Estrogens rapidly shape synaptic and intrinsic properties to regulate the temporal precision of songbird auditory neurons.

Sensory neurons parse millisecond-variant sound streams like birdsong and speech with exquisite precision. The auditory pallial cortex of vocal learners like humans and songbirds contains an unconventional neuromodulatory system: neuronal expression of the estrogen synthesis enzyme aromatase. Local forebrain neuroestrogens fluctuate when songbirds hear a song, and subsequently modulate bursting, gain, and temporal coding properties of auditory neurons. However, the way neuroestrogens shape intrinsic and synaptic properties of sensory neurons remains unknown. Here, using a combination of whole-cell patch clamp electrophysiology and calcium imaging, we investigate estrogenic neuromodulation of auditory neurons in a region resembling mammalian auditory association cortex. We found that estradiol rapidly enhances the temporal precision of neuronal firing via a membrane-bound G-protein coupled receptor and that estradiol rapidly suppresses inhibitory synaptic currents while sparing excitation. Notably, the rapid suppression of intrinsic excitability by estradiol was predicted by membrane input resistance and was observed in both males and females. These findings were corroborated by analysis of in vivo electrophysiology recordings, in which local estrogen synthesis blockade caused acute disruption of the temporal correlation of song-evoked firing patterns. Therefore, on a modulatory timescale, neuroestrogens alter intrinsic cellular properties and inhibitory neurotransmitter release to regulate the temporal precision of higher-order sensory neurons.

Effects of timed food availability on reproduction and metabolism in zebra finches: Molecular insights into homeostatic adaptation to food-restriction in diurnal vertebrates.

Food availability affects metabolism and reproduction in higher vertebrates including birds. This study tested the idea of adaptive homeostasis to time-restricted feeding (TRF) in diurnal zebra finches by using multiple (behavioral, physiological and molecular) assays. Adult birds were subjected for 1 week or 3 weeks to food restriction for 4 h in the evening (hour 8-12) of the 12 h light-on period, with controls on ad lib feeding. Birds on TRF showed enhanced exploratory behavior and plasma triglycerides levels, but did not show differences from ad lib birds in the overall food intake, body mass, and plasma corticosterone and thyroxine levels. As compared to ad lib feeding, testis size and circulation testosterone were reduced after first but not after third week of TRF. The concomitant change in the mRNA expression of metabolic and reproductive genes was also found after week 1 of TRF. Particularly, TRF birds showed increased expression of genes coding for gonadotropin releasing hormone (GnRH) in hypothalamus, and for receptors of androgen (AR) and estrogen (ER-alpha) in both hypothalamus and testes. However, genes coding for the deiodinases (Dio2, Dio3) and gonadotropin inhibiting hormone (GnIH) showed no difference between feeding conditions in both hypothalamus and testes. Further, increased Sirt1, Fgf10 and Ppar-alpha, and decreased Egr1 expression in the liver suggested TRF-effects on the overall metabolism. Importantly, TRF-effects on gene expressions by week 1 seemed alleviated to a considerable extent by week 3. These results on TRF-induced reproductive and metabolic effects suggest homeostatic adaptation to food-restriction in diurnal vertebrates.

Early life stress increases testosterone and corticosterone and alters stress physiology in zebra finches.

Early life stress has enduring effects on behavior and physiology. However, the effects on hormones and stress physiology remain poorly understood. In the present study, parents of zebra finches of both sexes were exposed to an increased foraging paradigm from 3 to 33days post hatching. Plasma and brains were collected from chicks at 3 developmental time points: post hatching days 25, 60 and adulthood. Plasma was assayed for testosterone (T), estradiol (E2), and corticosterone (CORT). The paraventricular nucleus of the hypothalamus was assessed for corticotrophin releasing factor (CRH) and glucocorticoid receptor (GR) expression. As expected, body mass was lower in nutritionally stressed animals compared to controls at multiple ages. Nutritionally stressed animals overall had higher levels of CORT than did control and this was particularly apparent in females at post hatching day 25. Nutritionally stressed animals also had a higher number of cells expressing CRH and GR in the paraventricular nucleus of the hypothalamus than did controls. There was an interaction, such that both measures were higher in control animals at PHD 25, but higher in NS animals by adulthood. Females, regardless of treatment, had higher circulating CORT and a higher number of cells expressing CRH than did males. Nutritionally stressed animals also had higher levels of T than did control animals, and this difference was greatest for males at post hatching day 60. There were no effects of nutritional stress on E2. These findings suggest that nutritional stress during development has long-lasting effects on testosterone and stress physiology.

Efferent innervation of turtle semicircular canal cristae: comparisons with bird and mouse.

In the vestibular periphery of nearly every vertebrate, cholinergic vestibular efferent neurons give rise to numerous presynaptic varicosities that target hair cells and afferent processes in the sensory neuroepithelium. Although pharmacological studies have described the postsynaptic actions of vestibular efferent stimulation in several species, characterization of efferent innervation patterns and the relative distribution of efferent varicosities among hair cells and afferents are also integral to understanding how efferent synapses operate. Vestibular efferent markers, however, have not been well characterized in the turtle, one of the animal models used by our laboratory. Here we sought to identify reliable efferent neuronal markers in the vestibular periphery of turtle, to use these markers to understand how efferent synapses are organized, and to compare efferent neuronal labeling patterns in turtle with two other amniotes using some of the same markers. Efferent fibers and varicosities were visualized in the semicircular canal of red-eared turtles (Trachemys scripta elegans), zebra finches (Taeniopygia guttata), and mice (Mus musculus) utilizing fluorescent immunohistochemistry with antibodies against choline acetyltransferase (ChAT). Vestibular hair cells and afferents were counterstained using antibodies to myosin VIIa and calretinin. In all species, ChAT labeled a population of small diameter fibers giving rise to numerous spherical varicosities abutting type II hair cells and afferent processes. That these ChAT-positive varicosities represent presynaptic release sites were demonstrated by colabeling with antibodies against the synaptic vesicle proteins synapsin I, SV2, or syntaxin and the neuropeptide calcitonin gene-related peptide. Comparisons of efferent innervation patterns among the three species are discussed.

Effects of carotenoid and vitamin E supplementation on oxidative stress and plumage coloration in house finches (Haemorhous mexicanus).

There has been much recent interest from both applied and basic scientists in the broad series of benefits that animals reap from acquiring high concentrations of dietary antioxidants, such as carotenoids and vitamins (e.g., vitamin E, or tocopherol). Most attention has been paid to separate effects of these compounds on, for example, coloration, health state, development, and vision, but because of possible interactions between these lipid-soluble molecules, we are in need of more studies that co-manipulate these substances and examine their possible synergistic impacts on animal physiology and phenotype. We capitalized on a model avian system (the house finch, Haemorhous mexicanus), where extensive information is available on the fitness roles of carotenoids, to test how variation in carotenoid and/or vitamin E concentrations in the diet impacts body accumulation of these compounds, factors related to oxidative damage (e.g., breast muscle and plasma oxidative-stress susceptibility, plasma nitric-oxide levels), and plumage color development. As in a previous study of ours on carotenoids and health in finches, we employed a 2×2 factorial experimental design on birds in both molting and non-molting conditions, to understand how seasonal shifts in carotenoid use (i.e., pigment incorporation into plumage) might alter the accumulation and roles of carotenoids and vitamins. As expected, lutein supplementation increased the level of circulating carotenoids in both experiments and the color of newly molted plumage. By contrast, vitamin E provisioning did not significantly affect plasma carotenoid levels or plumage coloration in either experiment. Interestingly, carotenoid provisioning decreased circulating vitamin E levels during molt, which suggests either molecular competition between carotenoids and tocopherol at the absorption/transport stages or that vitamin E serves as an antioxidant to offset harmful actions that carotenoids may have at very high concentrations. Finally, in both experiments, we found a reduction in breast-muscle oxidative damage for tocopherol-supplemented birds, which constitutes the first demonstration of a protective effect of vitamin E against oxidative stress in wild birds. Taken together, these findings provide an interesting contrast with our earlier work on season-specific physiological benefits of carotenoids in finches and point to complex associations between indicators of antioxidant and oxidative state in wild-caught animals.

Lipopolysaccharide injection induces rapid decrease of hypothalamic GnRH mRNA and peptide, but does not affect GnIH in zebra finches.

Lipopolysaccharide (LPS) is frequently used experimentally to mimic acute infection. Through activation of the host's immune response, an LPS injection has profound effects on the adrenocortical response to stress and on behaviors including reduction in activity, water and food intake, and libido. These behavioral changes occurring during infection are collectively called "sickness behavior." It is thought that adoption of sickness behavior reallocates energy from other fitness-enhancing activities, such as reproduction, for use in the immune response. Although the behavioral effects of LPS treatment are well-known, less information is available regarding the effects of LPS on the brain in terms of controlling reproductive behavior, specifically concerning a newly discovered neuropeptide, gonadotropin-inhibitory hormone (GnIH). This study investigated the effects of an LPS injection on the behavior and the hypothalamic neuropeptides controlling reproduction [GnIH and gonadotropin-releasing hormone (GnRH)] of zebra finches (Taeniopygia guttata). Overall, there was a decrease in activity in birds injected with LPS. The number of GnRH-immunoreactive neurons was significantly reduced in birds injected with LPS when compared to controls, while the number of GnIH-releasing neurons remained unchanged. At the level of gene expression, a similar pattern was found: there was reduced expression of GnRH mRNA in LPS-injected animals, whereas GnIH expression remained unchanged. Plasma testosterone did not change significantly in LPS-injected animals, nor did plasma corticosterone. Taken together, these results indicate a rapid (within 3h) inhibition of the reproductive axis during an immune challenge mimicking an infection, specifically acting on the GnRH system. The present study expands our knowledge on the interaction between the immune system and the reproductive system.

Deprivation of maternal care has long-lasting consequences for the hypothalamic-pituitary-adrenal axis of zebra finches.

Early-life stress caused by the deprivation of maternal care has been shown to have long-lasting effects on the hypothalamic-pituitary-adrenal (HPA) axis in offspring of uniparental mammalian species. We asked if deprivation of maternal care in biparental species alters stress responsiveness of offspring, using a biparental avian species--the zebra finch, Taeniopygia guttata. In our experiment, one group of birds was raised by both male and female parents (control), and another was raised by males alone (maternally deprived). During adulthood, offspring of both groups were subjected to two stressors (restraint and isolation), and corticosterone concentrations were measured. Additionally, we measured baseline levels of the two corticosteroid receptors--glucocorticoid receptor (GR) and mineralocorticoid receptor (MR)--in the hippocampus, hypothalamus and cerebellum. Our results suggest that maternally deprived offspring are hyper-responsive to isolation in comparison with controls. Furthermore, mRNA levels of both GR and MR receptors are altered in maternally deprived offspring in comparison with controls. Thus, absence of maternal care has lasting consequences for HPA function in a biparental species where paternal care is available.

A multifactorial test of the effects of carotenoid access, food intake and parasite load on the production of ornamental feathers and bill coloration in American goldfinches.

It has been well established that carotenoid and melanin pigmentation are often condition-dependent traits in vertebrates. Expression of carotenoid coloration in birds has been shown to reflect pigment intake, food access and parasite load; however, the relative importance of and the potential interactions among these factors have not been previously considered. Moreover, carotenoid and melanin pigmentation have been proposed to signal fundamentally different aspects of individual condition but few data exist to test this idea. We simultaneously manipulated three environmental conditions under which American goldfinches (Cardeulis tristis) grew colorful feathers and developed carotenoid pigmentation of their bills. Male goldfinches were held with either high or low carotenoid supplementation, pulsed or continuous antimicrobial drug treatment, or restricted or unlimited access to food. Carotenoid supplementation had an overriding effect on yellow feather coloration. Males given more lutein and zeaxanthin grew yellow feathers with hue shifted toward orange and with higher yellow chroma than males supplemented with fewer carotenoids. Parasites and food access did not significantly affect yellow feather coloration, and there were only minor interaction effects for the three treatments. By contrast, bill coloration was significantly affected by all three treatments. Carotenoid supplementation had a significant effect on yellow chroma of bills, drug treatment and food access both had a significant effect on bill hue, and food access had a significant effect on the yellow brightness of bills. Neither the size nor blackness of the black caps of male goldfinches was affected by any treatment. These results indicate that pigment intake, food access and parasite load can have complex and variable effects on color displays, and that feather and bill coloration signal different aspects of male condition.

Elevated aromatase activity in forebrain synaptic terminals during song.

The enzyme aromatase, which converts androgens into oestrogens, is expressed throughout the brain in zebra finches. Aromatase is enzymatically active in both cell bodies and synaptic terminals of neurones of the songbird brain, particularly within the forebrain motor and auditory networks. Aromatisation within synaptic terminals could thus provide localised and acute modulatory oestrogens within the forebrain during singing and/or audition. In male zebra finches, we tested the hypothesis that forebrain aromatase activity is elevated during singing behaviour and/or hearing male song. The present study reports that aromatase activity is elevated in males that were singing for 30 min compared to nonsinging males, and that this elevation occurs only within the cellular compartment that contains synaptic terminals. In a separate experiment, males that heard acoustic playback of song for 30 min exhibited no differences in aromatase activity or in aromatase mRNA levels, as revealed by quantitative polymerase chain reaction analysis. Therefore, these results indicate that activation of the motor pathway for song production is linked to local elevations in synaptic aromatase activity within the forebrain of male zebra finches. Future experiments could assess whether elevated synaptic aromatase activity during song is dependent on acute regulation of the aromatase protein.

Detection of two mRNA species at single-cell resolution by double-fluorescence in situ hybridization.

Here we describe a fluorescence in situ hybridization protocol that allows for the detection of two mRNA species in fresh frozen brain tissue sections. This protocol entails the simultaneous and specific hybridization of hapten-labeled riboprobes to complementary mRNAs of interest, followed by probe detection via immunohistochemical procedures and peroxidase-mediated precipitation of tyramide-linked fluorophores. In this protocol we describe riboprobes labeled with digoxigenin and biotin, though the steps can be adapted to labeling with other haptens. We have used this approach to establish the neurochemical identity of sensory-driven neurons and the co-induction of experience-regulated genes in the songbird brain. However, this procedure can be used to detect virtually any combination of two mRNA populations at single-cell resolution in the brain, and possibly other tissues. Required controls, representative results and troubleshooting of important steps of this procedure are presented. After tissue sections are obtained, the total length of the procedure is 2-3 d.

Collapsin response mediator protein-4 (CRMP-4) expression in posthaching development of song control nuclei in Bengalese finches.

CRMP-4 is regarded to play a role in neuronal differentiation, neurite growth and synapse formation. It has been shown to express in brain areas undergoing plastic changes or neuronal generation. Bird song is a learned, complex behavior. During song learning, some neural changes occur dramatically within song nuclei in neuron number, neuronal morphology, and synaptic formation or rearrangements. In order to get insights into the potential functions of CRMP-4 in the posthatching development of song nuclei during song learning, we examined the expression of CRMP-4 protein and mRNA in song control nuclei of Bengalese finch (Lonchura striata) from posthatching days (P) 10 to adulthood. Our study showed that cells positive for CRMP-4 protein and mRNA were distributed in song nuclei nearly in all the studied groups. The numbers of CRMP-4 cells in most of studied song nuclei changed significantly with age. They reached the peak at P15 in the lateral magnocellular nucleus of anterior nidopallium (LMAN) and the caudal medial nidopallium (NCM), or at P25 in HVC, Area X and the dorsolateral nucleus of the medial anterior thalamus (DLM). They then continued to decrease till adulthood. CRMP-4 protein and mRNA were both relatively high expressed during the post-hatch development of song control nuclei and song learning (P20-60), suggesting that CRMP-4 is involved in these activities. Although CRMP-4 protein and mRNA largely decreased at adulthood, they continued to express moderately, revealing that CRMP-4 may play a role in the maintenance of adult song nuclei.

Dietary retinoic acid affects song maturation and gene expression in the song system of the zebra finch.

Vitamin A, an essential nutrient, is required in its acidic form (retinoic acid) for normal embryogenesis and neuronal development, typically within well-defined concentration ranges. In zebra finches, a songbird species, localized retinoic acid synthesis in the brain is important for the development of song, a learned behavior sharing significant commonalities with speech acquisition in humans. We tested how dietary retinoic acid affects the development of song behavior and the brain's system for song control. Supplemental doses of retinoic acid given to juveniles during the critical period for song learning resulted in more variable or plastic-like songs when the birds reached adulthood, compared to the normal songs of vehicle-fed controls. We also observed that several genes (brinp1, nrgn, rxr-alpha, and sdr2/scdr9) had altered levels of expression in specific nuclei of the song system when comparing the experimental and control diet groups. Interestingly, we found significant correlations between gene expression levels in nuclei of the anterior forebrain pathway (lMAN and area X) and the degree of variability in the recorded songs. We observed, however, no major morphological effects such as changes in the volumes of song nuclei. Overall, our results lend further support to a fundamental role of retinoic acid in song maturation and point to possible molecular pathways associated with this action. The data also demonstrate that dietary content of Vitamin A can affect the maturation of a naturally learned complex behavior.

Do dietary antioxidants alleviate the cost of immune activation? An experiment with greenfinches.

Reactive oxygen and nitrogen species produced by metabolism and immune defenses can cause extensive damage to biomolecules. To counteract this damage, organisms rely on exogenous and endogenous antioxidants, although their relative importance in maintaining redox balance is unclear. We supplemented captive greenfinches with dietary antioxidants--carotenoids and vitamin E--and injected them with an inflammatory agent, phytohemagglutinin. Compared to controls, immune-challenged birds circulated more lipid peroxidation products but also increased total plasma antioxidativity. Carotenoid (but not vitamin E) supplementation generally reduced lipid peroxidation, but this did not compensate for the effects of immune activation. Levels of an endogenous antioxidant--uric acid--strongly contributed to plasma antioxidativity. We found no evidence that dietary antioxidants are immunostimulatory. These results demonstrate the antioxidant function of carotenoids in birds and show that simultaneous assessment of oxidative stress-driven damage, antioxidant barrier, and individual antioxidants is critical for explaining the potential costs of immune system activation.

FnTm2, a novel brain-specific transcript, is dynamically expressed in the song learning circuit of the zebra finch.

Zebra finch males learn their song by imitation, a process influenced by social variables. The neural pathways for acquisition and production of learned song are known, but the cellular and molecular underpinnings are not. Here we describe a novel gene named "FnTm2" ("Phantom 2") that is predicted to encode a small protein (220 aa) with a single fibronectin type III domain and a single transmembrane domain. This gene shows great variability in its expression in song system neurons of the anterior forebrain pathway (AFP), a circuit that influences song discrimination and is necessary for normal song acquisition. AFP nuclei that express FnTm2 include the nucleus HVC (its Area X-projecting neurons), Area X, and LMAN (core and shell). FnTm2 expression does not correlate with singing behavior like the immediate early gene ZENK. It is expressed variably during sleeping hours and is not dependent on an intact song circuit. FnTm2's expression is sensitive to hearing, because in deafened birds its expression is substantially reduced in the core of LMAN. Furthermore, a comparison of FnTm2 expression between mice and zebra finches revealed a conserved pattern of expression in the "limbic system." We suggest that FnTm2 may be sensitive to affective and/or attentional states and thus may provide insights on how social variables influence the production and discrimination of learned vocalizations.

Trace metal concentration in Great Tit (Parus major) and Greenfinch (Carduelis sinica) at the Western Mountains of Beijing, China.

We examined the concentrations of 11 trace metals in tissues from 10 body parts of Great Tits and Greenfinches collected at Badachu Park in the Western Mountains of Beijing, China to assess the metal accumulation level, distribution among body parts, and species and gender related variations. The highest concentrations of Hg, Ni, Zn, and Mn were found in the feather; Pb and Co in the bone; Cd, Cr, and Se in the kidney, and Cu in the liver and heart. Metal concentrations had substantial interspecific variation with Great Tits showing higher levels of Hg, Cr, Ni, and Mn than Greenfinches in tissues of most body parts. Gender related variations were body part and species specific. Meta-analyses using data from this study and other studies suggested that metal concentrations of Great Tits at our study site were relatively low and below the toxic levels.

The calmodulin pathway and evolution of elongated beak morphology in Darwin's finches.

A classic textbook example of adaptive radiation under natural selection is the evolution of 14 closely related species of Darwin's finches (Fringillidae, Passeriformes), whose primary diversity lies in the size and shape of their beaks. Thus, ground finches have deep and wide beaks, cactus finches have long and pointed beaks (low depth and narrower width), and warbler finches have slender and pointed beaks, reflecting differences in their respective diets. Previous work has shown that even small differences in any of the three major dimensions (depth, width and length) of the beak have major consequences for the overall fitness of the birds. Recently we used a candidate gene approach to explain one pathway involved in Darwin's finch beak morphogenesis. However, this type of analysis is limited to molecules with a known association with craniofacial and/or skeletogenic development. Here we use a less constrained, complementary DNA microarray analysis of the transcripts expressed in the beak primordia to find previously unknown genes and pathways whose expression correlates with specific beak morphologies. We show that calmodulin (CaM), a molecule involved in mediating Ca2+ signalling, is expressed at higher levels in the long and pointed beaks of cactus finches than in more robust beak types of other species. We validated this observation with in situ hybridizations. When this upregulation of the CaM-dependent pathway is artificially replicated in the chick frontonasal prominence, it causes an elongation of the upper beak, recapitulating the beak morphology of the cactus finches. Our results indicate that local upregulation of the CaM-dependent pathway is likely to have been a component of the evolution of Darwin's finch species with elongated beak morphology and provide a mechanistic explanation for the independence of beak evolution along different axes. More generally, our results implicate the CaM-dependent pathway in the developmental regulation of craniofacial skeletal structures.

Distribution of orexin/hypocretin immunoreactivity in the brain of a male songbird, the house finch, Carpodacus mexicanus.

Previous research has shown orexin/hypocretin immunoreactive (orexin-ir) neurons in domesticated Galliformes. However, these findings may not be representative of other birds and these studies did not include a distribution of orexin-ir projections throughout the brain. The present study was carried out in a wild-caught passerine, the house finch, Carpodacus mexicanus, and includes a detailed description of orexin-ir neurons and their projections. Orexin A and B-ir neurons were located in a single population centered on the paraventricular nucleus of the hypothalamus extending into the lateral hypothalamic area, consistent with other studies in birds. Orexin A and B-ir fibers were similarly visible across the brain, with the highest density within the preoptic area, hypothalamus and thalamus. Orexin-ir projections extended from the paraventricular nucleus rostrally to the preoptic area, laterally towards the medial striatum, nidopallium, and dorsally along the lateral ventricle towards the mesopallium. Caudally, the highest densities of orexin-ir fibers were found along the third ventricle. The periaqueductal grey, substantia nigra pars compacta and the locus coeruleus also showed a high density of orexin-ir fibers. This study showed a detailed fiber distribution previously unreported in birds and showed that orexin-ir neurons were located in similar areas regardless of phylogeny or domestication in birds. The apparently conserved neural distribution of orexins suggests that these peptides play similar roles among birds. The widespread distribution of the projections in brain areas serving various roles indicates the potential involvement of these peptides in multiple behavioral and physiological functions.

The combined effect of lead exposure and high or low dietary calcium on health and immunocompetence in the zebra finch (Taeniopygia guttata).

The widespread contamination by lead and the acidification of the environment ask for a better understanding of the effects of the interaction between lead and calcium on various aspects of health, including disease defense, in wildlife. Here, we investigated the effects of chronic exposure to sublethal levels of lead, combined with high or low dietary calcium, on health and several components of immunity in male and female zebra finches (Taeniopygia guttata). Thirty individuals of each sex were randomly assigned to three groups: a control group, a group exposed to lead with an additional calcium source (i.e. grit) and a group exposed to lead without access to an extra calcium source. Lead was administered as lead acetate via the drinking water (20 ppm) for 38 consecutive days. Exposure to lead increased significantly the concentrations of lead in kidney and bone in individuals of the experimental groups. Furthermore, the lack of a calcium supplement significantly enhanced the uptake of lead. Lead did not affect health indices such as hematocrit, spleen mass and body mass, nor the adrenal stress response. Cell-mediated immune responsiveness, assessed by a delayed-type hypersensitivity response to phytohaemagglutinin, was also not affected by lead exposure. On the other hand, lead exposure did significantly suppress the secondary humoral immune response towards sheep red blood cells in females, but only when the additional calcium source was not available. This effect was not found in males, suggesting sexual differences in susceptibility of humoral immunity to lead treatment in zebra finches.