The heart of an acrobatic bird.

The courtship behavior of some species of birds can be energetically demanding, but it is unknown if cardiovascular specializations enable such behaviors. While performing a highly acrobatic courtship dance, heart rate in male golden-collared manakins increases briefly to 1300 beats per minute, among the highest heart rates recorded in any bird or mammal. We hypothesize that male manakins have enhanced cardiovascular capabilities to meet these demands on the heart. Using histological and molecular techniques, we examined manakin heart structure as well as expression of genes involved in Ca2+ handling, action potential duration, steroidal signaling and cardiac growth. These measures were also made on the hearts of zebra finches, a similar-sized bird with limited cardiovascular demands. Compared to the zebra finch, the manakin had a significantly thicker left ventricular (LV) muscle (cross-sectional thickness of the free LV wall and septum) with a smaller LV chamber. In addition, compared to zebra finches, manakin hearts had significantly greater gene expression of ryanodine receptors as well as androgen receptors. Testosterone (T) treatment of non-breeding manakins (with low T) increased gene expression of the Ca2+ pump SERCA. These observations suggest that hearts of breeding male manakins require specialized Ca2+ handling and androgens may facilitate manakin cardiovascular function.

Energetics of the acrobatic courtship in male golden-collared manakins (Manacus vitellinus).

In lek mating systems, females choose mates through indicators of quality, which males may exhibit by their performance of courtship displays. In temperate regions, displaying seasons are brief (one to two months), whereas in the tropics courtship seasons may be prolonged. Moreover, in temperate-breeding animals lekking behaviour can be energetically demanding, but little is known about the energy costs of lekking in tropical animals. Daily, over the course of a nearly seven-month-long breeding season, male golden-collared manakins (Manacus vitellinus) of Panamanian rainforests perform acrobatic courtship displays that markedly elevate heart rates, suggesting that they require high energy investment. Typically, animals of tropical lowland forests (such as manakins) exhibit a 'slow pace of life' metabolic strategy. We investigated whether male manakin courtship is indeed metabolically costly or whether the birds retain a low daily energy expenditure (DEE), as seen in other tropical species. To assess these questions, we calibrated manakin heart rate against metabolic rate, examined daily lek activity and, using telemetry, obtained heart rates of individual wild, lekking male manakins. Although metabolic rates peak during courtship displays, we found that males actually invest minimal time (only approx. 5 min d(-1)) performing displays. As a consequence, the DEE of approximately 39 kJ d(-1) for male manakins is comparable to other lowland tropical species. The short, intense bursts of courtship by these birds make up only approximately 1.2% of their total DEE. Presumably, this cost is negligible, enabling them to perform daily at their arenas for months on end.

The genomic consequences of adaptive divergence and reproductive isolation between species of manakins.

The processes of adaptation and speciation are expected to shape genomic variation within and between diverging species. Here we analyze genomic heterogeneity of genetic differentiation and introgression in a hybrid zone between two bird species (Manacus candei and M. vitellinus) using 59 100 SNPs, a whole genome assembly, and Bayesian models. Measures of genetic differentiation (FST) and introgression (genomic cline center [α] and rate [ß]) were highly heterogeneous among loci. We identified thousands of loci with elevated parameter estimates, some of which are likely to be associated with variation in fitness in Manacus populations. To analyze the genomic organization of differentiation and introgression, we mapped SNPs onto a draft assembly of the M. vitellinus genome. Estimates of FST, α, and ß were autocorrelated at very short physical distances (< 100 bp), but much less so beyond this. In addition, average statistical associations (linkage disequilibrium) between SNPs were generally low and were not higher in admixed populations than in populations of the parental species. Although they did not occur with a constant probability across the genome, loci with elevated FST, α, and ß were not strongly co-localized in the genome. Contrary to verbal models that predict clustering of loci involved in adaptation and isolation in discrete genomic regions, these results are consistent with the hypothesis that genetic regions involved in adaptive divergence and reproductive isolation are scattered throughout the genome. We also found that many loci were characterized by both exceptional genetic differentiation and introgression, consistent with the hypothesis that loci involved in isolation are also often characterized by a history of divergent selection. However, the concordance between isolation and differentiation was only partial, indicating a complex architecture and history of loci involved in isolation.

Context-specific effects of estradiol on spatial learning and memory in the zebra finch.

Estradiol is known to impact cognitive function including spatial learning and memory, with studies focused largely on rodent models. Estrogens can be produced peripherally or centrally as neuroestrogens, and the specific role for neuroestrogens in memory processes remains unresolved. Many songbirds possess remarkable spatial memory capabilities and also express the estrogen synthetic enzyme aromatase abundantly in the hippocampus, suggesting that locally-produced estrogens may promote the acquisition or retrieval of spatial memories in these birds. We examined the effect of estradiol on spatial memory in three contexts in the zebra finch: retrieval after discrimination training, retrieval after familiarization but without discrimination training, and memory acquisition, using a combination of estradiol implants and oral dosing with the aromatase inhibitor fadrozole (FAD). Retrieval of spatial memory in both contexts was impaired when estradiol production was blocked. However, spatial memory acquisition was enhanced when estradiol production was inhibited whereas estradiol replacement impaired acquisition. These results provide evidence for a context-specific role of estradiol in songbird spatial memory, results that find accord with some mammalian studies but have not yet been observed in birds.

Sex-specific, rapid neuroestrogen fluctuations and neurophysiological actions in the songbird auditory forebrain.

Recent evidence shows that brain-derived steroids such as estrogens ("neuroestrogens") are controlled in a manner very similar to traditional neurotransmitters. The advent of in vivo microdialysis for steroids in songbirds has provided new information about the spatial and temporal dynamics of neuroestrogen changes in a region of the auditory cortex, the caudomedial nidopallium (NCM). Here, experiments using in vivo microdialysis demonstrate that neuroestradiol (E(2)) fluctuations occur within the auditory NCM during presentation of naturalistic auditory and visual stimuli in males but only to the presentation of auditory stimuli in females. These changes are acute (within 30 min) and appear to be specific to the NCM, because similar treatments elicit no changes in E(2) in a nearby mesopallial region or in circulating plasma. Further experiments coupling in vivo steroid microdialysis with extracellular recordings in NCM show that neuroestrogens rapidly boost auditory responses to song stimuli in females, similar to recent observations in males. We also find that the rapid actions of estradiol on auditory responses are fully mimicked by the cell membrane-impermeable estrogen biotinylestradiol, consistent with acute estrogen actions at the neuronal membrane. Thus we conclude that local and acute E(2) flux is regulated by convergent multimodal sensory input, and that this regulation appears to be sex-specific. Second, rapid changes in local E(2) levels in NCM have consequences for the modulation of auditory processing in females and males. Finally, the rapid actions of neuroestrogens on NCM auditory processing appear to be mediated by a nonclassical, membrane-bound estrogen receptor.

Neural sensitivity to sex steroids predicts individual differences in aggression: implications for behavioural evolution.

Testosterone (T) regulates many traits related to fitness, including aggression. However, individual variation in aggressiveness does not always relate to circulating T, suggesting that behavioural variation may be more closely related to neural sensitivity to steroids, though this issue remains unresolved. To assess the relative importance of circulating T and neural steroid sensitivity in predicting behaviour, we measured aggressiveness during staged intrusions in free-living male and female dark-eyed juncos (Junco hyemalis). We compared aggressiveness to plasma T levels and to the abundance of androgen receptor (AR), aromatase (AROM) and oestrogen receptor alpha (ORα) mRNA in behaviourally relevant brain areas (avian medial amygdala, hypothalamus and song control regions). We also asked whether patterns of covariation among behaviour and endocrine parameters differed in males and females, anticipating that circulating T may be a better predictor of behaviour in males than in females. We found that circulating T related to aggressiveness only in males, but that gene expression for ORα, AR and AROM covaried with individual differences in aggressiveness in both sexes. These findings are among the first to show that individual variation in neural gene expression for three major sex steroid-processing molecules predicts individual variation in aggressiveness in both sexes in nature. The results have broad implications for our understanding of the mechanisms by which aggressive behaviour may evolve.

Neurosteroids and brain sexual differentiation.

There is new evidence that the brain of developing songbirds can synthesize estradiol de novo. In males, this neurally derived estrogen might masculinize a connection within the neural song system. These results challenge traditional concepts about mechanisms of brain sexual differentiation and reveal a significant function for neurosteroids.

Neurosteroids and female reproduction: estrogen increases 3beta-HSD mRNA and activity in rat hypothalamus.

A central event in mammalian reproduction is the LH surge that induces ovulation and corpus luteum formation. Typically, the LH surge is initiated in ovariectomized rats by sequential treatment with estrogen and progesterone (PROG). The traditional explanation for this paradigm is that estrogen induces PROG receptors (PR) that are activated by exogenous PROG. Recent evidence suggests that whereas exogenous estrogen is necessary, exogenous PROG is not. In ovariectomized-adrenalectomized rats, estrogen treatment increases hypothalamic PROG levels before an LH surge. This estrogen-induced LH surge was blocked by an inhibitor of 3beta-hydroxysteroid dehydrogenase/delta5-delta4 isomerase (3beta-HSD), the proximal enzyme for PROG synthesis. These data indicate that estrogen induces de novo synthesis of PROG from cholesterol in the hypothalamus, which initiates the LH surge. The mechanism(s) by which estrogen up-regulates neuro-PROG is unknown. We investigated whether estrogen increases 1) mRNA levels for several proteins involved in PROG synthesis and/or 2) activity of 3beta-HSD in the hypothalamus. In ovariectomized-adrenalectomized rats, estrogen treatment increased 3beta-HSD mRNA in the hypothalamus, as measured by relative quantitative RT-PCR. The mRNAs for other proteins involved in steroid synthesis (sterol carrier protein 2, steroidogenic acute regulatory protein, and P450 side chain cleavage) were detectable in hypothalamus but not affected by estrogen. In a biochemical assay, estrogen treatment also increased 3beta-HSD activity. These data support the hypothesis that PROG is a neurosteroid, produced locally in the hypothalamus from cholesterol, which functions in the estrogen positive-feedback mechanism driving the LH surge.

Estrogen synthesis in vivo in the adult zebra finch: additional evidence that circulating estrogens can originate in brain.

Aromatase activity is abundant in limbic and nonlimbic neural structures in zebra finches, especially in the telencephalon near neural circuits that control singing behavior. Also, male songbirds can have high estrogen levels in blood. Because it is difficult to detect aromatase activity in other male tissues, we have postulated that the brain itself is the source of the estrogen present in the blood of males. Previously, we developed methods to measure estrogen synthesis in vivo by injecting [3H]androgen into the systemic circulation or directly into tissues and then determining the quantity of [3H]estrogen entering or leaving the brain. Our results support our hypothesis that the brain is the primary site of estrogen synthesis in males of this species. Here, we confirm that [3H]estrogen emanates from the brain after systemic [3H]androgen injection by showing that the presumptive estrogen in jugular plasma is significantly reduced by treatment with an aromatase inhibitor. In females, but not males, estrogens can be found in large amounts in carotid plasma after systemic androgen injection, presumably due to ovarian estrogen synthesis. However, carotid estrogen levels are variable in females, so it is difficult to determine if the female brain contributes estrogen to blood as is seen in males. The data presented here suggest that the adrenal of males is not an important site of aromatization, because [3H]estrogens are undetected in the adrenals of castrated or sham-castrated males after adrenal injections of [3H] androgen. Nevertheless, at least 2.8- to 17.3-fold more [3H]estrogen was present in the jugular than in the carotid of castrated males after systemic injection of [3H]androgen. Because androgen levels were in the physiological range, we conclude that the brain routinely enriches the estrogen content of blood in normal adult males of this species.

Plasma sex steroids and tissue aromatization in hatchling zebra finches: implications for the sexual differentiation of singing behavior.

One of the best examples for sex hormone regulation of brain development is found in songbirds. In zebra finches, only males sing because of striking sex differences in the neural circuitry that controls songs. Because developing females treated with estradiol (E2) develop a masculine song system, E2 is considered the normal masculinizing hormone. However, questions about the role of E2 in male development persist, because E2 treatments that masculinize song can demasculinize other sexual behaviors, and there exists contradictory evidence for high levels of circulating E2 in developing males. We remeasured plasma steriods in zebra finches during the first 13 days after hatching. E2 circulated at low levels, and there were no sex differences in circulating E2, estrone, testosterone, androstenedione, or dihydrotestosterone. We also measured aromatase activity [( 3H]androstenedione conversion to [3H]estrone and [3H]E2) in gonad, adrenal, brain, and other tissues of hatchlings. Aromatase was abundant in ovary, but was not definitively detected in testes, adrenals, or other nonneural tissues of males. Aromatase was also found in diencephalon and in high amounts in telencephalon, but sex differences were not detected in whole brain or cellular subfractions of telencephalon. Because ovarian steroidogenesis is high, it may be involved in differentiation of the female zebra finch, as in nonpasserine birds. By contrast, the functional estrogen necessary for masculinization of song is most likely derived from brain, supplied with substrate from the adrenals. The puzzle remains why the song system is not masculinized in females, who possess high levels of aromatizable androgens and telencephalic aromatase.

Aromatase activity in quail brain: correlation with aggressiveness.

Testosterone (T) triggers aggressive behavior in males of many vertebrate species; however, the neural and hormonal basis of individual differences in the frequency or intensity of aggressive behavior is still debated. Using the Japanese quail (Coturnix coturnix japonica), a species in which individuals exhibit a wide range of aggressiveness in nature and the laboratory, together with a newly devised test procedure for quantifying aggressiveness, we recently demonstrated that aggression is estrogen dependent. Here we extend these studies by testing the hypothesis that aromatization in brain is a rate-limiting step in the expression of individual differences in aggressiveness. Using procedures previously validated for this species, aromatase and 5 alpha- and 5 beta-reductase activities were estimated in selected brain regions of reproductively active male quail by measuring conversion of [3H]androstenedione to [3H]estrone, [3H]5 alpha-androstanedione, and [3H]5 beta-androstanedione, respectively. In Exp 1, behaviorally inexperienced test birds were killed 90 sec after a single behavioral test. Aggressiveness of individuals in this group, as determined by pecking and locomotor activity in response to visualization of a conspecific, ranged 3- to 4-fold from high to low. Aromatase activity in the posterior hypothalamus (PHYP) was significantly higher in males rated high for aggressiveness than in animals rated low (1.04 vs. 0.59 pmol/h.mg protein; P less than 0.02). Similar differences were observed in the anterior hypothalamus/preoptic area (AHPOA) but were not significant. In Exp 2, sexually mature males were behaviorally tested eight times over 22 days and killed 24 h after the final test. Aggressiveness varied 5-fold from high to low, although the rating in a given bird remained constant with time and repeat testing. Aromatase activity in the AHPOA was significantly greater in birds rated high for aggressiveness than in low aggressiveness birds (3.77 vs. 2.80 pmol/h.mg protein; P less than 0.02). In addition, when AHPOA aromatase in all birds was plotted against behavioral intensity, there was a 2-fold variation and a significant positive correlation (r = 0.556; P less than 0.02). Similar differences were observed in PHYP, but these were of borderline significance. By contrast, aromatase levels outside the AHPOA and PHYP were unrelated to behavior. Moreover, in both Exp 1 and 2, 5 alpha- and 5 beta-reductase activities in AHPOA, PHYP, and other brain regions; plasma T, 5 alpha-dihydrotestosterone, and total estrogens; and relative testicular weights were not consistently related to aggression.(ABSTRACT TRUNCATED AT 400 WORDS)

Zebra finch aromatase gene expression is regulated in the brain through an alternate promoter.

The learned singing behavior in songbirds is sex-steroid-dependent and sexually dimorphic. Estrogen plays a major role in masculinizing the song system in these songbirds. The songbird brain synthesizes large amounts of estrogen, which, in the case of zebra finches, have been found to enter the systemic circulation. Aromatase cytochrome P450 is the key enzyme catalyzing the conversion of androgens to estrogens. We have cloned a novel alternatively spliced form of aromatase cDNA expressed predominantly in the zebra finch brain. We have also isolated and characterized the gene coding for zebra finch aromatase which spans 20kb in length. The alternate forms of aromatase mRNA (ARO) differ in their 5'-untranslated regions encoded by either exon 1a or 1b. The putative promoter sequences controlling the regulation of the alternate forms of ARO in zebra finches contain consensus binding sites for various transcription factors. While both the promoters have binding sites for SRY-like transcription factor, a binding site for SF-1 is present only in the promoter 1b active in the ovary. Intriguingly, a 55bp segment within the promoter 1a sequence appears to be highly conserved among zebra finch, mouse and human aromatase promoters active in the brain.

An atlas of aromatase mRNA expression in the zebra finch brain.

Neural conversion of androgen to estrogen by aromatase is an important step in the development and expression of masculine behavior in mammals and birds. In contrast to the low telencephalic levels of aromatase in adult mammals and nonsongbirds, the zebra finch telencephalon possesses high aromatase activity. This study maps, by in situ hybridization, cells that express aromatase mRNA in the adult zebra finch telencephalon, diencephalon, midbrain, and pons. High aromatase mRNA expression was observed in the caudal neostriatum, limbic archistriatum, and hypothalamus. The hippocampus, parahippocampal area, and hyperstriatum accessorium contained cells expressing moderate amounts of aromatase message. Weakly labeled cells were found in the rostral neostriatum, lobus parolfactorius, and mesencephalic reticular formation. These findings are consistent with aromatase activity measurements of zebra finch tissue and document with anatomical precision both the widespread expression of aromatase mRNA in the brain and novel sites of brain aromatase. This study identifies the caudal neostriatum as a major site of telencephalic aromatase. A previous survey (Gahr et al., 1993: J. Comp. Neurol. 327:112-122) of several avian species found that the presence of estrogen receptors in parts of the caudal neostriatum is unique to songbirds, which are the only birds to possess the elaborated telencephalic song system. Together, these findings suggest that the heightened estrogen synthesis and estrogen sensitivity of the passerine caudal neostriatum may have some functional relation with the telencephalic circuits responsible for song.

Distribution and regulation of telencephalic aromatase expression in the zebra finch revealed with a specific antibody.

In songbirds, aromatase (estrogen synthase) activity and mRNA are readily detectable in the brain. This neural aromatization presumably provides estrogen to steroid-sensitive targets via autocrine, paracrine, and synaptic mechanisms. The location of immunoreactive protein, however, has been difficult to describe completely, particularly in distal dendrites, axons, and terminals of the forebrain. Here we describe the neuroanatomical distribution of aromatase in the zebra finch by using a novel antibody raised specifically against zebra finch aromatase. The distribution of aromatase-positive somata in the zebra finch brain is in excellent agreement with previous reports. Additionally, this antibody reveals elaborate, spinous dendritic arbors, fine-beaded axons, and punctate terminals of telencephalic neurons that may synthesize estrogen. Some of these axon-like fibers extend into the high vocal center (HVC) and the robust nucleus of the archistriatum (RA) in males and females, suggesting a role for presynaptic aromatization in cellular processes within these loci. Adult males have more aromatase-positive fibers in the caudomedial neostriatum (NCM) and the preoptic area (POA) compared to females, despite the lack of detectable sex differences in the number of immunoreactive somata at these loci. Thus, the compartmentalization of aromatase in dendrites and axons may serve a sexually dimorphic function in the songbird. Finally, in adult males, aromatase expression is down-regulated by circulating estradiol in the hippocampus, but not in the NCM or POA. The distribution of aromatase suggests a role for aromatization in the regulation of pre- and postsynaptic function in steroid sensitive areas of the songbird forebrain.

Aromatase, 5 alpha- and 5 beta-reductase in brain, pituitary and skin of the sex-role reversed Wilson's phalarope.

While intrasexual competition for mates is generally considered to be an androgen-dependent characteristic of reproductively active males, in the Wilson's phalarope (Phalaropus tricolor) it is the female that acquires the brighter nuptial plumage and aggressively competes for access to the less aggressive males. Despite this pronounced sex-role reversal, circulating sex steroid hormones of breeding phalaropes are similar to those of avian species displaying traditional male-female reproductive roles. To investigate whether these behavioural and morphological steroid-dependent differences may be due to differences in target organ metabolism of circulating androgen, [3H]androstenedione in the presence of an NADPH-generating system was incubated with homogenates of brain, pituitary and skin of male and female Wilson's phalaropes collected from a naturally breeding population. Oestrone, 5 alpha-androstanedione and 5 beta-androstanedione were measured as endpoints of aromatization, 5 alpha-reduction and 5 beta-reduction respectively. Aromatase activity in the anterior hypothalamus/preoptic area (AHPOA) and posterior hypothalamus was greater in breeding males with high circulating concentrations of testosterone than in females, and activity in the AHPOA was greater in breeding than in non-breeding males (with low circulating testosterone). Aromatase levels did not differ in septum, archistriatum, hyperstriatum or pituitary. 5 alpha- and 5 beta-reductase were detected in all neuroendocrine tissues sampled and although there were no significant male-female differences, 5 alpha-reductase was greater in the AHPOA of breeding than of nonbreeding males.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolation and characterization of a zebra finch aromatase cDNA: in situ hybridization reveals high aromatase expression in brain.

The brain regions controlling song are much larger in male than in female zebra finches. This sex difference is thought to arise developmentally when the male's brain is exposed to higher levels of estrogen. The synthesis of estrogen from androgen is catalyzed by aromatase, a key enzyme implicated in song development in the zebra finch. To study the role of estrogen synthesis in the organization of brain regions responsible for song, a cDNA encoding aromatase was isolated from a zebra finch ovarian cDNA library. The 3188 bp cDNA contains a 1527 bp open reading frame with 5' and 3' untranslated regions of 116 bp and 1545 bp, respectively. The deduced polypeptide is 509 amino acids in length, and is highly homologous to aromatases reported for chicken (92%), human (72%), mouse (70%), rat (69%) and trout (53%). Northern blot analysis revealed 5.4 kb, 4.8 kb and 3.2 kb aromatase mRNAs in brain and ovary. In situ hybridization histochemistry revealed the expression of aromatase mRNA in ovarian thecal cells of some, but not all, follicles, suggesting that aromatase gene expression is regulated during follicular maturation. In the adult brain, the distribution of aromatase mRNA was surprisingly widespread, and included the preoptic area, hypothalamus, hippocampus and neostriatum. By contrast, little aromatase mRNA expression was noted in the song nuclei (HVC, RA, area X). This study, the first description of aromatase mRNA expression by in situ hybridization in the brain of any species, identifies a surprisingly large number of cells that express aromatase mRNA in the zebra finch telencephalon. This pattern may be a unique feature of all songbirds.

Cyclic AMP phosphodiesterases in the zebra finch: distribution, cloning and characterization of a PDE4B homolog.

Songbirds are important animal models for studying neural mechanisms underlying learning and memory. While evidence has emerged that cAMP plays a significant role in invertebrate and mammalian learning, little is known about the role of cAMP pathways in regulating neuronal function in birds. With the goal of identifying important components of this pathway, we report the first cloning of a cAMP-specific, Type IV phosphodiesterase (PDE4) in a non-mammalian vertebrate. A combination of PCR analysis and cDNA library screening was used to show that homologs of the four known mammalian PDE4 genes also exist in zebra finch. A full-length cDNA representing the zebra finch homolog of PDE4B1 was isolated from a telencephalic library. Expression of this cDNA in human embryonic kidney 293 (HEK) cells yielded an enzyme that hydrolyzed cAMP with a low K(m) and was inhibited by micromolar concentrations of rolipram; these properties are typical of all known mammalian PDE4s. In brain, northern blots revealed transcripts of 3.6 and 4.4 kb in adults, but only the 3.6 kb transcript in juveniles, suggesting that PDE4 expression is developmentally regulated. In situ hybridization of tissue sections demonstrated that PDE4 message was distributed widely throughout the adult zebra finch brain, including regions controlling the learning of songs and the acquisition of spatial memories. These data suggest that PDE4 enzymes may influence a variety of brain functions in these birds and play a role in learning.

Rapid upregulation of aromatase mRNA and protein following neural injury in the zebra finch (Taeniopygia guttata).

The expression of aromatase (oestrogen synthase) within the vertebrate central nervous system (CNS) is key in the provision of local oestrogens to neural circuits. Aromatase expression appears to be exclusively neuronal under normal conditions. However, some in vitro studies suggest the presence of astrocytic aromatase in songbirds and mammals. Recently, aromatase in reactive astrocytes has been demonstrated in response to neural injury in the mammalian CNS. Since the glial aromatase expression first documented in cultures of the songbird telencephalon may reflect processes similar to those in response to mammalian neural injury, we investigated whether injury alters the pattern of aromatase-expression in the zebra finch, a species with very high levels of forebrain aromatase expression. Adult males received a penetrating neural injury to the right hemisphere and were killed either 24 or 72 h later. Controls were anaesthetized and otherwise unmanipulated. We determined the expression of aromatase mRNA and protein using in situ hybridization and immunocytochemistry, respectively. Both the transcription and translation of aromatase is dramatically upregulated around the lesion site in response to neural injury in the zebra finch forebrain. This effect is robust and rapid, occurring within 24 h of the injury itself. Cells that upregulate aromatase appear to be reactive astrocytes based upon morphology. The hemisphere contralateral to the injury and both hemispheres in control birds showed the normal, exclusively neuronal pattern of aromatase expression. The upregulation of aromatase in astrocytes may provide high levels of oestrogen available to modulate processes such as CNS repair. Injury-induced upregulation of astrocytic aromatase may be a general characteristic of the injured vertebrate brain.

5 beta-reductase and other androgen-metabolizing enzymes in primary cultures of developing zebra finch telencephalon.

Enzymes in the avian brain irreversibly catalyze the conversion of androgens into either active metabolites (aromatase and 5 alpha-reductase) or inactive metabolites (5 beta-reductase), 5 beta-reductase is thought to influence the formation of active metabolites by reducing the concentration of androgenic substrate available for these reactions. However, because these enzymes have different regional, cellular and subcellular distributions in brain, the traditional method to measure enzyme activity in brain homogenates may be inaccurate because of artificial mixing of enzymes. Recently, we have prepared primary cell cultures from the telencephalons of developing zebra finches. Cell cultures offer the advantage that enzyme activity can be measured in live cells in which the relative distribution of enzymes may more closely reflect that found in vivo. We have previously reported that aromatase is expressed at high levels in these cultures, and that it is active in both neurons and in glia. Here we report on the activities of 5 alpha- and 5 beta-reductase in these cell cultures. Along with aromatase, 5 beta-reductase was expressed at high levels in these mixed cell cultures, including cultures highly enriched in glia. This suggests that 5 beta-reductase is present in non-neuronal cells in brain, possibly co-localized with aromatase. However, despite the presence of 5 beta-reductase, aromatase was detected reliably in vitro even when the concentration of substrate was low. Thus, 5 beta-reductase does not prevent the synthesis of estrogen in the telencephalon of developing zebra finches. By contrast, 5 alpha-reductase activity was very low or absent in these cultures.(ABSTRACT TRUNCATED AT 250 WORDS)

Testicular hormones do not regulate sexually dimorphic Pavlovian fear conditioning or perforant-path long-term potentiation in adult male rats.

We recently reported that Pavlovian fear conditioning and hippocampal perforant-path long-term potentiation (LTP) are sexually dimorphic in rats. Males show greater contextual fear conditioning, which depends on the hippocampus, as well as greater hippocampal LTP. In order to examine the role of circulating gonadal hormones in adult male rats, animals were castrated in two experiments, and Pavlovian fear conditioning and in vivo perforant-path LTP were examined. It was found that sexually-dimorphic LTP and fear conditioning are not regulated by the activational effects of testicular hormones in adult male rats. That is, in every respect, castrated male rats were similar to intact male rats in Pavlovian fear conditioning and hippocampal LTP. It is likely that sexual dimorphism in this system is established earlier in development by the organizational effects of gonadal hormones.