. Selected sections were double labeled for aromatase (ARO) by a technique that uses alkaline phosphatase as the reporter enzyme and Fast blue as the chromogen. AR-ir material was detected in the nucleus of cells located in a variety of brain areas in the preoptic region and the hypothalamus including the medial preoptic (POM), the supraoptic, the paraventricular (PVN), and the ventromedial (VMN) nuclei, but also in the tuberculum olfactorium, the nucleus accumbens/ventral striatum, the nucleus taeniae, the tuberal hypothalamus, the substantia grisea centralis (GCt), and the locus ceruleus. Cells exhibiting a dense AR-ir label were also detected in the nucleus intercollicularis. Preincubation of the primary antibody with an excess of the synthetic peptide used for immunization completely eliminated this nuclear staining. A significant number of AR-ir cells in the POM, VMN, PVN, and tuberal hypothalamus also contained ARO-ir material in their cytoplasm. These data confirm and extend previous studies localizing AR in the avian brain, and raise questions about the possible regulation by androgens of the metabolizing enzyme aromatase.

Anatomical relationships between aromatase and tyrosine hydroxylase in the quail brain: double-label immunocytochemical studies.

The activation of male sexual behavior in Japanese quail (Coturnix japonica) requires the transformation of testosterone to 17beta-estradiol by the enzyme aromatase (estrogen synthetase). There are prominent sex differences in aromatase activity that may be regulated in part by sex differences in catecholaminergic activity. In this study, we investigate, with double-label immunocytochemistry methods, the anatomical relationship between the catecholamine synthesizing enzyme, tyrosine hydroxylase (TH) and aromatase (ARO) in the quail brain. The immunoreactivity observed for each antigen generally matched the previously described distribution. One exception is the observation that cells weakly labeled for aromatase were found widely distributed throughout the telencephalon. The presence of telencephalic aromatase was confirmed independently by radioenzymatic assays. There was an extensive overlap between the distribution of the two antigens in many brain areas. In all densely labeled aromatase-immunoreactive (ARO-ir) cell groups, including the preoptic medial nucleus, nucleus of the stria terminalis, mediobasal hypothalamus, and paleostriatum ventrale, ARO-ir cells were found in close association with TH-ir fibers. These TH-ir fibers often converged on an ARO-ir cell, and one or more TH-ir punctate structure(s) were found in close contact with nearly every densely labeled ARO-ir cell. In the telencephalon (mostly the neostriatum), all TH-ir fibers were found to be part of fiber groups that surrounded weakly immunoreactive aromatase cells. The few cells exhibiting an intracellular colocalization were detected in the anteroventral periventricular nucleus. These results are consistent with the hypothesis that catecholaminergic inputs regulate brain aromatase.

Systemic and intracerebroventricular injections of vasotocin inhibit appetitive and consummatory components of male sexual behavior in Japanese quail.

The authors investigated the behavioral actions of vasotocin (VT) in castrated testosterone-treated male Japanese quail. The appetitive and consummatory components of sexual behavior as well as the occurrence frequency of crows were inhibited, in a dose-dependent manner, by injections of VT. The authors observed opposite effects after injection of the V1 receptor antagonist, dPTyr(Me)AVP. Lower doses of VT were more active after central than after systemic injection, and effects of systemic injections of VT were blocked by a central injection of dPTyr(Me)AVP. The behavioral inhibition was associated with a modified diuresis after systemic but not central injection. These results provide direct evidence that VT affects male sexual behavior in quail by a direct action on the brain independent of its peripheral action on diuresis.

Copulation activates Fos-like immunoreactivity in the male quail forebrain.

It has been demonstrated using Fos immunocytochemistry that copulation activates specific cell populations in the mammalian brain. Prior to this study, no similar work has been carried out in birds. In mammals, Fos has identified brain circuits activated by genital (penile)/somatosensory and by olfactory/vomeronasal stimuli. Such inputs, of course, should play little or no role in birds (no penis, little or no role for olfaction) and a differential responsiveness could therefore be expected. Male Japanese quail (Coturnix japonica) were allowed to interact freely with adult females and the presence of active sexual behavior, including cloacal contact movements, was confirmed in each case. Control subjects were exposed to a domestic chick (same size as an adult quail) and no sexual behavior was observed. Copulation induced the appearance of Fos-like immunoreactive (FLI) cells in the preoptic area, the hyperstriatum ventrale, parts of the archistriatum, and the nucleus intercollicularis. Induction of FLI cells was observed throughout the rostral to caudal extent of the preoptic region of males from the level of the tractus septomesencephalicus to the level of the anterior commissure, and in the rostral part of the hypothalamus to the level of the supraoptic decussation. The FLI cells did not lie directly adjacent to the third ventricle, but were located 500-1000 microns from the ventricle wall at the level of the lateral edge of the medial preoptic nucleus or, in more caudal sections, in a position ventrolateral to the bed nucleus striae terminalis. It is unlikely that the Fos induction in males resulted from copulation-induced endocrine changes because copulation did not affect plasma levels of luteinizing hormone or testosterone. It is concluded that the responses were due to copulation-associated somatosensory inputs and/or to stimuli originating from the female.

Distribution of aromatase-immunoreactive cells in the forebrain of zebra finches (Taeniopygia guttata): implications for the neural action of steroids and nuclear definition in the avian hypothalamus.

Cells immunoreactive for the enzyme aromatase were localized in the forebrain of male zebra finches with the use of an immunocytochemistry procedure. Two polyclonal antibodies, one directed against human placental aromatase and the other directed against quail recombinant aromatase, revealed a heterogeneous distribution of the enzyme in the telencephalon, diencephalon, and mesencephalon. Staining was enhanced in some birds by the administration of the nonsteroidal aromatase inhibitor, R76713 racemic Vorozole) prior to the perfusion of the birds as previously described in Japanese quail. Large numbers of cells immunoreactive for aromatase were found in nuclei in the preoptic region and in the tuberal hypothalamus. A nucleus was identified in the preoptic region based on the high density of aromatase immunoreactive cells within its boundaries that appears to be homologous to the preoptic medial nucleus (POM) described previously in Japanese quail. In several birds alternate sections were stained for immunoreactive vasotocin, a marker of the paraventricular nucleus (PVN). This information facilitated the clear separation of the POM in zebra finches from nuclei that are adjacent to the POM in the preoptic area-hypothalamus, such as the PVN and the ventromedial nucleus of the hypothalamus. Positively staining cells were also detected widely throughout the telencephalon. Cells were discerned in the medial parts of the ventral hyperstriatum and neostriatum near the lateral ventricle and in dorsal and medial parts of the hippocampus. They were most abundant in the caudal neostriatum where they clustered in the dorsomedial neostriatum, and as a band of cells coursing along the dorsal edge of the lamina archistriatalis dorsalis. They were also present in high numbers in the ventrolateral aspect of the neostriatum and in the nucleus taeniae. None of the telencephalic vocal control nuclei had appreciable numbers of cells immunoreactive for aromatase within their boundaries, with the possible exception of a group of cells that may correspond to the medial part of the magnocellular nucleus of the neostriatum. The distribution of immunoreactive aromatase cells in the zebra finch brain is in excellent agreement with the distribution of cells expressing the mRNA for aromatase recently described in the finch telencephalon. This widespread telencephalic distribution of cells immunoreactive for aromatase has not been described in non-songbird species such as the Japanese quail, the ring dove, and the domestic fowl.

Effects of testosterone and its metabolites on aromatase-immunoreactive cells in the quail brain: relationship with the activation of male reproductive behavior.

The enzyme aromatase converts testosterone (T) into 17 beta-estradiol and plays a pivotal role in the control of reproduction. In particular, the aromatase activity (AA) located in the preoptic area (POA) of male Japanese quail is a limiting step in the activation by T of copulatory behavior. Aromatase-immunoreactive (ARO-ir) cells of the POA are specifically localized within the cytoarchitectonic boundaries of the medial preoptic nucleus(POM), a sexually dimorphic and steroid-sensitive structure that is a necessary and sufficient site of steroid action in the activation of behavior. Stereotaxic implantation of aromatase inhibitors in but not around the POM strongly decreases the behavioral effects of a systemic treatment with T of castrated males. AA is decreased by castration and increased by aromatizable androgens and by estrogens. These changes have been independently documented at three levels of analysis: the enzymatic activity measured by radioenzymatic assays in vitro, the enzyme concentration evaluated semi-quantitatively by immunocytochemistry and the concentration of its messenger RNA quantified by reverse transcription-polymerase chain reaction (RT-PCR). These studies demonstrate that T acting mostly through its estrogenic metabolites regulates brain aromatase by acting essentially at the transcriptional level. Estrogens produced by central aromatization of T therefore have two independent roles: they activate male copulatory behavior and they regulate the synthesis of aromatase. Double label immunocytochemical studies demonstrate that estrogen receptors(ER) are found in all brain areas containing ARO-ir cells but the extent to which these markers are colocalized varies from one brain region to the other. More than 70% of ARO-ir cells contain detectable ER in the tuberal hypothalamus but less than 20% of the cells display this colocalization in the POA. This absence of ER in ARO-ir cells is also observed in the POA of the rat brain. This suggests that locally formed estrogens cannot control the behavior and the aromatase synthesis in an autocrine fashion in the cells where they were formed. Multi-neuronal networks need therefore to be considered. The behavioral activation could result from the action of estrogens in ER-positive cells located in the vicinity of the ARO-ir cells where they were produced (paracrine action). Alternatively, actions that do not involve the nuclear ER could be important. Immunocytochemical studies at the electron microscope level and biochemical assays of AA in purified synaptosomes indicate the presence of aromatase in presynaptic boutons. Estrogens formed at this level could directly affect the pre-and post-synaptic membrane or could directly modulate neurotransmission namely through their metabolization into catecholestrogens (CE) which are known to be powerful inhibitors of the catechol- omicron - methyl transferase (COMT). The inhibition of COMT should increase the catecholaminergic transmission. It is significant to note, in this respect, that high levels of 2-hydroxylase activity, the enzyme that catalyzes the transformation of estrogens in CE, are found in all brain areas that contain aromatase. On the other hand, the synthesis of aromatase should also be controlled by estrogens in an indirect, transynaptic manner very reminiscent of the way in which steroids indirectly control the production of LHRH. Fibers that are immunoreactive for tyrosine hydroxylase (synthesis of dopamine), dopamine beta-hydroxylase (synthesis of norepinephrine) or vasotocine have been identified in the close vicinity of ARO-ir cells in the POM and retrograde tracing has identified the origin of the dopaminergic and noradrenergic innervation of these areas. A few preliminary physiological experiments suggest that these catecholaminergic inputs regulate AA and presumably synthesis.

Pre- and post-translational regulation of aromatase by steroidal and non-steroidal aromatase inhibitors.

Treatment of castrated quail with testosterone (T) reliably activates male copulatory behavior and, at the same time, increases the aromatase activity (AA), the number of aromatase-immunoreactive (ARO-ir) cells and the concentration of aromatase mRNA as measured by RT-PCR in the brain. All these effects can be mimicked by estrogens. The behavioral effects of T can be blocked by a variety of aromatase inhibitors and, in parallel, the AA is strongly inhibited in the preoptic area (POA). We showed recently that the steroidal inhibitor, 4-OH-androstenedione (OHA) markedly decreases the immunostaining density of brain ARO-ir cells while the non-steroidal inhibitor, R76713 (racemic Vorozole; VOR) unexpectedly increased the density of this staining, despite the fact that the enzyme activity was completely inhibited. To generalize these findings and try to identify the underlying mechanism, we compared here the effects of two steroidal (OHA and androstatrienedione [ATD]) and two non-steroidal (VOR and Fadrozole [FAD]) aromatase inhibitors on the aromatase immunostaining and aromatase mRNA concentration in the brain of castrated quail concurrently treated with T. The 4 inhibitors significantly blocked the activation by T of male copulation. The two steroidal inhibitors decreased the immunostaining of brain ARO-ir cells but both VOR and FAD markedly enhanced the density of this staining. In parallel, OHA and ATD completely blocked the T-induced increase in aromatase mRNA concentration, while VOR and FAD had no effect on these RNA concentrations in the POA-anterior hypothalamus and they decreased them only slightly in the posterior hypothalamus. Taken together these results suggest that the inhibition of AA by ATD or OHA and the subsequent removal of locally produced estrogens blocks the synthesis of aromatase presumably at the transcriptional level. By contrast, the two non-steroidal inhibitors tested here block AA but in parallel increase the aromatase immunostaining. This effect does not result from an enhanced transcription and it is therefore speculated that these compounds increase either the translation of the aromatase mRNA or the half-life of the protein itself.