A Quantification of the Injury-Induced Changes in Central Aromatase, Oestrogenic Milieu and Steroid Receptor Expression in the Zebra Finch.

In songbirds and mammals, brain injury results in the up-regulation of aromatase (oestrogen synthase) expression in astroglia. The resulting presumed synthesis of neural oestradiol (E2 ) has neuroprotective effects including a decrease in neurodegeneration, neuroinflammation and apoptosis. The development of therapeutic tools that exploit oestrogenic neuroprotection in the treatment of neurotrauma requires a precise quantification of the endogenous changes in neural aromatase and E2 following brain injury. Surprisingly, the expected increase in neural oestrogens following brain injury has not been demonstrated. Furthermore, we are just beginning to unravel the mechanisms behind the protective effects of centrally synthesised E2 . In the present study, levels of aromatase immunoprotein, neural E2 and steroid receptor mRNA were quantified in adult male and female zebra finches 48 h following a unilateral penetrating brain injury. Both aromatase and E2 were up-regulated in the injured hemisphere of the brain compared to the uninjured hemisphere, demonstrating for the first time a robust increase in neural E2 levels following injury. We did not detect an effect of injury on mRNA expression of the oestrogen receptors (ER)-α, ER-ß or GPER-1, but observed a significant decrease in androgen receptor transcription in the injured lobe relative to the contralateral uninjured hemisphere. We conclude that mechanical damage causes a dramatic increase in local aromatisation, and the resultant high levels of central E2 are available to modulate steroid sensitive targets. Studies using alternate methods of receptor detection and/or time points may be necessary to understand the complete suite of mechanisms underlying the neuroprotective effects of induced oestrogen synthesis in this animal model.

Induced synthesis of oestrogens by glia in the songbird brain.

Studies on birds have long provided landmarks and touchstones in the fields of neuroendocrinology, immunology and neuroplasticity. The passerine brain is an excellent model for studying the actions of hormones, including steroids, on a diversity of behavioural endpoints. Oestrogens, for example, have profound effects on avian neuroanatomy and neurophysiology throughout life and, importantly, are synthesised at high levels within neurones of the songbird brain. More recently, aromatisation in another set of neural cells has been identified. Specifically, aromatase expression is induced in astrocytes and radial glia following disruption of the neuropil by multiple forms of perturbation. The avian brain, therefore, can be provided with high levels of oestrogens constitutively or via induction, by aromatisation in neurones and glia, respectively. In this review, we begin with the initial discovery of aromatisation by non-neuronal cells and discuss the mechanisms underlying the induction of aromatase expression in glial cells. We then focus on the emerging interactions between the neuroendocrine and neuroimmune systems with respect to brain injury. Next, we briefly review the extensive literature on the influence of glial aromatisation on neuroplasticity, and end with some recent data on sex differences in the induction of glial aromatase in the zebra finch. Throughout this review, we consider the unanswered questions and future studies that may emerge from these findings.

Rapid effects of aggressive interactions on aromatase activity and oestradiol in discrete brain regions of wild male white-crowned sparrows.

Testosterone is critical for the activation of aggressive behaviours. In many vertebrate species, circulating testosterone levels rapidly increase after aggressive encounters during the early or mid-breeding season. During the late breeding season, circulating testosterone concentrations did not change in wild male white-crowned sparrows after an aggressive encounter and, in these animals, changes in local neural metabolism of testosterone might be more important than changes in systemic testosterone levels. Local neural aromatisation of testosterone into 17ß-oestradiol (E(2)) often mediates the actions of testosterone, and we hypothesised that, in the late breeding season, brain aromatase is rapidly modulated after aggressive interactions, leading to changes in local concentrations of E(2). In the present study, wild male white-crowned sparrows in the late breeding season were exposed to simulated territorial intrusion (STI) (song playback and live decoy) or control (CON) for 30 min. STI significantly increased aggressive behaviours. Using the Palkovits punch technique, 13 brain regions were collected. There was high aromatase activity in several nuclei, although enzymatic activity in the CON and STI groups did not differ in any region. E(2) concentrations were much higher in the brain than the plasma. STI did not affect circulating levels of E(2) but rapidly reduced E(2) concentrations in the hippocampus, ventromedial nucleus of the hypothalamus and bed nucleus of the stria terminalis. Unexpectedly, there were no correlations between aromatase activity and E(2) concentrations in the brain, nor were aromatase activity or brain E(2) correlated with aggressive behaviour or plasma hormone levels. This is one of the first studies to measure E(2) in microdissected brain regions, and the first study to do so in free-ranging animals. These data demonstrate that social interactions have rapid effects on local E(2) concentrations in specific brain regions.

Glial aromatization decreases neural injury in the zebra finch (Taeniopygia guttata): influence on apoptosis.

Emerging evidence suggests a neuroprotective role for oestrogens following damage to the vertebrate brain. Aromatase (oestrogen synthase) is rapidly transcribed and translated in glial cells around areas of neural damage in several vertebrates. However, the potential neuroprotection afforded by locally up-regulated glial aromatase immediately surrounding the injury remains to be tested. Towards this end, individual birds sustained penetrating mechanical injuries via a needle that contained either vehicle or the aromatase inhibitor fadrozole into contralateral hemispheres. Seventy-two hours later, the size of neural injury (as assessed by the extent of necrotic tissue) and the number of apoptotic cells around the injuries were evaluated. The size of injury in the hemisphere injected with fadrozole was significantly larger than the injury caused by vehicle injection. Furthermore, a greater number of apoptotic nuclei were found around the fadrozole-associated lesion relative to vehicle. Finally, constitutively expressed, neuronal aromatase close to the injury site did not differ between hemispheres. We conclude that local inhibition of glial aromatase immediately around the site of injury plays a neuroprotective role in the songbird brain and this protection involves apoptotic pathways. Local up-regulation of glial aromatase may play a pivotal role in the limitation of secondary damage and/or the acceleration of restorative processes following injury to the vertebrate brain.

Rapid upregulation of aromatase mRNA and protein following neural injury in the zebra finch (Taeniopygia guttata).

The expression of aromatase (oestrogen synthase) within the vertebrate central nervous system (CNS) is key in the provision of local oestrogens to neural circuits. Aromatase expression appears to be exclusively neuronal under normal conditions. However, some in vitro studies suggest the presence of astrocytic aromatase in songbirds and mammals. Recently, aromatase in reactive astrocytes has been demonstrated in response to neural injury in the mammalian CNS. Since the glial aromatase expression first documented in cultures of the songbird telencephalon may reflect processes similar to those in response to mammalian neural injury, we investigated whether injury alters the pattern of aromatase-expression in the zebra finch, a species with very high levels of forebrain aromatase expression. Adult males received a penetrating neural injury to the right hemisphere and were killed either 24 or 72 h later. Controls were anaesthetized and otherwise unmanipulated. We determined the expression of aromatase mRNA and protein using in situ hybridization and immunocytochemistry, respectively. Both the transcription and translation of aromatase is dramatically upregulated around the lesion site in response to neural injury in the zebra finch forebrain. This effect is robust and rapid, occurring within 24 h of the injury itself. Cells that upregulate aromatase appear to be reactive astrocytes based upon morphology. The hemisphere contralateral to the injury and both hemispheres in control birds showed the normal, exclusively neuronal pattern of aromatase expression. The upregulation of aromatase in astrocytes may provide high levels of oestrogen available to modulate processes such as CNS repair. Injury-induced upregulation of astrocytic aromatase may be a general characteristic of the injured vertebrate brain.

Direct innervation of GnRH neurons by encephalic photoreceptors in birds.

In nonmammalian vertebrates, photic cues that regulate the timing of seasonal reproductive cyclicity are detected by nonretinal, nonpineal deep brain photoreceptors. It has long been assumed that the underlying mechanism involves the transmission of photic information from the photoreceptor to a circadian system, and thence to the reproductive axis. An alternative hypothesis is that there is direct communication between the brain photoreceptor and the reproductive axis. In the present study, light and confocal microscopy reveal that gonadotropin releasing hormone (GnRH) neurons and processes are scattered among photoreceptor cells (identified by their opsin-immunoreactivity) in the lateral septum (SL). In the median eminence (ME), opsin and GnRH immunoreactive fibers overlap extensively. Single and double label ultrastructural immunocytochemistry indicate that in the SL and preoptic area (POA), opsin positive terminals form axo-dendritic synapses onto GnRH dendrites. In the ME, opsin and GnRH terminals lie adjacent to each other, make contact with tanycytes, or terminate on the hypophyseal portal capillaries. These results reveal thatbrain photoreceptors communicate directly with GnRH-neurons; this represents a means by which photoperiodic information reaches the reproductive axis.

Telencephalic aromatase but not a song circuit in a sub-oscine passerine, the golden collared manakin (Manacus vitellinus).

In oscine passerines, the telencephalon expresses high levels of the estrogen synthetic enzyme aromatase. In contrast, forebrain aromatase is limited to low levels at discrete limbic loci in non-passerines. The function of forebrain aromatase in oscines is unknown, however, estrogen-sensitive elements of the telencephalic song circuit (an oscine characteristic) may be influenced by local aromatization. Very few studies have investigated the neuroendocrine characteristics of sub-oscine passerines. Species of this passerine sub-order are taxonomically similar to oscines, but do not appear to learn how to sing as oscines, and show no evidence of a song circuit. The neural expression of aromatase in these birds is unknown. We asked whether the golden-collared manakin, a sub-oscine, (a) showed evidence of a song circuit, and (b) expressed aromatase in the telencephalon at high levels like the zebra finch (oscine passerine) or at low levels like the quail (non-passerine). Nissl stains and immunocytochemistry for microtubule associated proteins showed no evidence of a song circuit in manakins of either sex, whereas both techniques delineate all song nuclei in the zebra finch. However, biochemical and immunocytochemical measures reveal that in the manakin, several telencephalic loci, including the hippocampus, caudomedial neostriatum, nucleus taeniae, and the lateral neostriatum express aromatase. Assays run in parallel show low to undetectable levels of aromatase in the telencephalon of the quail (nonpasserine) and abundant levels in the zebra finch (oscine passerine), suggesting a dissociation between the presence of a song circuit and forebrain aromatase expression in this sub-oscine. These data suggest that forebrain aromatase may have evolved in sub-oscine songbirds before the evolution of a song circuit and singing behavior in oscines. Alternatively, forebrain aromatase may serve functions distinct from singing behavior.

Acute and chronic effects of an aromatase inhibitor on territorial aggression in breeding and nonbreeding male song sparrows.

Many studies have demonstrated that male aggression is regulated by testosterone. The conversion of testosterone to estradiol by brain aromatase is also known to regulate male aggression in the breeding season. Male song sparrows (Melospiza melodia morphna) are territorial not only in the breeding season, but also in the nonbreeding season, when plasma testosterone and estradiol levels are basal. Castration has no effect on nonbreeding aggression. In contrast, chronic (10 day) aromatase inhibitor (fadrozole) treatment decreases nonbreeding aggression, indicating a role for estrogens. Here, we show that acute (1 day) fadrozole treatment decreases nonbreeding territoriality, suggesting relatively rapid estrogen effects. In spring, fadrozole decreases brain aromatase activity, but acute and chronic fadrozole treatments do not significantly decrease aggression, although trends for some behaviors approach significance. In gonadally intact birds, fadrozole may be less effective at reducing aggression in the spring. This might occur because fadrozole causes a large increase in plasma testosterone in intact breeding males. Alternatively, estradiol may be more important for territoriality in winter than spring. We hypothesize that sex steroids regulate male aggression in spring and winter, but the endocrine mechanisms vary seasonally.

Distribution and regulation of telencephalic aromatase expression in the zebra finch revealed with a specific antibody.

In songbirds, aromatase (estrogen synthase) activity and mRNA are readily detectable in the brain. This neural aromatization presumably provides estrogen to steroid-sensitive targets via autocrine, paracrine, and synaptic mechanisms. The location of immunoreactive protein, however, has been difficult to describe completely, particularly in distal dendrites, axons, and terminals of the forebrain. Here we describe the neuroanatomical distribution of aromatase in the zebra finch by using a novel antibody raised specifically against zebra finch aromatase. The distribution of aromatase-positive somata in the zebra finch brain is in excellent agreement with previous reports. Additionally, this antibody reveals elaborate, spinous dendritic arbors, fine-beaded axons, and punctate terminals of telencephalic neurons that may synthesize estrogen. Some of these axon-like fibers extend into the high vocal center (HVC) and the robust nucleus of the archistriatum (RA) in males and females, suggesting a role for presynaptic aromatization in cellular processes within these loci. Adult males have more aromatase-positive fibers in the caudomedial neostriatum (NCM) and the preoptic area (POA) compared to females, despite the lack of detectable sex differences in the number of immunoreactive somata at these loci. Thus, the compartmentalization of aromatase in dendrites and axons may serve a sexually dimorphic function in the songbird. Finally, in adult males, aromatase expression is down-regulated by circulating estradiol in the hippocampus, but not in the NCM or POA. The distribution of aromatase suggests a role for aromatization in the regulation of pre- and postsynaptic function in steroid sensitive areas of the songbird forebrain.

Changes in brain gonadotropin-releasing hormone- and vasoactive intestinal polypeptide-like immunoreactivity accompanying reestablishment of photosensitivity in male dark-eyed juncos (Junco hyemalis).

In seasonally breeding, photoperiodic birds, the development of photorefractoriness is associated with decreased brain expression of gonadotropin-releasing hormone-like immunoreactivity (GnRH-li ir) and increased expression of vasoactive intestinal polypeptide-like immunoreactivity (VIP-li ir). Dissipation of photorefractoriness and reestablishment of photosensitivity are associated with increased GnRH-li ir brain production, but concurrent changes in VIP-li ir expression have not been investigated. To address this question, we compared the expression of VIP-li ir in the infundibulum (INF) of adult male dark-eyed juncos (Junco hyemalis) that were made photorefractory (PR) by prolonged exposure to long days with that of birds that were not photostimulated (PS), but had regained photosensitivity by exposure to short days for 5 (short-term-PS, ST-PS) or 13 (long-term-PS, LT-PS) consecutive months. Photosensitive males had smaller INF VIP-li ir cell bodies than PR males, but the numbers of INF VIP-li ir cells were independent of photoperiodic condition. Changes in infundibular VIP-li ir were correlated with changes in preoptic area (POA) GnRH-li expression. Specifically, photosensitive males had more and larger POA GnRH-li ir cells and more GnRH-li ir fibers in this region than PR males. Further, LT-PS males had more GnRH-li ir POA fibers and larger testes than ST-PS juncos. Thus, induction of photorefractoriness is associated with increased VIP and decreased GnRH brain expression whereas dissipation of photorefractoriness concurs with decreased VIP and increased GnRH brain expression. These results suggest a physiological role for VIP in the control of changes in GnRH expression as a function of the photosensitive condition.

Rapid effects of corticosterone on cache recovery in mountain chickadees (Parus gambeli).

Environmental perturbations increase adrenal activity in several vertebrates. Increases in corticosterone may serve as a proximate trigger whereby organisms can rapidly adapt their behavior to survive environmental fluctuations. In food-caching songbirds, inclement weather may present the need to alter caching and/or retrieval behaviors to ensure food supplies. We hypothesized that corticosterone may increase the rate of caching and/or retrieval behaviors in the mountain chickadee, a food-storing songbird, and tested if these potential effects were mediated by alterations in appetite, activity, or memory for cache sites. Corticosterone or vehicle was administered to subjects 5 min prior to either caching or recovery in a naturalistic laboratory paradigm during which we recorded the number of caching events, sites visited, and seeds eaten (caching) or caches recovered, total sites visited, cache-related visits, and non-cache-related visits (recovery). Data were analyzed using nested ANOVA for treatment within sequential trial. There was no effect on any caching behaviors following treatment. However, birds treated with corticosterone during retrieval recovered more seeds and tended to visit more cache-related sites than did controls. Since groups did not differ in the number of seeds eaten or the total number of sites visited, it seems unlikely that corticosterone affected appetite or activity. Rapid surges in corticosterone may increase the efficacy of an underlying memory process for cache sites which is reflected in higher cache recovery in corticosterone-treated birds than in controls. Thus, rapid alterations in plasma corticosterone following environmental change may alter memory-reliant behaviors which promote survival in the food-caching mountain chickadee.

Androgen metabolism in the juvenile oscine forebrain: a cross-species analysis at neural sites implicated in memory function.

Juvenile songbirds are useful models for studying the neural bases of memory. Memory-reliant behaviors demonstrated at this stage include song learning (most songbirds) and food caching (food-storing songbirds). Sex steroids are implicated in the modulation of memory processes in several vertebrates. The songbird forebrain expresses aromatase, 5alpha-reductase and 5beta-reductase, enzymes which convert testosterone to estradiol, 5alpha-, and 5beta-dihydrotestosterone, respectively. To explore the role of local androgen metabolism on memory processes, we documented the activities of these enzymes in the anterior neostriatum (NAN), caudomedial neostriatum (NCM), and hippocampus (HP) of four species of juvenile songbird, two of which are food storers. Areas were dissected, homogenized, and provided with radiolabeled substrate; and formed estrogens, and 5alpha- and 5beta-reduced androgens were measured. In the NAN, 5beta-reductase was the predominant enzyme, suggesting that local inactivation of testosterone may preserve the sensitive period of song acquisition. In the NCM, estrogens were formed in abundance despite high 5beta-reductase, suggesting that locally high estrogen synthesis may play a role in processes of song perception. In the HP, both estrogens and 5alpha reduced androgens were formed, suggesting that HP function may be modulated by both estrogens and androgens. Finally, a derived measure of steroid-differential reveals that food-storing songbirds differ from nonstorers in the steroidal milleiu within the HP, but not in the NAN or NCM. Thus, distinct loci within the juvenile songbird forebrain are exposed to different patterns of androgen metabolites. This local conversion may play a role in the neuroendocrine modulation of memory in these birds.

The passerine hippocampus is a site of high aromatase: inter- and intraspecies comparisons.

The vertebrate hippocampus (HP) plays a critical role in the organization of memory. Estrogens alter synaptic morphology and function in the mammalian HP and may potentiate memory performance. Previous studies suggest that the songbird HP itself is a site of significant aromatase expression, intimating that local estrogen synthesis may provide a source of this steroid to estrogen-sensitive neural circuits. To explore the potential role of local estrogen synthesis on HP structure and function, we have characterized aromatase message and activity in the zebra finch HP. Toward this end we have compared (a) HP aromatase mRNA with that at other neural loci, (b) HP aromatase activity between adults of both sexes, and (c) HP and hypothalamic preoptic area (HPOA) aromatase activity among songbirds and nonsongbirds. Finally we asked whether aromatase activity was intrinsic to the HP by maintaining it in culture, isolated from the rest of the telencephalon. The HP of every songbird studied expresses aromatase, with comparable levels across sexes. Notably, aromatase activity was found at higher levels in the songbird HP than in the HPOA. In both nonsongbird species investigated, however, HP aromatase activity was undetectable under identical assay conditions. Additionally, the developing songbird HP continues to express aromatase when cultured in isolation from the rest of the telencephalon. The data suggest that HP aromatase is characteristic of passeriformes and, as in the HPOA, may represent a mechanism whereby estrogen is made available to neural circuits. Passerines may prove invaluable animal models for investigations of the estrogenic modulation of HP structure and function.

Estrogen synthesis and secretion in the brown-headed cowbird (Molothrus ater).

Estrogens exert profound effects on vertebrate physiology and behavior. In most vertebrates, including birds, estrogens derived from ovarian tissue circulate at high levels during discrete periods of reproductive activity, and estrogen levels in males are low. In some songbirds (Passeriformes) plasma estrogens are high in both males and females. In the zebra finch, aromatase (estrogen-synthetase) is expressed abundantly in several regions of the male and female telencephalon and contributes to peripheral estrogen titers. To determine if this pattern of neural aromatase and estrogen synthesis is found in other songbirds, we have examined the patterns of estrogen synthesis in various tissues of another songbird, the common North American cowbird (Molothrus ater). Radiolabeled aromatizable androgenic substrate was injected in vivo or provided in vitro to telencephalic and gonadal tissue from adult male and female cowbirds. Estrogenic products were assayed in blood from the carotid artery and jugular vein, and in the telencephalon, ovary, and testes. Additionally, the presence of aromatase mRNA was studied in the brain using in situ hybridization. Radiolabeled androgenic substrate, injected in vivo, was readily converted to estrogens with higher amounts in the jugular compared to carotid blood, suggesting that the brain contains relatively high levels of aromatase. Further, radiolabeled androgens, provided in vitro to telencephalic, ovarian, and testicular tissue, resulted in the formation of radiolabeled estrogens. Aromatase mRNA is distributed widely in several areas of the cowbird telencephalon including the hippocampus, caudomedial neostriatum (including Field L), and nucleus taeniae. This pattern of neural aromatase expression resembles what we found previously in the zebra finch. Telencephalic aromatase may be characteristic of passerine songbirds and may function to provide local estrogenic cues to estrogen-sensitive neural loci, and/or contribute to peripheral estrogen titers in male and female songbirds.

Intraventricular prolactin inhibits hypothalamic vasoactive-intestinal polypeptide-expression in doves.

While the role of prolactin in promoting the development of the crop-sac in members of the pigeon family (Columbiformes) is well established, its action in the central nervous system is less well understood. In the present study, prolactin was administered intracerebroventricularily (i.c.v.) in ring doves, and central expression of vasoactive-intestinal polypeptide (VIP) and gonadotropin-releasing hormone (GnRH), and the display of sexual behavior was investigated. Ovine-prolactin (1 microgram in 2 microliters o-prl) was injected daily for six days through chronically implanted cannula either prior to a 2-h period of courtship, or late in incubation. Control subjects were given vehicle injections and were otherwise identical to experimental animals. Prolactin administered prior to courtship resulted in a reduction of sexual behavior, and in a decrease in testicular weight but had no detectable effect on the number of neurons expressing VIP or GnRH. In contrast, i.c.v. prolactin during incubation resulted in a reduced number of infundibular VIP-positive neurons and decreased crop weight. We conclude that during incubation prolactin regulates its own synthesis and/or release by modulating VIP expression in infundibular neurons.

A putative suprachiasmatic nucleus of birds responds to visual motion.

In mammals, the suprachiasmatic nucleus (SCN) is a pacemaker regulating daily rhythms. In birds, two retinorecipient nuclei have been called the avian SCN, one in the lateral hypothalamus and the other more medial and rostral. We asked whether the proto-oncogene c-fos is expressed in either nucleus after light exposure during subjective night, but not during subjective day, as is the case in the SCN of mammals. Chicks raised with one eye covered by a diffuser were exposed to vertically moving surroundings, after the diffuser had been switched to the other eye. Surprisingly, we saw strong Fos label only in the lateral nucleus contralateral to the eye newly exposed to visual motion, but not in the ipsilateral nucleus nor in either medial SCN. No label was seen in animals kept in darkness or if the diffuser was not switched. Fos labeling did not differ between subjective day and night. The sensitivity to "novel" motion is also seen in motion-processing nuclei of the accessory optic system and pretectum; this suggests either that the lateral SCN is not the SCN, but part of the motion pathway, or that the avian SCN may by motion-sensitive during both day and night.

Detection and transduction of daylength in birds.

Daylength is an important environmental cue used by temperate zone avian species to time the onset of seasonal reproductive activity. Photic cues are detected by extra-retinal, extra-pineal central nervous system elements, and are rapidly transduced to an efferent signal. In this paper, we describe the brain locus of putative encephalic photoreceptors in birds, and explore the pathway of information transfer from photic input to the reproductive axis. To this end, we examine how photoreceptors might communicate with the hypothalamic-pituitary axis, and how brain peptides vary seasonally. Recent studies indicate that brain photoreceptors lie in the lateral septum and in the tuberal hypothalamus, and co-express proteins characteristic of retinal photoreceptors, as well as vasoactive-intestinal polypeptide (VIP). At the light microscopic level, photoreceptor cells appear to communicate with gonadotropin-releasing hormone (GnRH) neurons, and vice versa. Expression of VIP-like immunoreactivity is highest in photorefractory animals while GnRH-like immunoreactivity is highest in photosensitive birds. Expression of these CNS peptides is correlated with changes in plasma prolactin and luteinizing hormone (LH), suggesting a mechanism mediating seasonal cyclicity.

Increased VIP and decreased GnRH expression in photorefractory dark-eyed juncos (Junco hyemalis).

Most temperate zone birds show dramatic seasonal cycles in responsiveness to light. In the spring the hypothalamo-pituitary-gonadal axis of photosensitive birds is stimulated by long days. In the late summer birds no longer respond to long days, their gonads regress, and they are said to be photorefractory. After several weeks of refractoriness birds regain photosensitivity. During refractoriness circulating concentrations of luteinizing hormone are low and prolactin levels are high. These fluctuations in peripheral hormones result from changes in the brain rather than in the pituitary and/or the gonads. In the present study we examined seasonal changes in expression of vasoactive-intestinal polypeptide (VIP) and gonadotropin-releasing hormone (GnRH) in the brain of dark-eyed juncos (Junco hyemalis). Birds were photosensitive and exposed to long photoperiod (20:4 LD) for 1 day, 45-60 days, or not at all, or they were photorefractory (housed in 20:4 LD). The results indicate that VIP expression was similar in all photosensitive birds. However, photorefractory birds had significantly higher numbers of VIP-positive neurons in the infundibulum compared to photosensitive birds. The number of GnRH-positive neurons in the preoptic area was significantly lower in photorefractory birds and significantly higher in long-term photostimulated birds. These results indicate that the inverse relationship between circulating prolactin (released by VIP) and luteinizing hormone (released by GnRH) during refractoriness may result from neural changes in VIP and GnRH expression, respectively.