Aromatase in axonal processes of early postnatal hypothalamic and limbic areas including the cingulate cortex

It has been shown that sexual dimorphic morphology of certain hypothalamic and limbic areas underlie gender-specific sexual behavior and neuroendocrine mechanisms. The key role played by locally formed estrogen in these developmental events has been revealed during a critical perinatal period. In this study, we aimed to document the presence of estrogen-synthetase (aromatase)-immunoreactive elements in the involved limbic system and hypothalamus of the developing rat brain. On postnatal day 5, animals of both sexes were perfusion-fixed, and sections from the forebrain and hypothalamus were immunolabelled for aromatase using an antiserum that was generated against a 20 amino acid sequence of placental aromatase. Aromatase-immunoreactivity was present in neuronal perikarya and axonal processes in the following limbic structures: the central and medial nuclei of the amygdala, stria terminalis, bed nucleus of the stria terminalis (BNST), lateral septum, medial septum, diagonal band of Broca, lateral habenula and all areas of the limbic (cingulate) cortex. In the hypothalamus, the most robust labelling was observed in the medial preoptic area, periventricular regions, ventromedial and arcuate nuclei. The most striking feature of the immunostaining with this antiserum was its intracellular distribution. In contrast to the heavy perikaryal labelling that can be observed with most of the currently available aromatase antisera, in the present experiments, immunoperoxidase was predominantly localized to axons and axon terminals. All the regions with fiber staining corresponded to the projection fields of neuron populations that have previously been found to express perikaryal aromatase. Our results confirm the presence of aromatase-immunoreactivity in developing limbic and hypothalamic areas. The massive expression of aromatase in axonal processes raises the possibility that estrogen formed locally by aromatase may not only regulate the growth, pathfinding and target recognition of its host neuronal processes, but may also exert paracrine actions on structures in close proximity, including the target cells.     

Photodynamic therapy for rheumatoid arthritis?

Songbirds have emerged as extremely important animals for investigating sex steroid hormone effects on the central nervous system. The masculinizing effects of exogenous estrogen on the neural circuits controlling song in female zebra finches are well documented. There is evidence that estrogens are necessary for the full activation of singing behavior in several species. These kinds of studies have led us and others to investigate the mechanisms whereby estrogens are made available to the brains of songbirds during development and adulthood. In this article, I review results of some of these studies examining the estrogen synthetic enzyme aromatase and its expression and activity in brain and in other tissues of songbirds. I will discuss some results and thoughts we have about the interactions of aromatase with the two remaining androgen-metabolizing enzymes in the avian brain, 5alpha-reductase, the enzyme that converts T into the active androgen 5alpha-dihydrotestosterone (DHT); and 5beta-reductase, the enzyme that converts T into the inactive 5beta-DHT. Finally, I will consider some ideas raised by these studies concerning potential sources of the androgen substrate for brain aromatization as well as some possible new functions that aromatase might be playing in the songbird telencephalon.     

Patterns of vertebrate neurogenesis and the paths of vertebrate evolution

Any substantial change in brain size requires a change in the number of neurons and their supporting elements in the brain, which in turn requires an alteration in either the rate, or the duration of neurogenesis. The schedule of neurogenesis is surprisingly stable in mammalian brains, and increases in the duration of neurogenesis have predictable outcomes: late-generated structures become disproportionately large. The olfactory bulb and associated limbic structures may deviate in some species from this general brain enlargement: in the rhesus monkey, reduction of limbic system size appears to be produced by an advance in the onset of terminal neurogenesis in limbic system structures. Not only neurogenesis but also many other features of neural maturation such as process extension and retraction, follow the same schedule with the same predictability. Although the underlying order of event onset remains the same for all of the mammals we have yet studied, changes in overall rate of neural maturation distinguish related subclasses, such as marsupial and placental mammals, and changes in duration of neurodevelopment distinguish species within subclasses. A substantial part of the regularity of event sequence in neurogenesis can be related directly to the two dimensions of the neuraxis in a recently proposed prosomeric segmentation of the forebrain [Rubenstein et al., Science, 266: 578, 1994]. Both the spatial and temporal organization of development have been highly conserved in mammalian brain evolution, showing strong constraint on the types of brain adaptations possible. The neural mechanisms for integrative behaviors may become localized to those locations that have enough plasticity in neuron number to support them.     

The role of estrogen in bone growth and maturation during childhood and adolescence

Twenty years ago it was believed that pubertal growth was stimulated by testicular androgen in boys and by adrenal androgen in girls. Estrogen, which was used to inhibit growth in excessively tall girls, was not thought to have growth-promoting effects. We hypothesized that estrogen has a biphasic effect on epiphyseal growth, with maximal stimulation at low levels. We showed that the administration of low doses of estrogen, corresponding to a serum estradiol level of about 4 pg/ml (15 pmol/l) caused more than a 60% increase over the prepubertal growth rate in both boys and girls. To test the hypothesis that estrogen is the principal mediator of the pubertal growth spurt in boys, we administered the aromatase inhibitor, testolactone, to boys with familial male-limited precocious puberty. Testolactone produced near normalization of both growth velocity and bone maturation, despite levels of serum testosterone that remained within the adult male range. The observation that low levels of estrogen stimulate growth and bone maturation suggested that estrogen might explain the more rapid epiphyseal maturation of prepubertal girls compared to boys. To determine whether prepubertal girls have higher estrogen levels than prepubertal boys, we developed an ultrasensitive recombinant cell bioassay for estrogen with a sensitivity of 0.02 pg/ml (0.07 pmol/l) estradiol equivalents. Prepubertal girls had approximately eight-fold higher levels of serum estradiol than did prepubertal boys (0.6 +/- 0.6 pg/ml (SD) (2.2 +/- 2.2 pmol/l) vs 0.08 +/- 0.2 pg/ml (0.29 +/- 0.73 pmol/l), P < 0.05). We concluded that the pubertal growth spurt of both sexes is driven primarily by estrogen, and that the more rapid epiphyseal maturation of prepubertal girls (vs boys) may be explained by their higher estradiol levels.     

Sex differences in the regulation of embryonic brain aromatase

Oestrogen formed from androgen by aromatization plays a critical role in the sexual differentiation of the male brain and behaviour. A question which has still to be answered is what regulates the gender-specific changes in aromatase activity forming oestrogen during sensitive periods of brain growth. Using a primary cell culture technique and sexed embryos, we have shown that in the fetal mouse brain, oestrogen formation in the male is neuronal rather than glial and aromatase activity is regionally localized, being higher in the hypothalamus than in the cortex. The aromatase activity measured from cells in culture has the same enzyme binding affinity (apparent Km approximately 40 nM) as intact brain samples. Neurones developing in the embryonic male brain (embryonic day (ED) 15) contain higher aromatase activity (Vmax, 895 fmol/h/mg protein) than the female (Vmax, 604). Although a sex difference exists at early stages of embryonic development (ED 13), the embryonic aromatase system is regulated by steroids later in fetal development. The developing aromatase-containing neuroblasts probably form processes which connect to other aromatase neurones. Immunoreactive staining with an aromatase polyclonal antibody identifies an increase in numbers of aromatase-immunoreactive hypothalamic neuronal cell bodies following testosterone treatment. Testosterone treatment also causes both stimulation of neurite growth and branching as well as functional maturation of aromatase neurones. In particular, there is an increase in aromatase activity per neurone as well as a dramatic increase in the number of neurones expressing the enzyme. Both the functional and morphological changes depend on androgen receptor stimulation for several days in vitro. This conclusion is supported by colocalization studies which reveal a high number of fetal hypothalamic aromatase neurones co-expressing androgen receptor. We conclude that testosterone influences the growth of male hypothalamic neurones containing aromatase at a sensitive period of brain development. Endogenous steroid inhibitors of aromatase, probably formed within the neuroglia, also play a role in the control of oestrogen production. An endogenous 5alpha-reduced metabolite of testosterone, 5alpha-androstanedione, is almost as potent in inhibiting neuronal hypothalamic aromatase activity (Ki = 23 nM) as the synthetic non-steroidal inhibitors such as the imidazole, fadrozole, and the triazoles, arimidex and letrozole. It is clear that the oestrogen-forming capacity of the male hypothalamus has the special characteristics and plasticity of regulation which could affect brain differentiation at specific steroid-sensitive stages in ontogeny.     

Brain formation of oestrogen in the mouse: sex dimorphism in aromatase development

Steroid sex hormones have an organizational role in gender-specific brain development. Aromatase, converting testosterone (T) to oestradiol-17 beta (E2), is a key enzyme in the brain and the regulation of this enzyme is likely to determine availability of E2 effective for neural differentiation. In rodents, oestrogens are formed very actively during male perinatal brain development. This paper reviews work on the sexual differentiation of the brain aromatase system in vitro. Embryonic day 15 mouse hypothalamic culture aromatase activity (AA: mean Vmax = 0.9 pmol/h/mg protein) is several times greater than in the adult, whereas apparent Km is similar for both (approximately 30-40 nM). Using microdissected brain areas and cultured cells of the mouse, sex differences in hypothalamic AA during both early embryonic and later perinatal development can be demonstrated, with higher E2 formation in the male than in the female. The sex differences are brain region-specific, since no differences between male and female are detectable in cultured cortical cells. AA quantitation and immunoreactive staining with an aromatase polyclonal antibody both identify neuronal rather than astroglial localizations of the enzyme. Kainic acid eliminates the gender difference in hypothalamic oestrogen formation indicating, furthermore, that this sex dimorphism is neuronal. Gender-specific aromatase regulation is regional in the brain. Oestrogen formation is specifically induced in cultured hypothalamic neurones of either sex by T, since androgen has no effect on cortical cells. Androgen is clearly involved in the growth of hypothalamic neurones containing aromatase. It appears that differentiation of the brain involves maturation of a gender-specific network of oestrogen-forming neurones.     

Sex differences and androgen-dependent regulation of aromatase (CYP19) mRNA expression in the developing and adult rat brain

Sex differences, androgen dependence and asymmetries of aromatase activity have been reported during ontogeny of the rat. It remains to be elucidated, however, whether the changes in aromatase activity are reflected by similar changes in specific mRNA levels. In addition, very little is known regarding mechanism(s) underlying such differential regulation of aromatase expression. To address these questions, we have employed the in situ hybridization (ISH) technique to examine specific mRNA levels in the brain of both male and female rats at selected stages of development. In prenatal stages of development, at gestational day (GD) 18 and 20, aromatase mRNA was detected in several hypothalamic and limbic brain regions. Semiquantitative analysis of aromatase mRNA did not reveal statistically significant sex differences in any of these regions (except in one experiment at GD20, when a sex difference was found in the medial preoptic nucleus). In contrast, clear sex differences were determined at postnatal day (PN) 2; male animals contained significantly more aromatase mRNA in the bed nucleus of the stria terminalis (BST) and the sexually dimorphic nucleus of the preoptic area (SDN) compared to female rats. Four days later in development, at PN6, sex differences of aromatase mRNA signals were observed in the BST, but were no longer detectable in the SDN. At PN15 and in adult animals, no sex differences could be determined. The effect of flutamide treatment (50 mg/kg/day) was investigated in GD20 fetuses as well as in adult rats. No statistically significant changes in aromatase mRNA expression were found in either case. In summary, our results suggest that differential regulation of aromatase mRNA expression during the critical period of sexual differentiation might, in part, account for the establishment of some of the many sexually dimorphic parameters of the rat brain. The role of androgens in the regulation of the sex-specific and developmental expression of aromatase mRNA in the rat brain remains to be clarified.     

Barley lipid-transfer protein complexed with palmitoyl CoA: the structure reveals a hydrophobic binding site that can expand to fit both large and small lipid-like ligands

The expression of cadherin-8 was mapped by in situ hybridization in the embryonic and postnatal mouse central nervous system (CNS). From embryonic day 18 (E18) to postnatal day 6 (P6), cadherin-8 expression is restricted to a subset of developing brain nuclei and cortical areas in all major subdivisions of the CNS. The anlagen of some of the cadherin-8-positive structures also express this molecule at earlier developmental stages (E12.5-E16). The cadherin-8-positive neuroanatomical structures are parts of several functional systems in the brain. In the limbic system, cadherin-8-positive regions are found in the septal region, habenular nuclei, amygdala, interpeduncular nucleus, raphe nuclei, and hippocampus. Cerebral cortex shows expression in several limbic areas at P6. In the basal ganglia and related nuclei, cadherin-8 is expressed by parts of the striatum, globus pallidus, substantia nigra, entopeduncular nucleus, subthalamic nucleus, zona incerta, and pedunculopontine nuclei. A third group of cadherin-8-positive gray matter structures has functional connections with the cerebellum (superior colliculus, anterior pretectal nucleus, red nucleus, nucleus of posterior commissure, inferior olive, pontine, pontine reticular, and vestibular nuclei). The cerebellum itself shows parasagittal stripes of cadherin-8 expression in the Purkinje cell layer. In the hindbrain, cadherin-8 is expressed by several cranial nerve nuclei. Results from this study show that cadherin-8 expression in the embryonic and postnatal mouse brain is restricted to specific developing gray matter structures. These data support the idea that cadherins are a family of molecules whose expression provides a molecular code for the regionalization of the developing vertebrate brain.     

Sex differences in the nervous system of reptiles

1. The study of sex differences in the brain and behavior of reptiles presents an excellent opportunity both to discern general principles of sexual differentiation in the nervous system and to explore the evolutionary history of this process in amniote vertebrates. 2. Findings in several reptiles suggest that some sex differences found in mammals and birds are conserved while others are not. Conserved features include areas in the limbic forebrain involved in the regulation of social and sexual behaviors. As in mammals and birds, it is rare to find differences in the distribution of sex steroid concentrating neurons in reptiles but common to find differences in the distribution of the various steroid hormone receptors and in their regulation. 3. This research has revealed that differences in social and sexual behavior are reflected better by the activity, not by the size, of hormone-sensitive limbic areas. 4. Finally, species differences in plasma levels of sex hormones are paralleled by differences in behavioral sensitivity to these hormones as well as by differences in the regulation of genes coding for steroid hormone receptors.     

New insights into the regulation and function of brain estrogen synthase (aromatase)

In the brain, conversion of androgens into estrogens by the enzyme aromatase (estrogen synthase) is a key mechanism by which testosterone regulates many physiological and behavioral processes, including the activation of male sexual behavior, brain sexual differentiation and negative feedback effects of steroid hormones on gonadotropin secretion. Studies on the distribution and regulation of brain aromatase have led to a new perspective on the control and function of this enzyme. A growing body of evidence indicates that the estrogen regulation of aromatase is, at least in part, trans-synaptic. Afferent catecholamine pathways appear to regulate aromatase activity in some brain areas and thereby provide a way for environmental cues to modulate this enzyme. The localization of aromatase in pre-synaptic boutons suggests possible roles for estrogens at the synapse.     

Possible role of cytochrome P450 in inactivation of testosterone in immortalized hippocampal neurons

The hippocampus as part of the limbic system is sensitive to gonadal hormones. The time-dependent expression of steroid receptors and the testosterone converting enzyme aromatase (CYP19) is well studied. In contrast, little is known about other cytochrome P450 enzymes in hippocampus which inactivate the gonadal hormones. For investigation of the total cytochrome P450 content and the expression of testosterone degrading CYP2B10 we used embryonic (E18) in comparison to postnatal (P21) immortalized hippocampal neurons. These embryonic neurons were demonstrated to react to hormones according a 'critical period' of sexual differentiation: testosterone treatment (1 microM to 5 microM in the culture medium) resulted in a decrease of beta-tubulin, as showed by immunocytochemistry and Western blotting. Measurements with reduced CO-difference spectrum elucidated that the P450 concentration in the embryonic neurons (10.2 pmol/mg protein; S.D. +/- 1.9) was twice as high as in the postnatal ones (5.2 pmol/mg protein; S.D. +/- 1.0). Correspondingly, a high value of the mitochondrial subfraction of approx. 141 pmol P450/mg protein was found in the embryonic neurons relative to the mitochondrial value of 37.7 pmol P450/mg protein in the postnatal neurons. Our results suggest a differential expression of cytochrome P450 during development. CYP2B10 was proved by electron microscopy and hormone degrading activity.     

Pharmacological analysis of the haemodynamic effects of 5-HT1B/D receptor agonists in the normotensive rat

Our previous findings in female rats suggest that the potent effects of sex steroids on mood and mental state may be mediated, in part, by the effect of estrogen on the 5-hydroxytryptamine2A receptor (5-HT2AR) in brain. The aim of the present study was to determine the effect of acute (approximately 32h) sex steroid manipulation on central 5-HT2AR in the adult male Wistar rat. Castration (under halothane anesthesia) decreased while testosterone or estrogen, but not 5alpha-dihydrotestosterone (5alpha-DHT), increased significantly the 5-HT2AR mRNA content in dorsal raphe nucleus and the density of 5-HT2AR binding sites in frontal, cingulate and primary olfactory cortex and nucleus accumbens. The lack of effect of 5alpha-DHT, a potent androgen which cannot be converted to estrogen, suggests that the action of testosterone depends upon its conversion to estrogen by aromatase. This may also explain why estrogen, but not testosterone or 5alpha-DHT, increased the density of 5-HT2AR binding sites in the caudate-putamen, a brain region where aromatase is scarce. These findings are discussed in relation to the possible role of the 5-HT2AR in depression, schizophrenia and Alzheimer's Disease.     

Immunolocalization of aromatase- and androgen receptor-positive neurons in the goldfish brain

Full expression of testosterone (T) actions in the brain requires both direct binding to androgen receptors (AR) and in situ aromatization to estradiol (E2). To determine the cellular basis of constitutively high aromatase and AR binding activities in teleost fish brain, and the neuroanatomic location and spatial relations of cells of each type, an immunocytochemical mapping study of goldfish (Carassius auratus) brain was carried out using antibodies to human placental aromatase and human/rat AR peptide and the avidin-biotin-peroxidase technique. Both antibodies specifically labeled cells that were neuronal in appearance and were most numerous in reproductive control centers: medial and ventral telencephalon (TEL) and preoptic and hypothalamic periventricular nuclei. Additional populations of aromatase- and AR-labeled cells were present in the olfactory bulbs, central telencephalon, and stratum periventriculare of the optic tectum. Anti-aromatase, but not anti-AR, labeled fiber tracts and fibrous layers in visual and auditory pathways, and perikarya and processes of premotor neurons known to integrate sensory input (reticulospinal neurons, Mauthner cells). Anti-AR selectively labeled lateral TEL regions, the nucleus ventromedialis thalami, and discrete cell clusters in the medial tegmental nucleus. Aromatase-immunoreactivity (-ir) was primarily cytoplasmic, whereas AR-ir was primarily nuclear, but relative intensity of nuclear vs cytoplasmic labeling with each antibody differed by brain region. Aromatase- and AR-ir cells were not obviously more numerous in goldfish brain than previously seen in birds and mammals, suggesting that enhanced expression occurs on a per cell basis. We conclude that T exerts its actions coordinately via direct and indirect pathways in most brain regions but independently via AR- or aromatase-mediated mechanisms in selected areas. These studies point to a wide role for androgen in modulating primary sensory signals as well as in classical reproductive processes.     

Chronic pain alters drug self-administration: Implications for addiction and pain mechanisms.

This review article focuses on the impact that the presence of pain has on drug self-administration in rodents, and the potential for using self-administration to study both addiction and pain, as well as their interaction. The literature on the effects of noxious input to the brain on both spinal and supraspinal neuronal activity is reviewed as well as the evidence that human and rodent neurobiology is affected similarly by noxious stimulation. The convergence of peripheral input to somatosensory systems with limbic forebrain structures is briefly discussed in the context of how the activity of one system may influence activity within the other system. Finally, the literature on how pain influences drug-seeking behaviors in rodents is reviewed, with a final discussion of how these techniques might be able to contribute to the development of novel analgesic treatments that minimize addiction and tolerance. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Interaction of cognitive balance and mood induction on self-descriptions: a Q methodological approach

Several neuropsychiatric disorders, including schizophrenia, are characterized by sensorimotor gating deficits. Prepulse inhibition of the acoustic startle response is an operational measure assessing sensorimotor gating and has been found to be reduced in schizophrenic patients. Much attention has therefore been paid to the neuronal mechanisms underlying the disruption of prepulse inhibition. The activity of limbic forebrain structures such as the septohippocampal system, the prefrontal cortex, and the nucleus accumbens has been the main focus of recent research into the regulation of prepulse inhibition in rats. We here provide a functional anatomical picture of forebrain structures probably involved in the regulation of prepulse inhibition. Stimulation of the ventral hippocampus with a subconvulsive dose of N-methyl-D-aspartate caused a significant and long-lasting disruption of prepulse inhibition. Immunostaining of the c-Fos protein revealed a characteristic pattern of neuronal activity in various forebrain areas, including the nucleus accumbens and different frontal cortical areas after hippocampal stimulation. Based on the present findings, we conclude that the overactivity within a network of cortico-limbic forebrain structures compromises the normal processing of sensory stimuli by disrupting a neuronal filter mechanism. Interestingly, there is a considerable overlap between the pattern of neuronal activity observed in our study and the brain pathology in schizophrenics reported in the literature.     

Neuroblastoma and Alzheimer's disease brain cells contain aromatase activity

Human brain steroidogenic mechanisms, particularly aromatase, have been investigated in healthy and diseased conditions. Aromatase activity was measured in differentiated and undifferentiated neuroblastoma cell lines from mouse (TMN) and human (5H SY5Y) and in human post mortem brain samples. Neuroblastomas show much higher aromatase activity than human brain samples. Homogenates of adult human male and female cortex and frontal and temporal areas of both Alzheimer's and control patients all show considerably lower activity. The temporal area has significantly higher aromatase activity than the frontal. Aromatisation activity in differentiated neuroblastoma cells of both species is lower than in undifferentiated cells. These results are consistent with an inverse relationship between brain estrogen formation and stage of neuronal differentiation and the hypothesis that aromatase may be involved in the early stages of neuronal growth. Significant but variable activities of other androgen-metabolising enzymes, such as 5 alpha-reductase, 3 alpha/beta-hydroxysteroid dehydrogenases, and 17 beta-hydroxysteroid dehydrogenase, which generate a spectrum of regulatory molecules, are also found.