Effects of intense storm events on dolphin occurrence and foraging behavior.

As storms become increasingly intense and frequent due to climate change, we must better understand how they alter environmental conditions and impact species. However, storms are ephemeral and provide logistical challenges that prevent visual surveys commonly used to understand marine mammal ecology. Thus, relatively little is known about top predators' responses to such environmental disturbances. In this study, we utilized passive acoustic monitoring to characterize the response of bottlenose dolphins to intense storms offshore Maryland, USA between 2015 and 2017. During and following four autumnal storms, dolphins were detected less frequently and for shorter periods of time. However, dolphins spent a significantly higher percentage of their encounters feeding after the storm than they did before or during. This change in foraging may have resulted from altered distributions and behavior of their prey species, which are prone to responding to environmental changes, such as varied sea surface temperatures caused by storms. It is increasingly vital to determine how these intense storms alter oceanography, prey movements, and the behavior of top predators.

Empirical evidence that large marine predator foraging behavior is consistent with area-restricted search theory.

When prey is patchily distributed, predators are expected to spend more time searching for food in proximity of recent prey captures before searching in other areas. This behavior, known as area-restricted search, results in predators remaining localized in areas where prey had been detected previously because of the higher probability of encountering additional prey. However, few studies have tested these predictions on marine species because of the difficulties of observing feeding behavior. In this study, we utilized passive acoustic detections of echolocating dolphins to identify foraging behavior. C-PODs (click train detectors) were deployed for two years with an acoustic recorder attached to the same mooring during the second year. The time series of feeding buzzes, indicative of foraging behavior, revealed that both bottlenose (Tursiops truncatus) and common dolphins (Delphinus delphis) were more likely to stay in the area longer when foraging activity was high at the beginning of the encounter. The probability of foraging was also higher following previous foraging activity. This suggests that dolphins were feeding on spatially patchy prey and previous foraging experience influenced their movement behavior. This is consistent with the predictions of area-restricted search behavior, a nonrandom foraging strategy.

Heavy rainfall triggers increased nocturnal flight in desert populations of the Pacific black duck (Anas superciliosa).

Understanding of avian nocturnal flight comes mainly from northern hemisphere species in seasonal temperate ecosystems where nocturnal flight is often precisely timed and entrained by annual photoperiod. Here we investigate patterns of nocturnal flight in waterbirds of Australian desert ecosystems that fly considerable distances to find temporary water bodies formed from rainfall which is highly unpredictable seasonally and spatially, and when there is sufficient water, they then breed. How they perform these feats of navigation and physiology remain poorly known. Using GPS tracking of 38 satellite tagged Pacific black ducks (Anas superciliosa) in two contrasting ecosystems, before and after heavy rainfall we revealed a key role for facultative nocturnal flight in the movement ecology of this species. After large rainfall events, birds rapidly increased nocturnal flight activity in the arid aseasonal ecosystem, but not in the mesic seasonal one. Nocturnal flights occurred throughout the night in both ecosystems. Long range flights (>50 km in 2 hours) occurred almost exclusively at night; at night the distance flown was higher than during the day, birds visited more locations, and the locations were more widely dispersed. Our work reveals that heavy rainfall triggers increased nocturnal flight activity in desert populations of waterbirds.

What do we actually see in intracellular SERS? Investigating nanosensor-induced variation.

Plasmonic nanoparticles (NPs), predominantly gold (AuNPs), are easily internalised into cells and commonly employed as nanosensors for reporter-based and reporter-free intracellular SERS applications. While AuNPs are generally considered non-toxic to cells, many biological and toxicity studies report that exposure to NPs induces cell stress through the generation of reactive oxygen species (ROS) and the upregulated transcription of pro-inflammatory genes, which can result in severe genotoxicity and apoptosis. Despite this, the extent to which normal cellular metabolism is affected by AuNP internalisation remains a relative unknown along with the contribution of the uptake itself to the SERS spectra obtained from within so called 'healthy' cells, as indicated by traditional viability tests. This work aims to interrogate the perturbation created by treatment with AuNPs under different conditions and the corresponding effect on the SERS spectra obtained. We characterise the changes induced by varying AuNP concentrations and medium serum compositions using biochemical assays and correlate them to the corresponding intracellular reporter-free SERS spectra. The different serum conditions lead to different extents of nanoparticle internalisation. We observe that changes in SERS spectra are correlated to an increasing amount of internalisation, confirmed qualitatively and quantitatively by confocal imaging and ICP-MS analysis, respectively. We analyse spectra and characterise changes that can be attributed to nanoparticle induced changes. Thus, our study highlights a need for understanding condition-dependent NP-cell interactions and standardisation of nanoparticle treatments in order to establish the validity of intracellular SERS experiments for use in all arising applications.

Dehydroepiandrosterone Heightens Aggression and Increases Androgen Receptor and Aromatase mRNA Expression in the Brain of a Male Songbird.

Dehydroepiandrosterone (DHEA) is a testosterone/oestrogen precursor and known modulator of vertebrate aggression. Male song sparrows (Melospiza melodia morphna) show high aggression during breeding and nonbreeding life-history stages when circulating DHEA levels are high, and low aggression during molt when DHEA levels are low. We previously showed that androgen receptor and aromatase mRNA expression are higher during breeding and/or nonbreeding in brain regions associated with reproductive and aggressive behaviour, although the potential role of DHEA in mediating these seasonal changes remained unclear. In the present study, nonbreeding male song sparrows were captured and held in the laboratory under short days (8 : 16 h light/dark cycle) and implanted with s.c. DHEA-filled or empty (control) implants for 14 days. DHEA implants increased aggression in a laboratory-based simulated territorial intrusion. Brains of DHEA-implanted birds showed higher aromatase mRNA expression in the preoptic area (POA) and higher androgen receptor mRNA expression in the periventricular nucleus of the medial striatum (pvMSt) and ventromedial nucleus of the hypothalamus. The DHEA-induced increases in aromatase expression in the POA and androgen receptor expression in the pvMSt are consistent with previously reported seasonal increases in these markers associated with naturally elevated DHEA levels. This suggests that DHEA facilitates seasonal increases in aggression in nonbreeding male song sparrows by up-regulating steroid signalling/synthesis machinery in a brain region-specific fashion.

Patterns of hypothalamic GnIH change over the reproductive period in starlings and rats.

Gonadotropin inhibitory hormone (GnIH) exerts powerful inhibitory effects on various levels of the vertebrate hypothalamic-pituitary-gonadal (reproductive) axis, yet little is known of how it might change naturally over the course of reproduction. We characterized patterns of hypothalamic GnIH cell abundance over the reproductive period in two popular models used for the study of reproductive endocrinology: European starlings (Sturnus vulgaris) and Sprague-Dawley rats (Rattus norvegicus). We also examined the effects on an unpredictable change in the environment on GnIH cell abundance during the reproductive period, specifically during the period of parental care, by simulating a nest predation event and removing eggs/pups. In both species, we report changes in GnIH cell abundance are occurring at similar reproductive time points but are not always directionally parallel; this may be due to a difference in life histories and physiology mediating parental care. We discovered that cells immunoreactive for the GnIH peptide in male and female starlings are most highly abundant on the first day of incubation and the first day after the first chick hatches. Conversely in rats, GnIH cell abundance decreases in dams on the first day after pups are born. In both male and female starlings and female rats, GnIH cell abundance increases in response to egg/pup loss, indicating that GnIH responds to an unpredictable change in the environment in a potentially conserved fashion. These changes in GnIH cell abundance during the reproductive period inspire further investigation of its adaptive role in reproductive physiological events and behaviors, especially parental care.

A mechanistic approach to understanding range shifts in a changing world: What makes a pioneer?

A species' range can be thought of as a manifestation of the ecological niche in space. Within a niche, evolution has resulted in traits that maximize fitness. Across millennia, natural oscillations in temperature have caused shifts in the geographic location of appropriate habitat and with corresponding changes in species' ranges. Contemporary climate change and human disturbance may lead to rapid range expansion or contractions with largely unknown consequences. Birds provide an excellent case study of this phenomenon with some taxa expanding range and others contracting even to the point of extinction. What leads some populations to expand while others contract? Are there physiological and behavioral attributes of "pioneers" at the forefront of a range shift/expansion? The concept of allostasis provides a framework with which to begin to evaluate when a species will be able to successfully expand into new habitat. This tool allows the integration of normal energetic demands (e.g. wear and tear of daily and seasonal routines) with novel challenges posed by unfamiliar and human altered environments. Allostasis is particularly attractive because it allows assessment of how individual phenotypes may respond differentially to changing environments. Here, we use allostasis to evaluate what characteristics of individuals and their environment permit successful range expansion. Understanding variation in the regulatory mechanisms that influence response to a novel environment will be fundamental for understanding the phenotypes of pioneers.

Corticosterone, prolactin and egg neglect behavior in relation to mercury and legacy POPs in a long-lived Antarctic bird.

Seabirds often have high loads of contaminants. These contaminants have endocrine disrupting properties but their relationships with some endocrine mechanisms are still poorly investigated in free-living organisms. This is the case for the stress response which shifts energy investment away from reproduction and redirects it towards survival. In birds, this stress response is achieved through a release of corticosterone and is also accompanied by a decrease in circulating prolactin, an anterior pituitary hormone widely involved in regulating parental cares. We measured blood concentrations of some legacy persistent organic pollutants (POPs) and mercury (Hg) and examined their relationships with the corticosterone and prolactin responses of known-age (9-46 years old) incubating snow petrels (Pagodroma nivea) to a standardized capture/handling stress protocol. In this Antarctic seabird, we also investigated whether high contaminant burden correlates with a higher occurrence of egg neglect, a frequently observed behavior in snow petrels. POPs and Hg were unrelated to age. Stress-induced corticosterone concentrations were positively related to POPs in both sexes, and stress-induced prolactin concentrations were negatively related to Hg in males. Egg-neglect behavior was not related to POPs burden, but males with higher Hg concentrations were more likely to neglect their egg. This suggests that in birds, relationships between age and contaminants are complex and that even low to moderate concentrations of POPs and Hg are significantly related to hormonal secretion. In this Antarctic species, exposure to legacy POPs and Hg could make individuals more susceptible to environmental stressors such as ongoing disturbances in Polar Regions.

Decreases in mineralocorticoid but not glucocorticoid receptor mRNA expression during the short Arctic breeding season in free-living Gambel's white-crowned sparrow (Zonotrichia leucophrys gambelii).

The acute stress response in vertebrates is a highly adaptive suite of physiological and behavioural mechanisms that promote survival in the face of deleterious stimuli from the environment. Facultative changes of physiology and behaviour are mediated through changes in circulating levels of glucocorticoids (corticosterone, cortisol) and their subsequent binding to the high-affinity mineralocorticoid receptor (MR) or the low-affinity glucocorticoid receptor (GR). Free-living male wild Gambel's white-crowned sparrows (Zonotrichia leucophrys gambelii) display annual fluctuations in the stress response with marked attenuation during the transition from the pre-parental to the parental stage. We investigated whether this rapid reduction in the stress response is mediated through changes in MR and GR mRNA expression in the brain using in situ hybridisation. MR mRNA expression was found to be significantly lower in the hippocampus as the male birds became parental. No changes were observed in GR mRNA expression in the paraventricular nucleus (PVN) or preoptic area (POA) at this time. No significant correlations were found between initial capture levels of corticosterone and GR or MR mRNA expression. No differences were found in basal levels of corticosterone between pre-parental and parental in birds collected for in situ hybridisation. Stress response data revealed no difference at baseline but reductions in peak levels of corticosterone as birds became parental. These data suggest that changes in MR expression may be important for the regulation of the stress response or behavioural stress sensitivity with respect to promoting parental care and investment.

Polychlorinated biphenyl exposure and corticosterone levels in seven polar seabird species.

The role of polychlorinated biphenyls (PCBs) on exposure-related endocrine effects has been poorly investigated in wild birds. This is the case for stress hormones including corticosterone (CORT). Some studies have suggested that environmental exposure to PCBs and altered CORT secretion might be associated. Here we investigated the relationships between blood PCB concentrations and circulating CORT levels in seven free-ranging polar seabird species occupying different trophic positions, and hence covering a wide range of PCB exposure. Blood ∑7PCB concentrations (range: 61-115,632 ng/g lw) were positively associated to baseline or stress-induced CORT levels in three species and negatively associated to stress-induced CORT levels in one species. Global analysis suggests that in males, baseline CORT levels generally increase with increasing blood ∑7PCB concentrations, whereas stress-induced CORT levels decrease when reaching high blood ∑7PCB concentrations. This study suggests that the nature of the PCB-CORT relationships may depend on the level of PCB exposure.

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.

Social and breeding status are associated with the expression of GnIH.

Discoveries of how social behavior can influence the plasticity of gonadotropin-releasing hormone (GnRH) have revolutionized the field of behavioral neuroendocrinology by providing new insights into the neural mechanisms controlling behavior. In 2000, the neuropeptide gonadotropin inhibitory hormone (GnIH) was discovered and is changing the way we view how the brain mediates reproduction and associated behaviors. GnIH acts as a reproductive 'pause button', momentarily inhibiting the activity of the reproductive system. However, how GnIH fluctuates naturally in response to social environment is unknown. We examine how the outcome of competition for limited resources needed for reproduction is associated with GnIH. We experimentally manipulated nesting opportunities for pairs of European starlings (Sturnus vulgaris) and examined brain GnIH mRNA and peptide content, as well as GnRH content and plasma testosterone and corticosterone. By limiting the number of nest boxes per enclosure and thus the number of social pairing and nesting opportunities, we observed that birds which outcompeted others for nest boxes ('winners') had significantly fewer numbers of GnIH peptide-producing cells than those without nest boxes ('losers') and this relationship changed with breeding stage. GnRH content, testosterone and corticosterone did not vary with nest box ownership. Thus, while birds appeared reproductively capable across treatments, our data indicate that GnIH may serve as a modulator of reproductive behaviors in response to social environment. Additionally, we provide some evidence of the adaptive value of this mechanism.

'Islands' and 'doctor's tool': the ethical significance of isolation and subordination in UK community pharmacy.

Empirical ethics research is increasingly valued in offering insights into how ethical problems and decision-making occur in healthcare. In this article, the findings of a qualitative study of the ethical problems and decision-making of UK community pharmacists are presented, and it is argued that the identified themes of pharmacists' relative isolation from others and their subordination to doctors are ethically significant. Semi-structured interviews were conducted with 23 community pharmacists in England, UK. Analysis of interviews revealed that isolation involved separation of pharmacists from their peers, other healthcare professionals, patients and customers. Such isolation is argued to be inimical to ethical practice - impeding ethical discourse as understood by Habermas, resulting in a form of anomie that inhibits the transmission of professional values, leading to a lack of proximity between pharmacist and patient or customer that may impede ethical relationships and resulting, psychologically, in less ethical concern for those who are less close. Pharmacists' subordination to doctors not only precipitated some ethical problems but also allowed some pharmacists to shift ethical responsibility to a prescribing doctor, as in the case of emergency hormonal contraception. The emergence of atrocity stories further supports a culture of subordination that may cause ethical problems. The study has implications for community pharmacy practice in terms of supervision issues, developments such as prescribing responsibilities and how ethical values can be taught and communicated. The potential for isolation and subordination in other healthcare professions, and resultant ethical problems, may also need to be addressed and researched.

Gonadotrophin-inhibitory hormone: a multifunctional neuropeptide.

Gonadotrophin-inhibitory hormone (GnIH) was discovered 8 years ago in birds. Its identification raised the possibility that gonadotrophin-releasing hormone (GnRH) is not the sole hypothalamic neuropeptide that directly influences pituitary gonadotrophin release. Initial studies on GnIH focused on the avian anterior pituitary as comprising the only physiological target of GnIH. There are now several lines of evidence indicating that GnIH directly inhibits pituitary gonadotrophin synthesis and release in birds and mammals. Histological studies on projections from hypothalamic GnIH neurones subsequently implied direct actions of GnIH within the brain and in the periphery. In addition to actions on the pars distalis via the median eminence, GnIH axons and terminals are present in multiple brain areas in birds, and the GnIH receptor is expressed on GnRH-I and -II neurones. Furthermore, we have demonstrated the presence of GnIH and its receptor in avian and mammalian gonads. Thus, GnIH can act directly at multiple levels: within the brain, on the pituitary and in the gonads. In sum, our data indicate that GnIH and its related peptides are important modulators of reproductive function at the level of the GnRH neurone, the gonadotroph and the gonads. Here, we provide an overview of the known levels of GnIH action in birds and mammals. In addition, environmental and physiological factors that are involved in GnIH regulation are reviewed.

A new key neurohormone controlling reproduction, gonadotrophin-inhibitory hormone in birds: discovery, progress and prospects.

In vertebrates, the neuropeptide control of gonadotrophin secretion is primarily through the stimulatory action of the hypothalamic decapeptide, gonadotrophin-releasing hormone (GnRH). Gonadal sex steroids and inhibin inhibit gonadotrophin secretion via feedback from the gonads, but a hypothalamic neuropeptide inhibiting gonadotrophin secretion was, until recently, unknown in vertebrates. In 2000, we discovered a novel hypothalamic dodecapeptide that directly inhibits gonadotrophin release in quail and termed it gonadotrophin-inhibitory hormone (GnIH). GnIH acts on the pituitary and GnRH neurones in the hypothalamus via a novel G-protein-coupled receptor for GnIH to inhibit gonadal development and maintenance by decreasing gonadotrophin release and synthesis. The pineal hormone melatonin is a key factor controlling GnIH neural function. GnIH occurs in the hypothalamus of several avian species and is considered to be a new key neurohormone inhibiting avian reproduction. Thus, the discovery of GnIH provides novel directions to investigate neuropeptide regulation of reproduction. This review summarises the discovery, progress and prospects of GnIH, a new key neurohormone controlling reproduction.

Latitudinal distribution, migration, and testosterone levels in birds.

Tropical bird species usually have lower testosterone (T) levels during breeding than temperate species. However, the potential mechanisms behind the positive interspecific correlation between T and latitude remain unexplored. In a comparative study of more than 100 bird species, we examined whether social constraints during male-male competition arising from migration and breeding synchrony are responsible for the latitude effects. Species that breed at higher latitudes are more likely to migrate and experience more intense intrasexual competition upon spring arrival than nonmigrant species from lower latitudes. Additionally, species from higher latitudes cope with shorter breeding seasons and thus with more synchronous breeding, which selects for high T titers via increased male-male conflicts. Accordingly, peak T levels were associated with migration and the duration of the egg laying period that reflects breeding synchrony. Because migration and breeding synchrony were related to latitudinal distribution, they appear to be important components of the latitude effects on T. A multivariate model controlling for covariation of predictor variables revealed that latitude remained the strongest predictor of peak T. Therefore, selection due to migration and breeding synchrony may partially cause the latitude effect, but other geographically varying factors may also play a role in mediating peak T levels at different latitudes.

Ethical decision-making, passivity and pharmacy.

BACKGROUND: Increasing interest in empirical ethics has enhanced understanding of healthcare professionals' ethical problems and attendant decision-making. A four-stage decision-making model involving ethical attention, reasoning, intention and action offers further insights into how more than reasoning alone may contribute to decision-making. AIMS: To explore how the four-stage model can increase understanding of decision-making in healthcare and describe the decision-making of an under-researched professional group. METHODS: 23 purposively sampled UK community pharmacists were asked, in semi-structured interviews, to describe ethical problems in their work and how they were resolved. Framework analysis of transcribed interviews utilised the four decision-making stages, together with constant comparative methods and deviant-case analysis. RESULTS: Pharmacists were often inattentive and constructed problems in legal terms. Ethical reasoning was limited, but examples of appeals to consequences, the golden rule, religious faith and common-sense experience emerged. Ethical intention was compromised by frequent concern about legal prosecution. Ethical inaction was common, typified by pharmacists' failure to report healthcare professionals' bad practices, and ethical passivity emerged to describe these negative examples of the four decision-making stages. Pharmacists occasionally described more ethically active decision-making, but this often involved ethical uncertainty. DISCUSSION: The four decision-making stages are a useful tool in considering how healthcare professionals try to resolve ethical problems in practice. They reveal processes often ignored in normative theories, and their recognition and the emergence of ethical passivity indicates the complexity of decision-making in practice. Ethical passivity may be deleterious to patients' welfare, and concerns emerge about improving pharmacists' ethical training and promoting ethical awareness and responsibility.

A new prescription for empirical ethics research in pharmacy: a critical review of the literature.

Empirical ethics research is increasingly valued in bioethics and healthcare more generally, but there remain as yet under-researched areas such as pharmacy, despite the increasingly visible attempts by the profession to embrace additional roles beyond the supply of medicines. A descriptive and critical review of the extant empirical pharmacy ethics literature is provided here. A chronological change from quantitative to qualitative approaches is highlighted in this review, as well as differing theoretical approaches such as cognitive moral development and the four principles of biomedical ethics. Research with pharmacy student cohorts is common, as is representation from American pharmacists. Many examples of ethical problems are identified, as well as commercial and legal influences on ethical understanding and decision making. In this paper, it is argued that as pharmacy seeks to develop additional roles with concomitant ethical responsibilities, a new prescription is needed for empirical ethics research in pharmacy-one that embraces an agenda of systematic research using a plurality of methodological and theoretical approaches to better explore this under-researched discipline.

Does extra corticosterone elicit increased begging and submissiveness in subordinate booby (Sula nebouxii) chicks?

We tested whether in two-chick broods of the blue-footed booby (Sula nebouxii) elevated circulating corticosterone in the socially subordinate broodmate facilitates submissive behavior and/or enhances food solicitation. Implanting corticosterone in 17 subordinate chicks (experimental broods) produced changes in the behavior of chicks and parents over the first two days, relative to 17 matched families (control broods) where subordinate chicks were implanted with empty capsules. Experimental broods showed increased activity/wakefulness of the dominant broodmate and, consequently, increased simultaneous activity of both broodmates, but there was scant evidence that subordinates submitted more readily when attacked. Implanted subordinates increased their rate of spontaneous submission over the total observation time, but this increase was mostly explained by the additional time when both broodmates were simultaneously active. There was little sign that extra corticosterone induced more begging, except possibly by eliciting increased activity. Experimental broods increased their rate of feeding, and most if not all of the increase was due to the increased activity and increased feeding rate of dominant broodmates. On the third and fourth days after implantation all effects of implanted corticosterone disappeared, except for the elevated activity and feeding rates of dominant chicks. At the end of four days, subordinates implanted with corticosterone showed no increase in circulating corticosterone and experimental broods showed no gain in mass or body size, relative to controls. Extra corticosterone, above the high level that normally circulates in subordinate chicks, apparently does not enhance submission to aggression or food solicitation, but provokes a cascade of changes in the behavior of broodmates and parents.

Hypothalamic pro-GnRH-GAP, GnRH-I and GnRH-II during the onset of photorefractoriness in the white-crowned sparrow (Zonotrichia leucophrys gambelii).

Gambel's white-crowned sparrow is a long distance migrant that undergoes spontaneous gonadal regression as a result of long day exposure. This termination of breeding is caused by the development of photorefractoriness and the birds become insensitive to long days, including continuous light. The present study investigated its possible mechanisms by examining the activity of the gonadotrophin-releasing hormone (GnRH) system under different photoperiodic regimes. We investigated the localisation and distribution of GnRH-I, its precursor pro-GnRH-GAP and GnRH-II in Gambel's white-crowned sparrow brain using immunocytochemistry with specific antibodies during photostimulation and the development of photorefractoriness. The study revealed that photoperiodic treatment, including the onset of photorefractoriness, had no significant effect on the size or number of GnRH-I, pro-GnRH-GAP or GnRH II immunoreactive cells, or the density of the GnRH-I, pro-GnRH-GAP immunoreactive fibres at the median eminence. GnRH-II was not found in the median eminence, suggesting that it does not regulate pituitary gonadotrophin secretion. GnRH-I measurement in hypothalamic extracts by radioimmunoassay did not reveal any significant difference between birds that were photostimulated or in the early stages of photorefractoriness. Furthermore, the action of the excitatory amino acid glutamate agonist N-methyl-D-aspartate on GnRH neurones in photorefractory birds was demonstrated by the significant blockade of luteinising hormone release with a specific GnRH antagonist. Taken together, these results suggest that, in Gambel's white-crowned sparrow, a decrease in GnRH-I secretion is the initial step for the onset of photorefractoriness and not a decrease in GnRH-I biosynthesis.