Genes located in a chromosomal inversion are correlated with territorial song in white-throated sparrows.

The genome of the white-throated sparrow (Zonotrichia albicollis) contains an inversion polymorphism on chromosome 2 that is linked to predictable variation in a suite of phenotypic traits including plumage color, aggression and parental behavior. Differences in gene expression between the two color morphs, which represent the two common inversion genotypes (ZAL2/ZAL2 and ZAL2/ZAL2(m) ), may therefore advance our understanding of the molecular underpinnings of these phenotypes. To identify genes that are differentially expressed between the two morphs and correlated with behavior, we quantified gene expression and terrirorial aggression, including song, in a population of free-living white-throated sparrows. We analyzed gene expression in two brain regions, the medial amygdala (MeA) and hypothalamus. Both regions are part of a 'social behavior network', which is rich in steroid hormone receptors and previously linked with territorial behavior. Using weighted gene co-expression network analyses, we identified modules of genes that were correlated with both morph and singing behavior. The majority of these genes were located within the inversion, showing the profound effect of the inversion on the expression of genes captured by the rearrangement. These modules were enriched with genes related to retinoic acid signaling and basic cellular functioning. In the MeA, the most prominent pathways were those related to steroid hormone receptor activity. Within these pathways, the only gene encoding such a receptor was ESR1 (estrogen receptor 1), a gene previously shown to predict song rate in this species. The set of candidate genes we identified may mediate the effects of a chromosomal inversion on territorial behavior.

Chromosome-wide linkage disequilibrium caused by an inversion polymorphism in the white-throated sparrow (Zonotrichia albicollis).

Chromosomal inversions have been of long-standing interest to geneticists because they are capable of suppressing recombination and facilitating the formation of adaptive gene complexes. An exceptional inversion polymorphism (ZAL2(m)) in the white-throated sparrow (Zonotrichia albicollis) is linked to variation in plumage, social behavior and mate choice, and is maintained in the population by negative assortative mating. The ZAL2(m) polymorphism is a complex inversion spanning > 100 Mb and has been proposed to be a strong suppressor of recombination, as well as a potential model for studying neo-sex chromosome evolution. To quantify and evaluate these features of the ZAL2(m) polymorphism, we generated sequence from 8 ZAL2(m) and 16 ZAL2 chromosomes at 58 loci inside and 4 loci outside the inversion. Inside the inversion we found that recombination was completely suppressed between ZAL2 and ZAL2(m), resulting in uniformly high levels of genetic differentiation (F(ST)=0.94), the formation of two distinct haplotype groups representing the alternate chromosome arrangements and extensive linkage disequilibrium spanning ~104 Mb within the inversion, whereas gene flow was not suppressed outside the inversion. Finally, although ZAL2(m) homozygotes are exceedingly rare in the population, occurring at a frequency of < 1%, we detected evidence of historical recombination between ZAL2(m) chromosomes inside the inversion, refuting its potential status as a non-recombining autosome.

Gonadotrophin-releasing hormone neurons in a photoperiodic songbird express fos and egr-1 protein after a single long day.

Birds use a variety of environmental cues, such as day length, temperature and social interactions, to time reproductive efforts. For most seasonally breeding birds, day length is the most important cue and takes precedence over all others. Experimental manipulation of day length has shown that, in a number of galliformes and passeriformes, exposure to a single long day induces a rise in plasma luteinising hormone (LH). The mechanisms underlying this response are only beginning to be understood. In Japanese quail and Zonotrichia sparrows, one long day causes striking up-regulation of the protein products of immediate early genes (IEGs) in the mediobasal hypothalamus, near gonadotrophin-releasing hormone (GnRH) axons and terminals. Photoperiodic induction of the same proteins in the GnRH somata themselves, however, has not been described in these species. In the present study, we used immunohistochemistry to assay the induction of two IEGs, Fos and Egr-1, in the GnRH somata of male and female white-throated sparrows (Zonotrichia albicollis) exposed to a single long day. We found that immunoreactivity for both proteins increased in a subset of the GnRH neurones of the septo-preoptic area by the morning after the long day. Photo-induced expression of Egr-1 or Fos protein in GnRH neurones was limited to a population of cells in the medial preoptic area. Males showed significantly greater induction of both proteins in this population of GnRH neurones than did females, which is consistent with the hypothesis that males may be more sensitive to photic cues. Overall, the results obtained suggest that photostimulation stimulates new protein synthesis in GnRH neurones on a relatively rapid time scale. Further research is required to determine whether the GnRH somata are themselves integrating photic cues, or whether they are responding rapidly to an increased demand for GnRH synthesis.

Immediate early gene response to hearing song correlates with receptive behavior and depends on dialect in a female songbird.

Stimulus-induced expression of the immediate early gene ZENK (egr-1) in the songbird's auditory forebrain presumably depends on the behavioral significance of the stimulus. Few studies, however, have quantified both the ZENK and behavioral responses to a stimulus in the same individuals. We played conspecific male song of either hatch (local) or foreign dialect to female white-crowned sparrows (Zonotrichia leucophrys oriantha) and quantified both the auditory ZENK response and their behavioral response, which is known to depend on dialect. Birds hearing hatch dialect showed greater ZENK induction in the caudomedial hyperstriatum ventrale and the dorsal portion of the caudomedial neostriatum than birds hearing foreign dialect, supporting previous work showing a relationship between ZENK and salience of the stimulus. In the dorsal portion of the caudomedial neostriatum, ZENK induction was correlated with the amount of non-vocal courtship behavior; however, in the caudomedial hyperstriatum ventrale, ZENK induction was more highly correlated with the females' own vocal behavior and thus may have been partly self-induced. Some females sang and showed a male-like pattern of ZENK induction in their song systems. This study provides the first evidence that the ZENK response in a sensory area to a social stimulus is proportional to the animal's preference for the stimulus.

Gonadal steroid receptor mRNA in catecholaminergic nuclei of the canary brainstem.

Steroid actions in the song system may be modulated by ascending inputs from catecholaminergic (CA) brain nuclei; however, whether these nuclei contain steroid receptors is unknown. Here, we compared the distribution of androgen receptor (AR) and estrogen receptor-alpha (ER-alpha) mRNA with that of tyrosine hydroxylase immunoreactivity (TH-IR) in the brainstems of male canaries. Areas containing AR and ER-alpha mRNA overlapped with areas containing TH-IR cell bodies in the locus ceruleus and the area ventralis of Tsai. The substantia nigra and the midbrain central gray contained both TH-IR and AR mRNA. The presence of AR and ER-alpha within CA cell groups suggests that sex steroid hormones may modulate song production at the site of CA synthesis.

Visual influences on the development and recovery of the vestibuloocular reflex in the chicken.

Whenever the head turns, the vestibuloocular reflex (VOR) produces compensatory eye movements to help stabilize the image of the visual world on the retina. Uncompensated slip of the visual world across the retina results in a gradual change in VOR gain to minimize the image motion. VOR gain changes naturally during normal development and during recovery from neuronal damage. We ask here whether visual slip is necessary for the development of the chicken VOR (as in other species) and whether it is required for the recovery of the VOR after hair cell loss and regeneration. In the first experiment, chickens were reared under stroboscopic illumination, which eliminated visual slip. The horizontal and vertical VORs (h- and vVORs) were measured at different ages and compared with those of chickens reared in normal light. Strobe-rearing prevented the normal development of both h- and vVORs. After 8 wk of strobe-rearing, 3 days of exposure to normal light caused the VORs to recover partially but not to normal values. In the second experiment, 1-wk-old chicks were treated with streptomycin, which destroys most vestibular hair cells and reduces hVOR gain to zero. In birds, vestibular hair cells regenerate so that after 8 wk in normal illumination they appear normal and hVOR gain returns to values that are normal for birds of that age. The treated birds in this study recovered in either normal or stroboscopic illumination. Their hVOR and vVOR and vestibulocollic reflexes (VCR) were measured and compared with those of untreated, age-matched controls at 8 wk posthatch, when hair cell regeneration is known to be complete. As in previous studies, the gain of the VOR decreased immediately to zero after streptomycin treatment. After 8 wk of recovery under normal light, the hVOR was normal, but vVOR gain was less than normal. After 8 wk of recovery under stroboscopic illumination, hVOR gain was less than normal at all frequencies. VCR recovery was not affected by the strobe environment. When streptomycin-treated, strobe-recovered birds were then placed in normal light for 2 days, hVOR gain returned to normal. Taken together, the results of these experiments suggest that continuous visual feedback can adjust VOR gain. In the absence of appropriate visual stimuli, however, there is a default VOR gain and phase to which birds recover or revert, regardless of age. Thus an 8-wk-old chicken raised in a strobe environment from hatch would have the same gain as a streptomycin-treated chicken that recovers in a strobe environment.

Effects of N-methyl-D-aspartate on luteinizing hormone release and Fos-like immunoreactivity in the male White-crowned sparrow (Zonotrichia leucophrys gambelii).

Seasonal breeding is terminated in the White-crowned sparrow by the onset of absolute photorefractoriness, a condition in which the reproductive system is switched off indefinitely until it is dissipated by short day lengths. Absolute photorefractoriness is controlled by the central nervous system; however, the mechanisms underlying GnRH quiescence in photorefractory birds have yet to be elucidated. Using the excitatory amino acid glutamate agonist N-methyl-D-aspartate (NMDA), plasma LH levels in White-crowned sparrows were significantly elevated regardless of the reproductive or photoperiodic condition. NMDA also significantly induced Fos-like immunoreactivity (FLI) within the infundibular nucleus and median eminence, regions previously shown to express FLI after a photoperiodically driven LH rise. NMDA did not induce FLI within GnRH I neurons; instead, it significantly activated cells within the organum vasculosum of the lamina terminalis in close proximity to GnRH I perikarya. These findings provide the first evidence that photorefractoriness is not due to depletion of GnRH stores, as LH and presumably GnRH were secreted in response to excitatory amino acid stimulation. NMDA activation of FLI in the region of the organum vasculosum of the lamina terminalis and the basal tuberal hypothalamus suggests that seasonal reproductive neuroendocrine control may be mediated via cells in the region of the GnRH I perikarya and terminals.

Effects of vasoactive intestinal peptide on plasma prolactin in passerines.

Vasoactive intestinal peptide (VIP) is a potent releaser of prolactin (PRL) in domestic fowl, turkey, and ring doves. However, few comparative studies have investigated this in wild species. We tested the effects of intravenously administered chicken VIP on plasma PRL concentrations in four passerine species: the white-crowned sparrow (Zonotrichia leucophrys gambelii), the dark-eyed junco (Junco hyemalis), the Florida scrub-jay (Aphelocoma coerulescens), and the western scrub-jay (A. californica). In the white-crowned sparrow, junco, and Florida scrub-jay, which were tested during the breeding season, VIP induced a rapid increase in plasma PRL. Serial plasma samples taken after VIP injection in the white-crowned sparrow show a 10-fold increase in PRL within 2 min of treatment, followed by a gradual decline. Effects of VIP, as compared to saline, remained significant for at least 20 min after treatment. Western scrub-jays did not respond to intravenous VIP with a significant rise in PRL secretion, possibly because they were tested after termination of the breeding season. This study indicates that VIP control of PRL release may be widespread among avian species, and that seasonal changes in plasma PRL may be mediated in part at the level of the pituitary. In addition, analysis of the control data revealed no increase in plasma PRL as a result of injection or restraint, suggesting that unlike in mammals, PRL is not released during acute stress in passerines.

Effects of ambient temperature on photo-induced prolactin secretion in three subspecies of white-crowned sparrow, Zonotrichia leucophrys.

We tested the effects of naturally relevant ambient temperatures (5, 20, and 30 degrees C) on photoinduced prolactin (PRL) secretion in three subspecies of white-crowned sparrow, Zonotrichia leucophrys. In all three subspecies, transfer from short to long days triggered an increase in plasma PRL typical of an avian seasonal breeder. In Z. l. gambelii, which breeds at high latitudes, temperature does not affect the rate of photoinduced gonadal maturation or luteinizing hormone (LH) secretion. In this subspecies, we found that changes in plasma PRL concentrations were similar in all temperature treatments. In Z. l. pugetensis, which breeds in the Pacific Northwest, high temperatures accelerate gonadal development in females but not males and does not affect LH secretion. In this subspecies, we found that like gonadal growth, photoinduced changes in PRL secretion in Z. l. pugetensis vary with ambient temperature in females but not males. In Z. l. oriantha, which breeds in alpine regions of the West, both males and females respond to temperature cues by modulating gonadal growth but not LH secretion. We found in Z. l. oriantha that ambient temperature affects PRL secretion in both sexes. These results suggest that PRL may be involved in the transduction of ambient temperature cues used to time reproductive development and the termination of seasonal breeding. Alternatively, temperature-mediated differences in plasma PRL may be a result rather than a cause of differences in gonadal development, since sex steroids affect PRL secretion in some species.

Neuroendocrine suppression of female courtship in a wild passerine: corticotropin-releasing factor and endogenous opioids.

During emergencies in their natural environments, vertebrates initiate coping mechanisms that redirect behavior away from nonessential activities and towards survival. Reproductive behaviors are suppressed. Evidence from field studies on passerine birds shows that this inhibition may not depend on the suppression of gonadal sex steroids, since during the breeding season they remain elevated despite activation of the stress response. We hypothesize that an alternate, central mechanism mediates the inhibition of reproductive behavior during stress in passerines. In this study, we tested the intracerebroventricular effects of endogenous opioids and corticotropin-releasing factor (CRF), neuropeptides implicated in the stress response, on courtship behavior in wild-caught female white-crowned sparrows. Beta-endorphin (beta-EN) significantly inhibited copulation solicitation, an estrogen-dependent courtship display, 30 min after treatment. Naloxone, an opioid antagonist, enhanced the behavior. CRF caused a suppression of solicitation that was reversible by pretreatment with naloxone, suggesting an intermediary role for endogenous opioids in CRF-induced suppression of courtship. The effects of beta-EN and CRF on solicitation appear to be independent of any general effects on locomotor activity. These results support our hypothesis that stress neuropeptides orchestrate coping behaviors in wild birds.

Central opioid control of feeding behavior in the white-crowned sparrow, Zonotrichia leucophrys gambelii.

Many behavioral responses to stress do not appear to be mediated by glucocorticoids, suggesting another mechanism. We tested the effects of intracerebroventricular administration of beta-endorphin, a neuropeptide implicated in the stress response, on feeding behavior in captive, wild-caught white-crowned sparrows (Zonotrichia leucophrys gambelii). The amount of time spent feeding and the number of feeding bouts were higher after infusion with beta-endorphin than after saline infusion. Beta-endorphin decreased the latency to feed compared with saline. Naloxone, an opioid antagonist, suppressed feeding behavior and increased latency to feed. These results support our hypothesis that neuropeptides associated with stress may initiate adaptive responses to natural stressors in wild species.

Central administration of chicken gonadotropin-releasing hormone-II enhances courtship behavior in a female sparrow.

Like most vertebrates, birds have two forms of gonadotropin-releasing hormone (GnRH). Chicken GnRH-I (cGnRH-I) is released at the median eminence to elicit gonadotropin release; chicken GnRH-II (cGnRH-II) is thought to be non-hypophysiotropic and its function is unclear. Both forms are hypothesized to act as neurotransmitters in the control of reproductive behavior. In the present study, we implanted chronic cannulae aimed at the third ventricle in female white-crowned sparrows (Zonotrichia leucophrys gambelii) to test the effects of both forms of GnRH on copulation solicitation, a female courtship behavior. This behavior can be elicited in captive, estrogen-primed females by playing a recording of male song. We quantified the behavioral response to recorded song 30, 60, and 90 min after intracerebroventricular infusion of cGnRH-I, -II, or saline. cGnRH-II, but not cGnRH-I, increased solicitation behavior compared with saline 30 min after infusion. Under control conditions, responses to the playback diminish from the 30-min to the 90-min time point. Responses after cGnRH-II infusion followed a similar pattern, whereas after cGnRH-I, there was no significant response decrement. cGnRH-I appears to maintain the level of solicitation seen at 30 min after infusion. Our results suggest a behavioral role for cGnRH-II that may be independent of cGnRH-I.

Intraventricular infusion of arginine vasotocin induces singing in a female songbird.

Arginine vasotocin (AVT) has been implicated in the activation of courtship and aggressive behaviors in many vertebrate taxa. Here, we tested its effect on singing and other vocal behavior in a songbird. Female white-crowned sparrows (Z. l. gambelii) were implanted with chronic cannulae aimed at the third ventricle. Infusions of AVT dramatically increased the number of songs and other vocalizations during a 40 min period following infusion. Half of the subjects sang full song following AVT treatment. No bird sang after treatment with saline; any type of vocalization after saline treatment was rare. Female white-crowned sparrows are known to sing in both spring and winter in the wild; this behavior is thought to be aggressive, functioning in dominance interactions and territoriality. Central infusion of AVT induced singing and other vocalizations in estrogen-primed, photostimulated subjects as well as in non-reproductive subjects housed on short photoperiods. Thus, the effects of AVT on vocal behavior may not require breeding levels of gonadal steroids and are probably not seasonal. We hypothesize that both in the breeding and non-breeding seasons, AVT increases motivation to sing.