Estrogen mediation of injury-induced cell birth in neuroproliferative regions of the adult zebra finch brain

Estrogens influence neuronal differentiation, migration, and survival in intact brains. In injured brains, estrogens can also be neuroprotective. In Experiment 1, following a unilateral penetrating injury to the hippocampus (HP), adult female zebra finches were injected once with BrdU to label mitotic cells then sacrificed 2 h, 1 day, or 7 days postinjection. Cell proliferation was dramatically enhanced in the ipsilateral HP, as well as in neuroproliferative areas including the subventricular zone (SVZ) proximal to the injury. This increase was seen at all time points investigated. Ovariectomy (OVX) substantially suppressed proliferation bilaterally especially in the SVZ indicating that gonadal hormones influenced cell proliferation in both the intact and injured hemisphere. To determine if estrogens were directly involved, estrogen was depleted in Experiment 2 through either OVX or administration of the aromatase inhibitor fadrozole (FAD). Birds were implanted with estradiol or blank followed 2 weeks later by a unilateral penetrating lesion to the HP. Injury-induced substantial proliferation, which was again significantly suppressed bilaterally in both OVX and FAD birds. Estrogen replacement reversed this effect in FAD but not OVX birds therefore the suppression following OVX may be due in part to nonestrogenic influences. Suppression of cell birth in FAD birds was indeed due to the removal of endogenous sources of estrogen. Results therefore indicate that estrogens are directly involved in the brain's response to injury and may be acting to provide a rich environment for the production and perhaps protection of new cells.     

Aromatase expression and cell proliferation following injury of the adult zebra finch hippocampus

Estrogens can be neuroprotective following traumatic brain injury. Immediately after trauma to the zebra finch hippocampus, the estrogen-synthetic enzyme aromatase is rapidly upregulated in astrocytes and radial glia around the lesion site. Brain injury also induces high levels of cell proliferation. Estrogens promote neuronal differentiation, migration, and survival naturally in the avian brain. We suspect that glia are a source of estrogens promoting cell proliferation after neural injury. To explore this hypothesis, we examined the spatial and temporal relationship between glial aromatase expression and cell proliferation after neural injury in adult female zebra finches. Birds were ovariectomized and given a blank implant or one filled with estradiol; some birds were also administered an aromatase inhibitor or vehicle. All birds received penetrating injuries to the right hippocampus. Twenty-four hours after lesioning, birds were injected once with BrdU to label mitotically active cells and euthanized 2 h, 24 h, or 7 days later. The brains were processed for double-label BrdU and aromatase immunocytochemistry. Injury-induced glial aromatase expression was unaffected by survival time and aromatase inhibition. BrdU labeling was significantly reduced at 24 h by ovariectomy and by aromatase inhibition; effects were partially reversed by E2 replacement. Irrespective of ovariectomy, the densities of aromatase immunoreactive astrocytes and BrdU-labeled cells at known distances from the lesion site were highly correlated. These data suggest that injury-induced glial aromatization may influence the reorganization of injured tissue by providing a rich estrogenic environment available to influence cellular incorporation.     

Expression of estrogen receptor and aromatase mRNAs in embryonic and posthatch zebra finch brain

Male zebra finches sing and females normally do not. This sexually dimorphic behavior is mediated by a sexually dimorphic series of interconnected nuclei that are larger and more developed in males. Estradiol administered to females as early as the day of hatching (P1) causes profound masculinization of this song system. The exact timing of estrogen action is unknown, and there is little information concerning the times and sites of expression of estrogen receptors and aromatase before P5. We measured the expression of mRNAs encoding these proteins in brain during late embryogenesis and on P1 to determine if estrogen synthesis or receptor-mediated actions on the song system, as part of the program of sexual differentiation, might be possible during this period. Using highly sensitive and specific in situ hybridization procedures for mRNAs encoding ERalpha, ERbeta, and aromatase, we detected mRNA for ERs in archistriatal regions as early as embryonic stage 34, and in diencephalic regions as early as embryonic stage 30. ERalpha mRNA was also detected in the dorsal mesencephalon at P1. Aromatase mRNA expression was present as early as embryonic stage 30 in diencephalic and mesencephalic regions. No obvious sex differences in the spatio-temporal pattern of mRNA expression were detected. Our results suggest that estrogen can influence cell growth and differentiation in zebra finch brain well before hatching and into posthatching life. The results fail to provide support for the hypothesis that sexual differentiation of the song system is mediated by sex differences in the expression of these mRNAs at these ages.     

Aromatase is pre-synaptic and sexually dimorphic in the adult zebra finch brain

Oestrogens organize and activate circuits within the vertebrate central nervous system. Oestrogen synthesis occurs via the expression of aromatase, a P450 enzyme detected in microsomes and more recently in pre-synaptic boutons. Synaptic aromatase has only been described in brain regions that express aromatase in many subcellular compartments, so its function remains poorly understood. To more thoroughly study the role of oestrogen synthesis at synaptic terminals, we examined the ultrastructural compartmentalization of aromatase in the zebra finch; a species in which high aromatase activity can be measured in brain areas that do not contain somal aromatase. Here, we report the presence of aromatase in pre-synaptic boutons in the hippocampus and the high vocal centre brain areas with low and undetectable somal aromatase, respectively, in addition to areas with abundant somal aromatase such as the preoptic area and caudomedial nidopallium. At these brain areas, males had more total synapses, more aromatase pre-synaptic boutons and importantly, the proportion of total synaptic profiles that expressed aromatase was significantly higher in males relative to females. Aromatase-positive pre-synaptic boutons were always observed innervating aromatase-negative post-synaptic elements. We conclude that oestrogen may be provided to discrete oestrogen-sensitive targets by synaptic aromatization. Further, some targets may be exposed to more oestrogen in males. The expression of aromatase in individual synapses of projection neurons represents a unique mechanism of neuroendocrine action. Neurons with steroidogenic capability may modulate distant targets with the specificity of axonal innervation.     

Neuronal recruitment in adult zebra finch brain during a reproductive cycle

Previous studies suggest that adult neurogenesis and neuronal replacement are related to the acquisition of new information. The present study supports this hypothesis by showing that there is an increase in new neuron recruitment in brains of adult male and female zebra finches that coincides with the need to memorize vocalizations of nestlings before they fledge. We counted [(3)H]-Thymidine labeled neurons 40 days after [(3)H]-Thymidine injections. These counts were made in the parents' brains at the time eggs hatched, at the time juveniles fledged and still needed parental care, and at the time juveniles were already independent. We focused on nidopallium caudale (NC), a brain region which plays a role in sound processing. Recruitment of new NC neurons increased at the time the young fledged, followed by a significant decrease when the young reached independence. We suggest that this increase enables parents to recognize their own young when they are still dependent on parental feeding, yet easily lost among other fledglings in the colony. We saw no such increase in neuronal recruitment in the olfactory bulb, suggesting anatomical specificity for the effect seen in NC. We also found a preliminary, positive correlation between number of fledglings and number of new NC neurons in the parents' brain at fledging, suggesting that the number of neurons recruited is sensitive to the number of young fledged.     

Molecular cloning and characterization of the germline-restricted chromosome sequence in the zebra finch

The zebra finch (Taeniopygia guttata) germline-restricted chromosome (GRC) is the largest chromosome and has a unique system of transmission in germ cells. In the male, the GRC exists as a single heterochromatic chromosome in the germline and is eliminated from nuclei in late spermatogenesis. In the female, the GRC is bivalent and euchromatic and experiences recombination. These characteristics suggest a female-specific or female-beneficial function of the GRC. To shed light on the function of GRC, we cloned a portion of the GRC using random amplified polymorphic DNA–polymerase chain reaction and analyzed it using molecular genetic and cytogenetic methods. The GRC clone hybridized strongly to testis but not blood DNA in genomic Southern blots. In fluorescent in situ hybridization analysis on meiotic chromosomes from synaptonemal complex spreads, the probe showed hybridization across a large area of the GRC, suggesting that it contains repetitive sequences. We isolated a sequence homologous to the GRC from zebra finch chromosome 3 and a region of chicken chromosome 1 that is homologous to zebra finch chromosome 3; the phylogenetic analysis of these three sequences suggested that the GRC sequence and the zebra finch chromosome 3 sequence are most closely related. Thus, the GRC sequences likely originated from autosomal DNA and have evolved after the galliform–passeriform split. The present study provides a foundation for further study of the intriguing GRC.     

Neurogenesis in the adult Drosophila brain

Neurodegenerative diseases such as Alzheimer’s and Parkinson’s currently affect ∼25 million people worldwide. The global incidence of traumatic brain injury (TBI) is estimated at ∼70 million/year. Both neurodegenerative diseases and TBI remain without effective treatments. We are utilizing adult Drosophila melanogaster to investigate the mechanisms of brain regeneration with the long-term goal of identifying targets for neural regenerative therapies. We specifically focused on neurogenesis, i.e., the generation of new cells, as opposed to the regrowth of specific subcellular structures such as axons. Like mammals, Drosophila have few proliferating cells in the adult brain. Nonetheless, within 24 hours of a penetrating traumatic brain injury (PTBI) to the central brain, there is a significant increase in the number of proliferating cells. We subsequently detect both new glia and new neurons and the formation of new axon tracts that target appropriate brain regions. Glial cells divide rapidly upon injury to give rise to new glial cells. Other cells near the injury site upregulate neural progenitor genes including asense and deadpan and later give rise to the new neurons. Locomotor abnormalities observed after PTBI are reversed within 2 weeks of injury, supporting the idea that there is functional recovery. Together, these data indicate that adult Drosophila brains are capable of neuronal repair. We anticipate that this paradigm will facilitate the dissection of the mechanisms of neural regeneration and that these processes will be relevant to human brain repair.     

Sexual differentiation of brain and behavior in the zebra finch: Critical periods for effects of early estrogen treatment

In order to determine the critical period(s) during which estrogen alters sexually dimorphic behavior and neuroanatomy in zebra finches (Poephila guttata), nestlings were injected daily 20 μg estradiol benzoate (EB) during posthatching week 1, week 2, week 3, or weeks 1, 2, and 3. At 7 months of age, birds were implanted with testosterone propionate and tested with female partners for singing, dancing, and copulatory mounting. Brains were subsequently processed for morphometry, and the volumes of the song system nuclei HVC, area X, and RA and the soma sizes and densities of neurons in RA were determined. Males given EB during week 1 failed to mount. Females given EB during week 1 were fully masculinized with respect to dancing and RA neuron soma size and density, and were partially masculinized with respect to song nuclei volumes and singing. Treatment beginning after week 1 was ineffective or less effective for all measures. Only for RA neuron measures was treatment for all three weeks more effective than week 1 treatment. Thus the first post-hatching week is the most influential period of those tested for effects of exogenous estrogen on sexual differentiation in this species, and is a period during which both masculinization of females and demasculinization of males is possible. 1994 John Wiley & Sons, Inc.     

Interactions between nerve growth factor binding and estradiol in early development of the zebra finch telencephalon

The zebra finch telencephalon exhibits rapid and substantial development in the first few weeks after hatching. In parallel, the rate of estradiol synthesis is very high in the zebra finch forebrain, and estradiol can have potent neurotrophic effects in specific telencephalic regions, including those that control the learning and production of song. In an attempt to elucidate mechanisms regulating telencephalic development, potentially including a role for the large capacity for estrogen production, ¹²⁵I–nerve growth factor (NGF) binding was measured in homogenates of telencephalon from zebra finches age 3, 15, 30, 60, and 120 days. The highest density of low‐ and high‐affinity ¹²⁵I‐NGF binding sites was observed in 3‐day‐old finches. Using an aromatase inhibitor, Fadrozole, to reduce estradiol levels in 1 to 4‐day‐old zebra finches significantly decreased both high‐ and low‐affinity ¹²⁵I‐NGF binding sites. Conversely, treating adult or 8 to 14‐day‐old hatchlings with estradiol increased high‐affinity ¹²⁵I‐NGF binding sites. These results are consistent with the hypothesis that estradiol influences the level of NGF receptors, and suggest one mechanism through which the steroid could affect brain development. The data also indicate that estradiol and NGF activity may be important for very early development of the telencephalon. © 1999 John Wiley & Sons, Inc. J Neurobiol 40: 149–157, 1999     

Regulation of aromatase, 5α- and 5β-reductase in primary cell cultures of developing zebra finch telencephalon

Sex steroids act on the developing and adult telencephalon of songbirds to organize and activate the neural circuits required for the learning and production of song. Presumably, the availability of active androgens and estrogens to steroid-sensitive neural circuits controlling song is modulated by the local expression of androgen-metabolizing enzymes. Two enzymes, 5α- and 5β-reductase, are expressed widely in the songbird telencephalon, as they are in the telencephalons of other avian species. These enzymes convert circulating testosterone (T) into the active and inactive metabolites, 5α- and 5β-dihydrotestosterone (DHT), respectively. A third enzyme, aromatase, converts T into estradiol (E₂) and is expressed at unusually high levels in several regions of the songbird telencephalon. In many tissues, including the brain, the regulation of expression of one or more of these enzymes can be a critical feature of their ability to control the production of active sex steroids. We have used primary cell cultures to examine factors that might regulate the expression of these enzymes in developing zebra finch telencephalon. Cultures were treated for 0-72 h with sex steroids (T, E₂, 5α-DHT, and 5β-DHT) or with dibutyryl cAMP. Afterward, activities of aromatase, 5α-, and 5β-reductase were determined or total RNA was extracted for Northern analysis. Treatments with cAMP increased both aromatase activity and aromatase mRNA levels by 220%. E₂ significantly reduced aromatase activity by an average of 65%, whereas 5α- and 5β-DHT had no effect on aromatase activity. Compared to untreated controls, E₂ treatment decreased aromatase mRNA levels by 56%. None of these treatments consistently affected either 5α- or 5β-reductase activities. These results suggest that telencephalic E₂ may regulate its own synthesis by repression of aromatase expression, whereas factors that upregulate cAMP in the telencephalon can increase the local concentrations of E₂. © 1998 John Wiley & Sons, Inc. J Neurobiol 36: 30–40, 1998     

Lack of a synergistic effect between estradiol and dihydrotestosterone in the masculinization of the zebra finch song system

Previous studies have suggested that both major active metabolites of testosterone, estradiol (E₂) and dihydrotestosterone (DHT), are needed for complete masculinization of the brain regions that control song in passerine birds. However, DHT treatment of hatchling female zebra finches has only small masculinizing effects on the song system. To assess whether E₂ and DHT have a synergistic effect on the masculinization of the zebra finch song system, female zebra finches were given Silastic implants of E₂ on the day of hatching (day 1) either without any additional hormone treatment or in combination with DHT on days 1, 14, or 70. At 105 to 110 days of age, we measured the volumes of Area X, higher vocal center (HVC), robust nucleus of the archistriatum (RA), soma sizes in HVC, RA, and the lateral magnocellular nucleus of the neostriatum (lMAN), and neuron density and number in RA. E₂ masculinized all of the measures in the song system with the exception of the number of neurons in RA. DHT did not synergize with E₂ to produce any additional masculinization of the attributes measured. These data demonstrate that the combination of E₂ and DHT did not result in the complete masculinization of the song control nuclei and argue against the importance of androgen in sexual differentiation of the song system. © 1995 John Wiley & Sons, Inc.     

成年斑胸草雀在体HVC-RA突触传递的电生理特性

鸣禽高级发声中枢（high vocal center,HVC）至弓状皮质栎核（robust nucleus ofthe arcopallium,RA）的突触传递是鸣唱运动通路中的关键部分.本文运用在体场电位电生理记录的方法,研究了成年雄性斑胸草雀（Taeniopygia guttata）HVC-RA突触的电生理特性.实验结果显示,刺激HVC,在RA内所记录到的诱发场电位幅度较小.配对脉冲检测发现,HVC-RA突触传递具有明显的配对脉冲易化特性.当以强直刺激作用于HVC,RA内诱发场电位随即显著减小,并在15 min内逐渐恢复,表明HVC-RA突触传递在强直刺激过后出现了短时抑制.该通路的突触传递特性可能与其在发声控制中的作用有关.以上的实验结果为进一步研究发声运动过程中的突触可塑性提供了资料.     

Neonatal nutrition,adult antioxidant defences and sexual attractiveness in the zebra finch

Early nutrition has recently been shown to have pervasive, downstream effects on adult life-history parameters including lifespan, but the underlying mechanisms remain poorly understood. Damage to biomolecules caused by oxidants, such as free radicals generated during metabolic processes, is widely recognized as a key contributor to somatic degeneration and the rate of ageing. Lipophilic antioxidants (carotenoids, vitamins A and E) are an important component of vertebrate defences against such damage. By using an avian model, we show here that independent of later nutrition, individuals experiencing a short period of low-quality nutrition during the nestling period had a twofold reduction in plasma levels of these antioxidants at adulthood. We found no effects on adult external morphology or sexual attractiveness: in mate-choice trials females did not discriminate between adult males that had received standard- or lower-quality diet as neonates. Our results suggest low-quality neonatal nutrition resulted in a long-term impairment in the capacity to assimilate dietary antioxidants, thereby setting up a need to trade off the requirement for antioxidant activity against the need to maintain morphological development and sexual attractiveness. Such state-dependent trade-offs could underpin the link between early nutrition and senescence.     

Increasing stereotypy in adult zebra finch song correlates with a declining rate of adult neurogenesis

Adult neurogenesis is often correlated with learning new tasks, suggesting that a function of incorporating new neurons is to permit new memory formation. However, in the zebra finch, neurons are added to the song motor pathway throughout life, long after the initial song motor pattern is acquired by about 3 months of age. To explore this paradox, we examined the relationship between adult song structure and neuron addition using sensitive measures of song acoustic structure. We report that between 4 and 15 months of age there was an increase in the stereotypy of fine-grained spectral and temporal features of syllable acoustic structure. These results indicate that the zebra finch continues to refine motor output, perhaps by practice, over a protracted period beyond the time when song is first learned. Over the same age range, there was a decrease in the addition of new neurons to HVC, a region necessary for song production, but not to Area X or the hippocampus, regions not essential for singing. We propose that age-related changes in the stereotypy of syllable acoustic structure and HVC neuron addition are functionally related.