Baseline and stress-induced glucocorticoids during reproduction in the variable flying fox, Pteropus hypomelanus (Chiroptera: Pteropodidae)

Baseline and stress-responsive glucocorticoid (GC) levels were assessed during early pregnancy, late pregnancy, and lactation in female variable flying foxes (Pteropus hypomelanus) and in males over the same time period. Animals were maintained in a breeding colony in captivity. High levels of both cortisol and corticosterone were detected, with total plasma GC levels being among the highest documented in vertebrates (up to 3000 ng/ml in individual animals, with cortisol being the primary GC, accounting for approximately 78% of total GCs), and significantly greater in males than in females. Plasma levels of cortisol and corticosterone showed nearly identical profiles within each sex, with the exception of females in late pregnancy, in which corticosterone, but not cortisol, increased significantly. Baseline levels of plasma cortisol were highest in September (when pups were between 1 and 2 months of age) in both sexes, which may be related to the approaching onset of the mating period. There was a continuum in the magnitude of the response to stress (handling and sampling) over time in females, with the greatest stress response in early pregnancy, a dampened response during late pregnancy, and no significant stress response during lactation. Surprisingly, males failed to exhibit elevated GCs after this stress, but did have significant stress-induced hyperglycemia and suppression of plasma testosterone levels. This may be due to their high (perhaps maximal) baseline levels, which suggests that being in a breeding group was chronically stressful for males.     

Distribution and developmental change in [3H]MK-801 binding within zebra finch song nuclei.

In many songbirds, vocal learning depends upon appropriate auditory experience during a sensitive period that coincides with the formation and reorganization of song-related neural pathways. Because some effects of early sensory experience on neural organization and early learning have been linked to activation of N-methyl-D-aspartate (NMDA) receptors, we measured binding to this receptor within the neural system controlling song behavior in zebra finches. Quantitative autoradiography was used to measure binding of the noncompetitive antagonist [3H]MK-801 (dizocilpine) in the brains of both adult and juvenile male zebra finches, focusing on four telencephalic regions implicated in song learning and production. Overall, the pattern of MK-801 binding in zebra finches was similar to the pattern found in rats (Monaghan and Cotman, 1985, J. Neurosci. 5:2909-2919; Sakurai, Cha, Penney, and Young, 1991, Neuroscience 40:533-543). That is, binding was highest in the telencephalon, intermediate in thalamic regions, and virtually absent from the brain stem and cerebellum. The telencephalic song areas exhibited intermediate levels of binding, and binding in the juveniles was not significantly different from adult levels in most song nuclei. However, in the lateral magnocellular nucleus of the anterior neostriatum (IMAN), binding at 30 days of age was significantly higher than binding in adults. Given the established role of NMDA receptors in other developing neural systems, both their presence in song control nuclei and their developmental regulation within a region implicated in song learning suggest that NMDA receptors play a role in mediating effects of auditory experience on the development of song behavior.     

Rationale and methodology for testing auditory cognition in songbirds

Songbirds, and in particular zebra finches, present a wonderful opportunity to study cognition in species that have evolved specialized abilities and brain structures for auditory cognition. The authors describe the rationale, methods, and apparatus used to test the auditory perceptual and cognitive abilities of songbirds. They have developed an operant conditioning system for conducting discrimination experiments simultaneously with several songbirds. The system uses specialized single-board computers, standard personal computers, CD-ROMs, and custom-written software to present stimuli, control training, and record responses. Also, the authors describe software to produce high-quality synthesized and naturally occurring acoustic stimuli for use in studies of auditory cognition. Typical results from a challenging frequency-range discrimination are included.     

Distribution and developmental change in [3H]MK-801 binding within zebra finch song nuclei

In many songbirds, vocal learning depends upon appropriate auditory experience during a sensitive period that coincides with the formation and reorganization of song-related neural pathways. Because some effects of early sensory experience on neural organization and early learning have been linked to activation of N-methyl-d-aspartate (NMDA) receptors, we measured binding to this receptor within the neural system controlling song behavior in zebra finches. Quantitative autoradiography was used to measure binding of the noncompetitive antagonist [³H]MK-801 (dizocilpine) in the brains of both adult and juvenile male zebra finches, focusing on four telencephalic regions implicated in song learning and production. Overall, the pattern of MK-801 binding in zebra finches was similar to the pattern found in rats (Monaghan and Cotman, 1985, J. Neurosci. 5:2909–2919; Sakurai, Cha, Penney, and Young, 1991, Neuroscience 40:533–543). That is, binding was highest in the telencephalon, intermediate in thalamic regions, and virtually absent from the brain stem and cerebellum. The telencephalic song areas exhibited intermediate levels of binding, and binding in the juveniles was not significantly different from adult levels in most song nuclei. However, in the lateral magnocellular nucleus of the anterior neostriatum (IMAN), binding at 30 days of age was significantly higher than binding in adults. Given the established role of NMDA receptors in other developing neural systems, both their presence in song control nuclei and their developmental regulation within a region implicated in song learning suggest that NMDA receptors play a role in mediating effects of auditory experience on the development of song behavior.     

The acoustic expression of stress in a songbird: Does corticosterone drive isolation-induced modifications of zebra finch calls?

Animal vocalizations convey multiple pieces of information about the sender. Some of them are stable, such as identity or sex, but others are labile like the emotional or motivational state. Only a few studies have examined the acoustic expression of emotional state in non-human animals and related vocal cues to physiological parameters. In this paper, we examined the vocal expression of isolation-induced stress in a songbird, the zebra finch (Taeniopygia guttata). Although songbirds use acoustic communication extensively, nothing is known to date on how they might encode physiological states in their vocalizations. We tested the hypothesis that social isolation in zebra finches induces a rise of plasma corticosterone that modifies the vocal behavior. We monitored plasma corticosterone, as well as call rate and acoustic structure of calls of males in response to the playback of female calls of varied saliences (familiar versus stranger) in two situations: social isolation and social housing. Social isolation induced both a rise in plasma corticosterone, and a range of modifications in males' vocal behavior. Isolated birds showed a lower vocal activity, an abolition of the difference of response between the two stimuli, and evoked calls with longer duration and higher pitch. Because some of these effects were mimicked after oral administration of corticosterone in socially housed subjects, we conclude that corticosterone could be partly responsible for the isolation-related modifications of calls in male zebra finches. To our knowledge, this is the first demonstration of the direct implication of glucocorticoids in the modulation of the structure of vocal sounds.     

Zebra Finch Mates Use Their Forebrain Song System in Unlearned Call Communication

Unlearned calls are produced by all birds whereas learned songs are only found in three avian taxa, most notably in songbirds. The neural basis for song learning and production is formed by interconnected song nuclei: the song control system. In addition to song, zebra finches produce large numbers of soft, unlearned calls, among which “stack” calls are uttered frequently. To determine unequivocally the calls produced by each member of a group, we mounted miniature wireless microphones on each zebra finch. We find that group living paired males and females communicate using bilateral stack calling. To investigate the role of the song control system in call-based male female communication, we recorded the electrical activity in a premotor nucleus of the song control system in freely behaving male birds. The unique combination of acoustic monitoring together with wireless brain recording of individual zebra finches in groups shows that the neuronal activity of the song system correlates with the production of unlearned stack calls. The results suggest that the song system evolved from a brain circuit controlling simple unlearned calls to a system capable of producing acoustically rich, learned vocalizations.     

Localization of androgen receptor mRNA-containing cells in avian respiratory-vocal nuclei: An in situ hybridization study

Song development and song pattern formation in oscine songbirds are influenced by steroid hormones such as estrogens and androgens, and the control of vocal pattern generation is mediated via a network of interconnected vocal and respiratory nuclei. The main components of the respiratory part of the network are the expiratory and inspiratory premotor nuclei, known as retroambigualis (RAm) and the rostral ventral respiratory group (rVRG), respectively. These respiratory components play an integral role in song production either by providing the expiratory pulses of air required for each and every song syllable, or by controlling inspiration between syllables in the form of minibreaths, and between phrases for major replenishments of air. Here we analyze the distribution of androgen receptors (AR) and estrogen receptors (ER) in the midbrain and hindbrain of male and female zebra finches, and male canaries and green finches, using in situ hybridization with cRNA probes of the zebra finch AR and ER. ERmRNA was not expressed in any of the respiratory-vocal nuclei of the midbrain or hindbrain, but ARmRNA was expressed in the tracheosyringeal motor nucleus (nXIIts) and in RAm and rVRG. The size of the ARmRNA defined RAm and rVRG was similar in male and female zebra finches, but the size of ARmRNA defined nXIIts was slightly sexual dimorphic. Previously undescribed areas of ARmRNA expression outside the respiratory-vocal network in the brain stem were the nucleus semilunaris and layers 10–12 of the optic tectum. ARmRNA expression in the respiratory-vocal nuclei of adult male songbirds, adult female zebra finches, and juvenile zebra finches suggests that the temporal pattern of learned and unlearned vocalizations is sensitive to androgen-dependent mechanisms mediated by RAm and rVRG. © 1997 John Wiley & Sons, Inc. J Neurobiol 33: 865–876, 1997     

Influence of pinealectomy and pineal stalk deflection on circadian gastrointestinal tract melatonin rhythms in zebra finches (Taeniopygia guttata).

The authors examined levels of melatonin in the plasma and various tissues in intact, pinealectomized, and pineal stalk-deflected zebra finches kept under 12:12 LD to determine if the melatonin found in the gastrointestinal tract is secreted in a circadian manner. In intact and pineal stalk-deflected birds, there is a clear day-night rhythm in melatonin content of the plasma, pineal gland, eyes, proventriculus, crop, duodenum, jejunum/ileum, colon, heart, and liver. In contrast, pinealectomy abolished the day-night rhythm. These results indicate that most of the melatonin present in the gastrointestinal tract of zebra finches is of pineal origin. However, some melatonin remained. This suggests that this melatonin may be locally synthesized and has paracrine and/or autocrine functions. Nonetheless, the results do not lend support to the contention that this putative melatonin secretion by the gastrointestinal tract is circadian.     

Food, stress, and reproduction: Short-term fasting alters endocrine physiology and reproductive behavior in the zebra finch

Stress is thought to be a potent suppressor of reproduction. However, the vast majority of studies focus on the relationship between chronic stress and reproductive suppression, despite the fact that chronic stress is rare in the wild. We investigated the role of fasting in altering acute stress physiology, reproductive physiology, and reproductive behavior of male zebra finches (Taeniopygia guttata) with several goals in mind. First, we wanted to determine if acute fasting could stimulate an increase in plasma corticosterone and a decrease in corticosteroid binding globulin (CBG) and testosterone. We then investigated whether fasting could alter expression of undirected song and courtship behavior. After subjecting males to fasting periods ranging from 1 to 10 h, we collected plasma to measure corticosterone, CBG, and testosterone. We found that plasma corticosterone was elevated, and testosterone was decreased after 4, 6, and 10 h of fasting periods compared with samples collected from the same males during nonfasted (control) periods. CBG was lower than control levels only after 10 h of fasting. We also found that, coincident with these endocrine changes, males sang less and courted females less vigorously following short-term fasting relative to control conditions. Our data demonstrate that acute fasting resulted in rapid changes in endocrine physiology consistent with hypothalamo-pituitary-adrenal axis activation and hypothalamo-pituitary-gonadal axis deactivation. Fasting also inhibited reproductive behavior. We suggest that zebra finches exhibit physiological and behavioral flexibility that makes them an excellent model system for studying interactions of acute stress and reproduction.     

Effects of testosterone and corticosterone on immunocompetence in the zebra finch

The original immunocompetence handicap hypothesis (ICHH) suggested that testosterone has a handicapping effect in males by both promoting the development of sexual signals and suppressing immune function. A modified version, the stress-linked ICHH, has recently proposed that testosterone is immunosuppressive indirectly by increasing production of corticosterone. To test both the original and stress-mediated versions of the ICHH, we implanted male zebra finches taken from lines selected for divergent maximum stress-induced levels of corticosterone (high, low and control) with either empty or testosterone-filled implants. Their humoral and cell-mediated immune responses were then assessed by challenge with diphtheria:tetanus vaccine and phytohemagglutinin respectively. We found no effect of the hormone manipulations on either PHA or tetanus antibody responses, but found a significant interaction between titers of both testosterone and corticosterone on diphtheria secondary antibody response; antibody response was greatest in individuals with high levels of both hormones. There was also a significant interactive effect between testosterone treatment group and corticosterone titer on body mass; the body mass of males in the elevated testosterone treatment group decreased with increasing corticosterone titer. These results suggest that, contrary to the assumption of the stress-mediated version of the ICHH, high plasma levels of corticosterone are not immunosuppressive, but are in fact immuno-enhancing in the presence of high levels of plasma testosterone. Equally, the central assumption of the ICHH that testosterone is obligately immunosuppressive is also not supported. The same individuals with the highest levels of both hormones and consequently the most robust antibody response also possessed the lowest body mass.     

Flight performance in the altricial zebra finch: Developmental effects and reproductive consequences

The environmental conditions animals experience during development can have sustained effects on morphology, physiology, and behavior. Exposure to elevated levels of stress hormones (glucocorticoids, GCs) during development is one such condition that can have long‐term effects on animal phenotype. Many of the phenotypic effects of GC exposure during development (developmental stress) appear negative. However, there is increasing evidence that developmental stress can induce adaptive phenotypic changes. This hypothesis can be tested by examining the effect of developmental stress on fitness‐related traits. In birds, flight performance is an ideal metric to assess the fitness consequences of developmental stress. As fledglings, mastering takeoff is crucial to avoid bodily damage and escape predation. As adults, takeoff can contribute to mating and foraging success as well as escape and, thus, can affect both reproductive success and survival. We examined the effects of developmental stress on flight performance across life‐history stages in zebra finches (Taeniopygia guttata). Specifically, we examined the effects of oral administration of corticosterone (CORT, the dominant avian glucocorticoid) during development on ground‐reaction forces and velocity during takeoff. Additionally, we tested for associations between flight performance and reproductive success in adult male zebra finches. Developmental stress had no effect on flight performance at all ages. In contrast, brood size (an unmanipulated variable) had sustained, negative effects on takeoff performance across life‐history stages with birds from small broods performing better than birds from large broods. Flight performance at 100 days posthatching predicted future reproductive success in males; the best fliers had significantly higher reproductive success. Our results demonstrate that some environmental factors experienced during development (e.g. clutch size) have stronger, more sustained effects than others (e.g. GC exposure). Additionally, our data provide the first link between flight performance and a direct measure of reproductive success.     

Singing Under the Influence: Examining the Effects of Nutrition and Addiction on a Learned Vocal Behavior

The songbird model is widely established in a number of laboratories for the investigation of the neurobiology and development of vocal learning. While vocal learning is rare in the animal kingdom, it is a trait that songbirds share with humans. The neuroanatomical and physiological organization of the brain circuitry that controls learned vocalizations has been extensively characterized, particularly in zebra finches (Taeniopygia guttata). Recently, several powerful molecular and genomic tools have become available in this organism, making it an attractive choice for neurobiologists interested in the neural and genetic basis of a complex learned behavior. Here, we briefly review some of the main features of vocal learning and associated brain structures in zebra finches and comment on some examples that illustrate how themes related to nutrition and addiction can be explored using this model organism.     

Age effect of deafening on stereotyped song maintenance in adult male bengalese finches Lonchura striata domestica

Birdsong is a complex learned vocal behavior that relies on auditory experience for development.However,it appears that among different species of close-ended songbirds,there are some variations in the necessity of auditory feedback for maintaining stereotyped adult song.In zebra finches,the deterioration of adult songs following deafness depends on the birds' age.It is unknown whether this age effect is a general rule in other avian species as well.Therefore,we chose Bengalese finches,whose songs show more...     

Effects of experimentally elevated testosterone on plasma glucocorticoids, body mass, and recapture rates in yellow-pine chipmunks, Tamias amoenus

In male yellow-pine chipmunks plasma levels of glucocorticoids (GCs) are low while plasma testosterone (T) levels peak during the mating season, suggesting that T suppresses GC levels. To test this hypothesis, free-living, post-reproductive males were implanted during summer with either a T-filled (T-males) or an empty silastic implant (controls or C-males). Body mass and plasma levels of corticosterone, cortisol, and T were measured immediately before and 1 month after implantation. Exogenous testosterone increased T to high physiological levels typical of reproductively active males. By 1 month after implantation, T-males decreased their mean body mass and plasma GC levels, while C-males maintained their mean body mass and GC levels. Even though T-males lost mass, recapture success 1 month after implantation for T-males (71%) was equal to that of C-males (71%). However, the overwinter recapture rate of C-males (83%) was significantly greater than that of T-males (20%). The results support the hypothesis that high plasma T of males during mating has a suppressive effect on plasma GC levels. Additionally, experimentally elevated T significantly reduced the rate of recapture during the following spring, and this may reflect a reduction in local overwinter survival. The suppression of adrenocortical activity by T may contribute to the reductions in prehibernation body mass and post-emergence recapture success. J. Exp. Zool. 287:378-383, 2000.     

Carotenoids, immunocompetence, and the information content of sexual colors: an experimental test

Many male birds use carotenoid pigments to acquire brilliant colors that advertise their health and condition to prospective mates. The direct means by which the most colorful males achieve superior health has been debated, however. One hypothesis, based on studies of carotenoids as antioxidants in humans and other animals, is that carotenoids directly boost the immune system of colorful birds. We studied the relationship between carotenoid pigments, immune function, and sexual coloration in zebra finches (Taeniopygia guttata), a species in which males incorporate carotenoid pigments into their beak to attract mates. We tested the hypotheses that increased dietary carotenoid intake enhances immunocompetence in male zebra finches and that levels of carotenoids circulating in blood, which also determine beak coloration, directly predict the immune response of individuals. We experimentally supplemented captive finches with two common dietary carotenoid pigments (lutein and zeaxanthin) and measured cell-mediated and humoral immunity a month later. Supplemented males showed elevated blood-carotenoid levels, brighter beak coloration, and increased cell-mediated and humoral immune responses than did controls. Cell-mediated responses were predicted directly by changes in beak color and plasma carotenoid concentration of individual birds. These experimental findings suggest that carotenoid-based color signals in birds may directly signal male health via the immunostimulatory action of ingested and circulated carotenoid pigments.     

Immediate early gene (ZENK) responses to song in juvenile female and male zebra finches: effects of rearing environment

Accurate song perception is likely to be as important for female songbirds as it is for male songbirds. Male zebra finches (Taeniopygia guttata) show differential ZENK expression to conspecific and heterospecific songs by day 30 posthatch in auditory perceptual brain regions such as the caudomedial nidopallium (NCM) and the caudomedial mesopallium (CMM). The current study examined ZENK expression in response to songs of different qualities at day 45 posthatch in both sexes. Normally reared juvenile zebra finches showed higher densities of immunopositive nuclei in both the dorsal and ventral areas of NCM and CMM (formerly cmHV), but not HA, a visual area, in response to normal song over untutored song or silence. Male and female patterns of ZENK expression did not differ. We next compared responses of birds reared without exposure to normal song (untutored) to those of normally reared birds. Untutored birds did not show higher responses to normal song than to untutored song in the three song perception areas. Furthermore, untutored birds of both sexes showed lower densities of immunopositive nuclei in all four areas than did normally reared birds. In addition, ZENK expression was greater in untutored females than in males in the dorsal portion of NCM and in CMM. Our findings suggest that at least some neural mechanisms of song perception are in place in socially reared female and male finches at an early age. Furthermore, early exposure to song tutors affects responses to song stimuli.     

Ontogeny of Adaptive Antibody Response to a Model Antigen in Captive Altricial Zebra Finches

Based on studies from the poultry literature, all birds are hypothesized to require at least 4 weeks to develop circulating mature B-cell lineages that express functionally different immunoglobulin specificities. However, many altricial passerines fledge at adult size less than four weeks after the start of embryonic development, and therefore may experience a period of susceptibility during the nestling and post-fledging periods. We present the first study, to our knowledge, to detail the age-related changes in adaptive antibody response in an altricial passerine. Using repeated vaccinations with non-infectious keyhole limpet hemocyanin (KLH) antigen, we studied the ontogeny of specific adaptive immune response in altricial zebra finches Taeniopygia guttata. Nestling zebra finches were first injected at 7 days (7d), 14 days (14d), or 21 days post-hatch (21d) with KLH-adjuvant emulsions, and boosted 7 days later. Adults were vaccinated in the same manner. Induced KLH-specific IgY antibodies were measured using ELISA. Comparisons within age groups revealed no significant increase in KLH-specific antibody levels between vaccination and boost in 7d birds, yet significant increases between vaccination and boost were observed in 14d, 21d, and adult groups. There was no significant difference among age groups in KLH antibody response to priming vaccination, yet KLH antibody response post-boost significantly increased with age among groups. Post-boost antibody response in all nestling age groups was significantly lower than in adults, indicating that mature adult secondary antibody response level was not achieved in zebra finches prior to fledging (21 days post-hatch in zebra finches). Findings from this study contribute fundamental knowledge to the fields of developmental immunology and ecological immunology and strengthen the utility of zebra finches as a model organism for future studies of immune ontogeny.     

Acoustic characteristics, early experience, and endocrine status interact to modulate female zebra finches' behavioral responses to songs

Female songbirds use male songs as an important criterion for mate selection. Properties of male songs are thought to indicate the male's quality as a potential mate. Song preferences in female zebra finches are known to be influenced by two factors--early auditory experience and the acoustic characteristics of males' songs. Studies often investigate song preferences by priming females with estrogen. However, estrogenic influences on song preferences have not been studied. We investigated the relative influence of early auditory experience, acoustic features of songs, and estrogen availability on song responsiveness in female zebra finches. Juvenile female zebra finches were tutored for 10 days with 40 songs per day with one of three acoustically different song types--simple songs, long-bout songs or complex songs. A fourth group of females was untutored. Aside from this brief song exposure, females were raised and maintained without exposure to male songs. During adulthood, females' behavioral responses to the three song types were tested under three hormone conditions--untreated, estradiol-treated and 1,4,6-androstatriene-3,17-dione (ATD)-treated (to lower endogenous estrogen). Based on the results of our study, four conclusions can be drawn. First, song responsiveness in female zebra finches is strongly affected by minimal early acoustic experience. Second, inexperienced female zebra finches are inherently biased to respond more to complex songs over other song types Third, although female zebra finches are inherently biased to respond more to complex songs, early acoustic experience may either reinforce or weaken this inherent responsiveness to complex songs. Fourth, estrogen selectively accentuates song responsiveness in acoustically-experienced female zebra finches.     

Glucocorticoids exacerbate the deleterious effects of gp120 in hippocampal and cortical explants

Glucocorticoids (GCs), the adrenal steroids secreted during stress, can compromise the ability of hippocampal neurons to survive numerous necrotic insults. We have previously observed that GCs worsen the deleterious effects of gp120, the glycoprotein of the acquired immune deficiency syndrome virus, which can indirectly damage neurons and which is thought to play a role in the neuropathological features of human immunodeficiency virus infection. Specifically, GCs augment gp120-induced calcium mobilization, ATP depletion, decline in mitochondrial potential, and neurotoxicity in fetal monolayer cultures from a number of brain regions. In the present report, we demonstrate a similar gp120/GC synergy in adult hippocampal and cortical explants. We generated explants from rats that were either adrenalectomized, adrenally intact, or intact and treated with corticosterone to produce levels seen in response to major stressors. Metabolic rates in explants were then indirectly assessed with silicon microphysiometry, and cytosolic calcium concentrations were assessed with fura-2 fluorescent microscopy. We observed that basal levels of GCs tonically augment the disruptive effects of gp120 on metabolism in the CA1 cell field of the hippocampus and in the cortex. Moreover, raising GC concentrations into the stress range exacerbated the ability of gp120 to mobilize cytosolic calcium in a number of hippocampal cell fields. Finally, we observed that the synthetic GC prednisone had similarly exacerbating effects on gp120. Thus, GCs can worsen the deleterious effects of gp120 in a system that is more physiologically relevant than the fetal monolayer culture and in a region-specific manner.