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Seasonal reproduction is common across temperate zone avian species. In these species, physiological and behavioral adaptations have evolved to change according to day length (i.e., seasonally) in order to maximize reproductive output. The hormone prolactin regulates many aspects of parental care, a critical component of reproductive success. It's secretion in birds has been shown to be under photoperiodic control, with the highest levels measured in the spring and summer months, when birds breed and show parental care. However, to date, no study has tested whether the densities of central prolactin binding sites vary seasonally, which may also account for prolactin's effect on parental care. To test this, we collected brains from free-ranging adult male dark-eyed juncos, Junco hyemalis, a biparental songbird, in the spring, summer, and fall, and used quantitative in vitro autoradiography to compare the densities of specific prolactin binding sites across 20 different brain regions. Prolactin binding sites were found in regions that regulate parental behavior in other avian species. During the summer, several hypothalamic regions that regulate parental care, including the preoptic area and tuberal nucleus, contained lower densities of prolactin binding sites, suggesting exposure to higher endogenous prolactin levels, than at other times. This observation is consistent with the fact that circulating prolactin is highest during summer, when males would be providing care to young. Overall, these data suggest that prolactin binding sites are relatively conserved in the avian brain and that central prolactin activity supports parental care efforts in juncos and other avian species., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Prolactin acts on target cells in the central nervous system (CNS) to stimulate behavioral changes associated with parental care in birds, but the signaling mechanisms that mediate these actions have not been characterized. In mammals, the Janus Kinase 2-Signal Transducer and Activator of Transcription 5 (JAK2-STAT5) signaling pathway mediates many of the actions of prolactin. To assess the importance of this pathway in prolactin-sensitive target cells in the avian brain, we measured changes in activated (phosphorylated) STAT5 (pSTAT5) in the forebrain of female ring doves sampled as plasma prolactin levels change during the breeding cycle and in prolactin-treated, non-breeding females. The anatomical distribution of cells exhibiting pSTAT5 immunoreactivity in dove brain closely paralleled the distribution of prolactin receptors in this species. The density of pSTAT5 immunoreactive (pSTAT5-ir) cells was highest in the preoptic area, the suprachiasmatic, paraventricular, and ventromedial hypothalamic nuclei, the lateral and tuberal hypothalamic regions, the lateral bed nucleus of the stria terminalis, and the lateral septum. Mean pSTAT5-ir cell densities in these eight brain areas were several fold higher in breeding females during late incubation/early post-hatching when plasma prolactin levels have been observed to peak than in non-breeding females or breeding females sampled at earlier stages when prolactin titers have been reported to be lower. Similar differences were observed between prolactin-treated and vehicle-treated females in all three of the forebrain regions that were compared. We conclude that JAK2-STAT5 signaling is strongly activated in response to prolactin stimulation in the ring dove brain and could potentially mediate some of the centrally-mediated behavioral effects of this hormone., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Many birds and mammals show changes in the hypothalamo-pituitary-gonadal (HPG) axis in response to social or sexual interactions between breeding partners. While alterations in GnRH neuronal activity play an important role in stimulating these changes, it remains unclear if acute behaviorally-induced alterations in GnRH release are accompanied by parallel changes in GnRH synthesis. To investigate this relationship, we examined changes in the activity of GnRH neurons in the brains of male ring doves following brief periods of courtship interactions with females. Such interactions have been previously shown to increase plasma LH in courting male doves at 24 h, but not at 1 h, after pairing with females. In the first study, males allowed to court females for 2 h had 60% more cells that showed immunocytochemical labeling for GnRH-I in the preoptic area (POA) of the hypothalamus than did control males that remained isolated from females. To determine whether an increase in GnRH gene expression preceded this increase in GnRH immunoreactivity in the POA, changes in the number of cells with detectable GnRH-I mRNA in the POA were measured by in situ hybridization following a 1 h period of courtship interactions with females. In this second study, courting males exhibited 40% more cells with GnRH-I in this region than did isolated control males. GnRH-immunoreactive neurons in two other diencephalic regions failed to show these courtship-induced changes. Plasma LH was not elevated after 1 or 2 h of courtship. These results demonstrate that the release of GnRH-I in the POA that is presumably responsible for courtship-induced pituitary and gonadal activation is accompanied by a rapid increase in GnRH synthesis that occurs before plasma LH levels increase. We suggest that this increase in GnRH synthesis is necessary to support the extended period of HPG axis activation that is seen in this species during the 5-10 day period of courtship and nest building activity.

Like lactating mammals, male and female ring dove parents increase their food consumption to meet the energetic challenges of provisioning their young. To clarify the neurochemical mechanisms involved, the present study investigated the relationship between parental hyperphagia and changes in activity of the potent orexigen neuropeptide Y (NPY) in the hypothalamus of breeding doves. Changes in NPY-immunoreactive (NPY-ir) cell numbers in the tuberal hypothalamus of male and female doves were examined by immunocytochemistry at six stages of the breeding cycle. Parallel NPY mRNA measurements were recorded in mediobasal hypothalamus (which includes the tuberal hypothalamus) by semiquantitative reverse transcription-polymerase chain reaction using 18S rRNA as the internal standard. NPY mRNA changes were also measured in the mediobasal hypothalamus of nonbreeding doves following intracranial administration of prolactin, an orexigenic hormone that is elevated in the plasma of parent doves, and in response to food deprivation, which mimics the negative energy state that develops in parents as they provision their growing young. NPY-ir cell numbers in the tuberal hypothalamus and NPY mRNA levels in the mediobasal hypothalamus were significantly higher in breeding males and females during the period of parental hyperphagia after hatching than during the late incubation period when food intake remains unchanged. In nonbreeding doves, food deprivation and prolactin treatment increased NPY mRNA in this region by two- to three-fold, which suggests that NPY expression is sensitive to hormonal and metabolic signals associated with parenting. We conclude that NPY synthesis is increased in the mediobasal hypothalamus during the posthatching period, which presumably supports increased NPY release and resulting parental hyperphagia. NPY-ir and mRNA were also high in the mediobasal hypothalamus prior to egg laying when food intake remained unchanged. Several lines of evidence suggest that this elevation in NPY supports the increased gonadal activity that accompanies intense courtship and nest building interactions in breeding doves.

Neuronal activation was examined by fos immunohistochemistry in ring doves (Streptopelia risoria) reunited with their young after overnight separation. In an initial study, squab-exposed parents showed more fos immunoreactivity (ir) in the preoptic area (POA) and lateral hypothalamus (LH) than squab-deprived parents. In a 2nd study, parents allowed free access to young and those separated from young by a wire mesh partition showed more fos-ir in the POA, LH, and lateral septum than box-exposed controls. Contact with young also increased fos-ir in the medial preoptic nucleus and bed nucleus of the stria terminalis, but noncontact exposure did not. Conversely, nontactile squab exposure stimulated more fos-ir in the POA than did free access to young, which suggests POA involvement in appetitive aspects of parenting., (Copyright 2006 APA, all rights reserved.)

Hyperphagia is a prominent component of the parental behavior repertoire in male and female ring doves and is necessary in order for parents to successfully provision their growing young. Although previous studies implicate both prolactin and the endogenous glucocorticoid, corticosterone, in parental hyperphagia, the functional interactions between these two hormones in regulating changes in feeding activity have not been characterized. These studies examined the possibility that prolactin's orexigenic effects are mediated through the increased secretion of corticosterone. Twice-daily intracerebroventricular (icv) injection of prolactin increased plasma corticosterone concentration in non-breeding doves of both sexes, with males exhibiting more pronounced effects than females. To further test the importance of glucocorticoid signaling in prolactin-induced feeding responses, changes in food intake were investigated in icv prolactin-treated, non-breeding doves following icv infusion of the glucocorticoid receptor antagonist RU38486 or propylene glycol vehicle. No attenuation of prolactin-induced hyperphagia was observed in either sex following co-administration of RU38486 at a dose shown previously to block dexamethasone-induced feeding in doves. These findings suggest that elevated corticosterone titers in blood may contribute to the hyperphagia observed in response to prolactin, but corticosterone signaling through a mammalian-type glucocorticoid receptor is not essential.

Ring doves (Streptopelia risoria) exhibit marked increases in food consumption and decreases in body weight when they are provisioning their young. This study examined changes in hypothalamic immunostaining for agouti-related peptide (AGRP), an endogenous melanocortin receptor antagonist and appetite stimulant, during the ring dove breeding cycle. Because prolactin is orexigenic in doves, and is elevated in blood at the onset of parental hyperphagia, we also explored the possibility that prolactin-induced hyperphagia is associated with AGRP changes. The numbers of AGRP-immunoreactive (ir) cells within the tuberal hypothalamus were maximal during the prelaying period of the breeding cycle but decreased dramatically during early incubation. AGRP-ir cell numbers began to increase again during late incubation and reached a peak during the early and late posthatching stages. Because posthatching elevations in AGRP-ir were temporally associated with marked elevations in parental food intake, and because AGRP is orexigenic in doves, these findings suggest that increased AGRP activity in the dove tuberal hypothalamus may contribute to parental hyperphagia. Rising prolactin secretion during late incubation and early posthatching may initiate this increase in AGRP-ir, since intracerebroventricular administration of prolactin significantly elevated food intake and AGRP-ir cell numbers in the tuberal hypothalamus of nonbreeding doves. Prolactin-induced elevations in AGRP-ir cell numbers persisted when the confounding effects of weight gain that accompany prolactin-induced hyperphagia in nonbreeding doves were eliminated by a food restriction procedure, thereby suggesting that prolactin can directly influence AGRP activity under neutral energy state conditions.

The melanocortin-4 receptor (MC4-R) is an important mediator of the effects of two melanocortin system ligands, alpha melanocyte stimulating hormone (alpha-MSH) and agouti-related peptide (AGRP), on feeding behavior and energy balance in mammals. Although an avian homologue of the mammalian MC4-R has recently been identified, there is little information on the role of this receptor and the melanocortin system in avian feeding and body weight regulation. In these studies, we measured changes in feeding behavior in ring doves (Streptopelia risoria) following intracerebroventricular (i.c.v.) injection of various melanocortin receptor agonists and antagonists. The selective MC4-R antagonist HS014 elevated food intake within 4 h at all three doses tested (0.02, 0.2, and 2 nmol). A 1 nmol dose of the endogenous antagonist AGRP also stimulated feeding but only after a post-injection interval of 10 h. Surprisingly, the MC3-R and MC4-R antagonist SHU9119 not only failed to stimulate food intake at the same doses as HS014, but actually inhibited food intake at 8 h after injection. Whether this was due to toxicity effects or differences in the pharmacology of avian and mammalian melanocortin receptors remains to be determined. Food-deprived doves showed a fourfold increase in the number of AGRP-immunoreactive cells in the tuberal region of the hypothalamus and 5 ng of the MC3-R and MC4-R agonist MTII significantly attenuated the amount of food consumed by food-deprived birds that were allowed to re-feed. These data support a role for the melanocortin system and the melanocortin-4 receptor in the ring dove feeding behavior.

These studies explored the possibility that glucocorticoids promote parental care in ring doves by mediating, at least in part, the pronounced increase in food consumption that parent doves exhibit while provisioning their young. Plasma concentrations of the endogenous glucocorticoid corticosterone were found to be significantly higher in breeding females during the posthatching phase than during the incubation period. These differences were not observed in male breeding partners, but sex differences in daily activity rhythms are well documented in breeding doves, and blood sampling at different times of day would be required to adequately characterize the pattern of corticosterone in males during these breeding stages. In studies on nonbreeding doves, twice-daily intracerebroventricular (icv) injections of the synthetic glucocorticoid dexamethasone (DEX) increased food intake by 25-50% in both sexes, and further studies in males revealed that the increase was directly related to the dose of DEX administered. The highest dose of DEX given icv (1.0 microg/day) was not effective in stimulating feeding when given systemically, thereby suggesting that the hyperphagic action of DEX is exerted directly on the central nervous system. The icv infusion of the selective glucocorticoid receptor antagonist RU38486 blocked the hyperphagic effects of twice-daily icv injections of DEX in both sexes. Collectively, these data support the hypothesis that corticosterone contributes to the parental hyperphagia exhibited by breeding doves during the posthatching period. They also suggest that these orexigenic effects are mediated in part by CNS binding sites that resemble mammalian glucocorticoid receptors.

Although neuropeptide-Y (NPY) has been widely reported to be a potent stimulator of feeding activity and regulator of energy homeostasis, most of the supportive evidence for such effects has been gathered in mammalian species. This study characterized the orexigenic potency of NPY in an avian species, the ring dove, and measured changes in hypothalamic NPY-immunoreactive (NPY-ir) cell numbers in response to energy state fluctuations or intracranial administration of the potent orexigenic hormone prolactin. Food intake was significantly elevated in male doves at 1 h after intracerebroventricular (i.c.v.) injection of 0.25 and 0.5 microg NPY but not after injection of a higher dose (1.0 microg). In time course studies, food intake was increased at 1 h after i.c.v. injection of 0.5 microg NPY but was not elevated at 2, 3, or 4 h. The number of NPY-ir cell bodies in the infundibular region of the dove hypothalamus increased two to four-fold following acute food deprivation, chronic food restriction, or repeated i.c.v. injections of prolactin. No additive effects were observed when food restriction and prolactin treatment were combined. These findings suggest that NPY is involved in energy homeostasis in doves and are consistent with the hypothesis that prolactin-induced hyperphagia is mediated in part by NPY.

Prolactin (Prl) increases food consumption in ring doves (Streptopelia risoria) and may promote the hyperphagia exhibited by parent doves when provisioning young. These experiments tested whether Prl also enhances appetitive aspects of feeding behavior. Prl elevated pecking rates in food-restricted doves on a variable-interval (VI) reinforcement schedule and supported continued responding when doves were returned to ad-lib feeding. Prl-treated doves learned the key-pecking response when food intake was clamped at ad-lib levels exhibited before Prl treatment but not when given free access to food. Median break points on a progressive ratio schedule were 2-3 times greater in food-restricted doves than Prl-treated, food-clamped doves even though response rates were similar on VI schedules. These results indicate that Prl enhances appetitive aspects of feeding, although food restriction at the level imposed in this study was more effective in this regard.

Prolactin (PRL)- and growth-hormone (GH)-containing perikarya and fibers independent of the anterior pituitary gland have been reported to exist in the central nervous system of several mammalian species. The specific distributions of PRL- or GH-like neurons in the avian forebrain and midbrain, however, have not been reported. The objective of the study was to identify GH- and PRL-containing neurons in the hypothalamus and a few extrahypothalamic areas of two avian species. Brain and peripheral blood samples were collected from laying and broody turkey hens and ring doves. Broody turkey hens and doves had significantly higher plasma PRL concentrations compared with laying hens. Coronal brain sections were prepared and immunostained using anti-turkey GH and anti-chicken synthetic PRL antibodies. In turkey hens, the most dense GH-immunoreactive (ir) perikarya and fibers were found in hippocampus (Hp), periventricular hypothalamic nucleus, paraventricular nucleus, inferior hypothalamic nucleus, infundibular hypothalamic nucleus, medial and lateral septal area, and external zone of the median eminence (ME). In the ring dove, a similar pattern of distribution of GH-ir neurons was noticed at the brain sites listed above except that GH-ir fibers and granules were found only in the internal zone of ME and not in the external zone. In both turkeys and doves, the most immunoreactive PRL-ir perikarya and fibers were found in the medial and lateral septal area, Hp (turkey only), and bed nucleus of the stria terminalis pars magnocellularis. There were no apparent differences in the staining pattern of GH- or PRL-ir neurons between the laying and broody states in either species. However, the presence of GH-ir- and PRL-ir perikarya and fibers in several hypothalamic nuclei indicates that GH and PRL may influence parental behavior, food intake, autonomic nervous system function, and/or reproduction.

In addition to stimulating crop "milk" formation in ring doves, prolactin (PRL) may promote the parental regurgitation behavior that transfers this "milk" to the young at the time of hatching. Although earlier studies suggest that previous breeding experience is an important modulator of PRL-induced parental regurgitation behavior in ring doves, the ways in which experience, hormones, and stimuli from young interact to promote parental behavior have not been well characterized in this species. In the first study, untreated, nonbreeding female doves with and without previous breeding experience were given 10 daily parental behavior tests (2 h/day) with a hungry 5- to 10-day-old foster squab. Experienced females exhibited a higher incidence of regurgitation behavior, defensive behavior, and crouching or sitting in the nest than did inexperienced females. In a second study, nonbreeding females were given 10 daily tests for parental behavior while they received sc injections of ovine PRL or vehicle. Prolactin reduced squab-directed aggression and increased the incidence of regurgitation feeding behavior of foster squabs in both experienced and inexperienced females. However, the average number of regurgitation feeding acts displayed by those PRL-treated females that showed the behavior was over eight times higher in experienced females than in inexperienced females. Previous experience also enhanced the stimulatory effects of PRL on defensive behavior and crouching or sitting in the nest. The parental behavior exhibited by nonbreeding, PRL-treated experienced females was qualitatively and quantitatively similar to that observed in normally breeding females during a single test with their own hungry 5- to 10-day-old squabs. These findings indicate that PRL and previous breeding experience both enhance the parental responsiveness of nonbreeding female doves and that under optimal hormonal, experiential, and squab exposure conditions, nonbreeding doves exhibit levels of parental activity that rival those of normally breeding parents., (Copyright 1999 Academic Press.)

Intracerebroventricular (ICV) injections of prolactin (PRL) exert potent antigonadal and antigonadotropic effects in ring doves (Streptopelia risoria) at doses that are insufficient to stimulate prolactin-dependent crop growth. To explore the physiological basis of these effects, we tested the ability of ICV-injected PRL to influence pituitary responsiveness to chicken gonadotropin-releasing hormone-I (cGnRH-I) and to alter GnRH content and concentration in the preoptic area (POA) and median eminence (ME). cGnRH-I-induced changes in plasma LH were monitored by radioimmunoassay (RIA) in photostimulated male doves after they received five daily ICV injections of ovine PRL (1 microg/2 microl) or saline vehicle. Although PRL treatment reduced basal plasma LH levels and testes weight, it did not reduce the amount or alter the pattern of LH released in response to a bolus injection of cGnRH-I. This suggests that ICV PRL does not suppress LH by reducing pituitary responsiveness to GnRH. In two subsequent studies, GnRH content (ng/region) and concentration (pg/microg protein) in the POA and ME were measured in male doves by RIA and by competitive enzyme immunoassay after 5 days of ICV PRL or vehicle treatment. Although ICV PRL reduced plasma LH levels in both studies, no significant PRL-induced alterations in GnRH content or concentration were apparent. In a final study, PRL-treated female doves had lower plasma LH levels than vehicle-treated control females at 12 and 24 h after a single ICV injection. GnRH content of the POA was also lower in PRL-treated females than in controls at 24 h. However, the two treatment groups did not differ in POA or ME GnRH content at earlier postinjection sampling intervals. Analysis of GnRH concentration data revealed no treatment group differences in either region at any sampling interval (1, 6, 12, or 24 h post-PRL injection). Collectively, these results are consistent with the idea that ICV-injected PRL acts at the level of the CNS to inhibit the reproductive system. However, the nature of the alterations involved remains to be clarified. Plausible hypotheses are (1) that ICV PRL suppresses the gonadal axis by influencing the activity of GnRH neurons at brain sites other than the POA or ME or (2) that PRL alters the synthesis, storage, degradation, and/or release of GnRH in the POA or ME, but the dynamic changes involved are not reflected in integrated, steady-state measures such as peptide content or concentration in tissue., (Copyright 1999 Academic Press.)

Prolactin (PRL) strongly stimulates feeding activity and body weight gain in ring doves, and of the brain loci tested to date, the ventromedial hypothalamus (VMH) is the most effective site of PRL action in promoting these changes. To determine if the VMH is essential for this response, we examined the effects of VMN destruction on spontaneous feeding and on changes in food intake induced by intracerebroventricular (i.c.v.) injections of PRL. Male birds were selectively destroyed by radiofrequency lesions (n = 6). A group of sham-lesioned males (n = 6) served as controls. Lesioned birds exhibited a transient increase in food intake that peaked around the seventh postoperative day and declined to baseline levels by day 12. In contrast to this pattern, body weights of lesioned birds increased in parallel with food intake, but remained elevated throughout the 3-week postoperative period. During the peak period of hyperphagia in the lesioned group, food intake and body weight increases were two to three times greater in lesioned birds than in controls. After postoperative feed intake had stabilized, each bird received 5 daily i.c.v. injections of ovine PRL. Food intake and body weight increased dramatically in both groups in response to PRL treatment, and no group differences were observed in response to magnitude. We conclude that VMH destruction strongly perturbs feeding and body weight regulation in doves. However, VMH integrity is not essential for the expression of PRL-induced hyperphagia.

Numerous studies have shown that parental behaviors are mediated by prolactin (PRL), while testosterone (T) interferes with their full expression. The limited data available suggest that reduced parental behavior induced by T is not mediated by reduced concentrations of plasma PRL. We hypothesized that T reduces parental behaviors by reducing PRL receptor binding activity at central neural sites that promote the expression of parental behaviors. To test this hypothesis we implanted male dark-eyed juncos (Junco hyemalis) with testosterone-filled or empty implants and measured T and PRL levels, paternal behavior, and specific binding of radio-labeled PRL at selected brain regions that have been implicated in the mediation of parental behaviors. Our findings concurred with previous studies in that T-treated males reduced their parental contributions, had higher levels of T, and had equivalent levels of PRL compared with controls. We found no differences in the capacity to bind 125I-oPRL in three brain regions previously implicated in the mediation of parental care in birds, i.e., the preoptic area, ventromedial nucleus of the hypothalamus, and paraventricular nucleus of the hypothalamus. Thus our findings do not support the hypothesis that T interferes with the expression of parental behavior by reducing PRL receptor binding activity at central sites.

Wilson's Phalarope (Phalaropus tricolor) is a sex role-reversed species in which incubation of eggs and care of young is performed exclusively by the male. Plasma levels of prolactin (PRL), the hormone most associated with parental care in birds, are higher in incubating males than in nonincubating males or females. Conversely, plasma testosterone levels are reduced in males during incubation. In an attempt to characterize the physiological basis of this unusual parental care system we used quantitative film autoradiography and densitometry to measure the specific binding in vitro of 125I-ovine PRL to 12 brain regions in females, nonincubating males, and incubating males during the normal breeding season. We also measured hypothalamic chicken gonadotropin-releasing hormone I (cGnRH-I) in three brain areas in these same birds, as well as plasma levels of PRL and testosterone. Analysis revealed that cGnRH-I concentrations in the preoptic area and plasma testosterone levels were significantly lower in incubating males than in nonincubating males. Specific binding of 125I-ovine PRL was detected in choroid plexus and in several diencephalic brain regions of both sexes, with highest binding activity recorded in the dorsolateral thalamus, medial habenula, nucleus subrotundus, and preoptic area. When adjustments were made for the large number of comparisons performed, specific binding did not vary significantly by sex or breeding stage in any single brain region. However, average specific binding values in nonincubating males exceeded those of incubating males in 9 of the 11 PRL-sensitive regions examined. Increased occupancy of the receptor by endogenous PRL during incubation could have contributed to this result, since plasma PRL levels were elevated in incubating males. In addition, PRL binding activity in several of these brain regions tended to correlate negatively with plasma PRL. The two exceptions to this general pattern were the preoptic area and the lateral septum, where mean specific binding was 14-15% higher in incubating males than in nonincubating males. This raises the interesting possibility that PRL sensitivity is up-regulated during incubation in some regions of the male phalarope brain, such as the preoptic area and lateral septum, that have been implicated in PRL-modulated changes in behavior and reproductive activity during this breeding stage.

Previous radioimmunoassay (RIA) data indicate that plasma prolactin (PRL) is elevated during the late incubation and the early posthatching periods of the ring dove breeding cycle. Although these changes are temporally associated with changes in PRL-dependent crop sac growth, the precise relationship between immunoreactive and bioactive PRL has not been directly examined. To investigate this question and to further explore the relationship between sitting behavior and PRL secretion, we used rat Nb2 lymphoma cell proliferation to estimate the concentration of bioactive PRL-like activity (PLA) in the plasma of breeding ring doves. Serial dilutions of dove pituitary homogenate and dove plasma stimulated mitogenic responses that were parallel to those observed with purified ovine PRL. Changes in plasma PLA during the breeding cycle closely resembled changes in PRL that have been previously reported by RIA, although the relative changes in PLA were more pronounced. In both sexes, PLA remained at basal levels prior to egg laying and during early incubation (Day 4-5) but then abruptly increased to reach peak values near the time of hatching (Day 14-15). Activity remained high for 3-4 days after hatching, declined gradually thereafter, and returned to baseline values by Posthatching Days 14-17. Plasma PLA levels of birds sampled at the end of incubation were correlated with those of their breeding partners. In the majority of pairs, females had higher PLA levels than their mates at this stage even though no significant overall sex differences in PLA levels were observed. Plasma PLA declined precipitously in birds that were nest deprived on the last day of the incubation period. Nevertheless, plasma PLA levels of normally breeding birds at the end of incubation were not correlated with the average time spent in the nest during the incubation period. However, day-to-day variability in time spent in the nest correlated negatively with plasma PLA in incubating males, and females exhibited a similar trend that approached significance. These data suggest (1) that published RIA estimates of PRL are reasonably accurate reflections of changes in bioactive PLA in dove plasma and (2) that while sitting duration itself is not strongly related to plasma PLA, large day-to-day fluctuations in nest occupation time are associated with reduced PLA levels in incubating doves.

The role of the preoptic area (POA) in mediating PRL-induced parental responsiveness toward young was investigated in nonbreeding-doves with previous breeding experience. Birds received microinjections of the axon-sparing excitotoxin ibotenic acid to destroy neuronal cell bodies in the POA. Sham-lesioned controls received POA injections of vehicle. Upon recovery, birds received subcutaneous injections of ovine PRL for 7 days, followed by a 2.5-hr test with a foster squab to assess parental responsiveness. Lesioned doves displayed significantly fewer parental feeding invitations, parental regurgitation feeding bouts, and total regurgitation movements than did sham-lesioned animals. As a result, nestlings given to sham-lesioned birds gained significantly more weight than those given to lesioned birds. A significant negative correlation was obtained between the extent of POA damage induced by ibotenic acid infusion and the amount of parental behavior displayed by lesioned animals. These deficits in parental behavior were apparently a selective effect of POA damage because another PRL-mediated behavior, hyperphagia, was unaffected. The results suggest that the POA is an important component of the neural circuitry underlying the expression of PRL-induced parental feeding behavior in non-breeding ring doves. Because elevated PRL levels are temporally associated with parental activity during the breeding cycle, these results also implicate the POA in the spontaneous display of parental feeding activity that occurs naturally at the time of hatching.

Although binding sites for prolactin (PRL) have been mapped and partially characterized in the brains of several species, there is as yet no direct evidence that the effects of intracranial PRL on brain function are receptor-mediated events. We addressed this question by testing whether antibodies generated against rat liver PRL receptors can effectively antagonize the ability of PRL to enhance feeding behavior in male ring doves (Streptopelia risoria). Both agents were administered directly to the ventromedial hypothalamic nucleus (VMN), which is an effective site of PRL action in promoting hyperphagia in this species. In the initial study, affinity-purified gamma-globulin (IgG) from the receptor antiserum preparation was tested for its ability to compete with 125I-ovine PRL for binding to receptors in rat liver, dove choroid plexus, and 6 PRL-sensitive dove brain regions using in vitro quantitative film autoradiography. Although the binding affinity of the anti-PRL receptor antibodies was at least 50 times lower in dove brain than in rat liver, a 40-50% inhibition of specifically bound 125I-ovine PRL was observed in choroid plexus and in 5 of 6 brain regions with anti-receptor IgG concentrations of 5.8 x 10(-7) M and 1.2 x 10(-6) M, using sections incubated with normal rabbit serum (NRS) IgG as a control. In a second study, anti-PRL receptor IgG or NRS IgG (2.4 micrograms) was injected unilaterally into the VMN at 45-60 min prior to VMN injection of ovine PRL (50 ng) or saline vehicle. This procedure was repeated at twice-daily intervals for 5 days. When compared to the feeding behavior of PRL-injected birds given NRS IgG, antireceptor antibody-treated animals showed a marked reduction in PRL-induced hyperphagia. The magnitude of this reduction was calculated to be approximately 50% after corrections were made for a mild hypophagia induced by the anti-receptor IgG treatment alone, as reflected in the feeding behavior of the anti-PRL receptor IgG + vehicle-treated group. These results suggest that PRL receptors in dove brain and rat liver exhibit structural similarities as well as differences and that the hyperphagia induced by intracranial injections of PRL is mediated, at least in part, by interactions with PRL receptors in the brain.

Prolactin stimulates the growth and development of specialized epithelial cells lining the cropsac of pigeons and doves (family Columbidae), leading to formation of "cropmilk," which is fed to the newly hatched squab. This system of milk feeding is unique among birds. To support the feeding of cropmilk, a complex array of behavioral adaptations are also supported by high levels of prolactin secretion in columbids during parenting. These specializations include elevated food intake (hyperphagia), nest attendance, and regurgitation feeding of the squab. Although prolactin is clearly important for these behavioral adaptations, the precise physiological and mechanistic bases for these behavioral effects remain controversial. The molecular mechanisms of prolactin action in the cropsac epithelium have been studied by cloning prolactin-induced genes, by cloning and expressing the pigeon prolactin receptor, and by analyzing the transcription factors that are activated after prolactin treatment. The avian (pigeon) prolactin receptor is a member of the cytokine receptor superfamily and uniquely contains a complete duplication of the extracellular ligand-binding domain. One of the early signal-transducing actions of prolactin in cropsac epithelium is the activation of signal transducer and activator of transcription (STAT) proteins via tyrosine phosphorylation. This fundamental signaling pathway is shared with mammalian prolactin target tissues. The convergent evolution of milk feeding and the behaviors that support parenting in columbids and mammals has depended on adaptation of both conserved mechanisms and divergent physiological processes.

Microinjections of prolactin (PRL) into the ventromedial nucleus of the hypothalamus (VMN) or the preoptic area (POA) have been previously shown to increase food intake and body weight in ring doves. In an attempt to corroborate these results and to provide a more complete map of PRL-sensitive brain sites mediating the orexigenic action of PRL, a microinjection procedure was employed in the present study that delivered PRL or saline vehicle in extremely small volumes (10 nl/injection) to a variety of diencephalic sites in dove brain that had been previously demonstrated to contain high concentrations of PRL receptors. Estimates obtained from one female subject given a single 10 nl injection of [125I]ovine PRL into the VMN supported the claim that such injection volumes resulted in limited diffusion, as 80% of the tissue radioactivity was found within a 280 mm area surrounding the injection site at 30 min after injection. Food intake of cannulated male doves in the mapping study was monitored daily during a 6 day baseline period, an initial 4 day treatment period, a 6-12 day post-treatment recovery period, and a second 4 day treatment period. Approximately half of the birds received PRL injections (50 ng/10 nl twice daily) and saine vehicle injections (10 nl twice daily) during the first and second treatment periods, respectively, while remaining birds received these treatments in the reverse order. No significant changes in food intake across baseline, vehicle, post-treatment, or PRL treatment periods were observed in birds with injection sites in the lateral POA, paraventricular nucleus of the hypothalamus (PVN), or the medial-basal hypothalamic region between the tuberal hypothalamus (TU) and VMN. In contrast, injections of PRL into the VMN area, medial POA, or TU resulted in average daily food intake values that significantly exceeded those recorded during other periods. The most robust feeding response was seen in the VMN group, where PRL injections resulted in a 58% increase in food intake over that recorded during injection of vehicle. This increase was significantly greater than that observed following PRL injections into the mPOA (26%) or the TU (32%). These findings suggest that the VMN may be a primary site of PRL action in promoting hyperphagia in this species, although PRL effects at other diencephalic loci, such as the mPOA and TU, may also contribute to the orexigenic action of this hormone.

Film autoradiography and densitometry were employed to obtain a detailed map of prolactin (PRL) receptor distribution in ring dove brain and to characterize the affinity, capacity, specificity, and in vivo saturability of 125I-ovine PRL binding in individual brain regions. Significant specific binding was detected in choroid plexus and in a variety of forebrain and midbrain sites, with highest levels observed in the preoptic area, tuberoinfundibular region, lateral hypothalamic area, and the suprachiasmatic, paraventricular, and ventromedial hypothalamic nuclei. A comparison of PRL receptors in the choroid plexus, preoptic area, ventromedial nucleus, suprachiasmatic nucleus, and tuberal hypothalamus revealed significant regional differences in binding capacity, but not differences in binding affinity (Kd = 0.4-0.9 nM; Bmax = 6-14 fmol/mg protein). Unlabelled ovine PRL proved to be an effective competitor of labelled ovine PRL at concentrations of 43 pM, 0.43 nM, and 4.3 nM in the choroid plexus and in 5 diencephalic areas. In contrast, no significant competition was observed in any of these brain areas in tests with unlabelled turkey GH, ovine GH, or ovine LH at similar concentrations. Unlabelled ovine PRL (37 nmol) significantly reduced the uptake of 125I-ovine PRL (37 pmol) in the preoptic area, tuberal hypothalamus, and bed nucleus of the pallial commissure at 2 h after intravenous injection. In addition, specific binding of 125I-ovine PRL in vitro was reduced by over 67% in these brain areas and in several other brain regions following a 7 day period of systemic ovine PRL administration. These results indicate that PRL receptors in different regions of the dove brain exhibit similar binding characteristics. In addition, they suggest that at least some of these PRL-sensitive brain loci are accessible to blood-borne hormone.

Systemic administration of ovine prolactin (PRL) has been previously reported to stimulate parental feeding behavior toward 7-day-old foster squabs by nonbreeding ring doves with previous breeding experience. The first of the present experiments reexamined this claim in experienced, nonbreeding birds given test squabs of different ages. Each visually isolated male and female dove was given twice-daily subcutaneous injections of ovine PRL or vehicle for 7 days and then tested for parental responses toward a single 1-3, 6-8, or 11- to 13-day-old foster squab. Prolactin significantly increased the incidence or frequency of parental regurgitation-feeding episodes in tests with all three squab age groups and, in addition, increased the incidence of parental feeding invitations (squab-oriented bill openings) in tests with 6- to 8-day-old squabs. A second study explored the degree to which PRL can act directly on the central nervous system to facilitate parental activity in the absence of peripheral cues generated from PRL-induced changes in other target organs, such as those associated with crop sac growth and distension. In this experiment, 6- to 8-day-old test squabs were used to determine if parental behavior is enhanced by twice-daily intracerebroventricular (ICV) injections of PRL in doses below those required to stimulate peripheral target organs. Injection schedules and behavior testing procedures were the same as those used in Experiment 1. However, half of the ICV PRL-treated and ICV vehicle-treated birds were food deprived for 16 hr before and during the test in order to control for PRL-induced hyperphagia and resulting crop sac distension, which could confound the results by generating peripheral stimuli conductive to the display of regurgitation-feeding behavior. Intracranial injections of prolactin significantly increased the incidence of feeding behavior, parental feeding invitations, and crouching or sitting in the nest in food-deprived doves but not in freely fed animals. Empty crop sac weights of freely fed and food-deprived PRL-treated birds were not increased above control values, thus indicating that ICV PRL treatment did not result in significant stimulation of peripheral target organs. These results demonstrate a facilitative action of PRL on regurgitation-feeding responses and associated parental behaviors that is not restricted to squabs of one particular age range. They also indicate that PRL is capable of acting directly on the brain to promote these activities in the absence of PRL-induced changes in the crop sac and other peripheral target organs.

Ovine prolactin, labelled with 125I by either lactoperoxidase or a mild chloramine T method, bound to receptors from the pigeon crop sac mucosa cells of prolactin-injected pigeons. Binding was demonstrated in a crude homogenate of mucosal cells removed from the crop by scraping and in a subcellular fraction in which 5'-nucleotidase activity was enhanced two- to threefold. The binding was specific, dependent on time, temperature and the concentration of receptors and had a dissociation constant of 7 X 10(-10) mol/l. The binding capacity of the crop tissue was 71 fmol/mg membrane protein. Nine purified preparations of prolactin from four species were assayed by local pigeon crop sac bioassay and by radioreceptor assay. The two methods were highly correlated (r = 0.934). The regression equation was radioreceptor assay = 1.22 bioassay--0.18 indicating a 1 : 1 correspondence between the two methods for prolactin purified from sheep, rat, horse and pig anterior pituitary glands.

The role of prolactin in the maintenance of incubation behaviour in ring doves was re-examined and the dose-response relationships for behavioural, target tissue and body weight changes induced by injections of prolactin were compared in doves tested during the incubation phase of the breeding cycle. Doves given injections of prolactin twice a day starting on day 4 of incubation, during a 10-day period of isolation from their mates and nests, showed a higher persistence of incubation behaviour than doves injected with saline vehicle. However, the prolactin treatment failed to maintain incubation behaviour to the same extent as that observed in non-isolated untreated breeding pairs. Liver and body weights were higher and testicular weights lower in birds treated with high doses of prolactin than in non-isolated birds which had been incubating for 14 days. Good dose-response relationships were established between body, liver, crop and testes weights and the dose of prolactin administered. However, only a weak dose-response relationship was observed between prolactin and the maintenance of incubation behaviour. Overall, females injected with prolactin displayed more quiet sitting behaviour, less body weight gain and more gonadal regression than males injected with prolactin. Males in untreated breeding pairs had higher liver weights and lower crop weights than females. It is concluded that prolactin plays a role in maintaining readiness to incubate in doves, but that other factors may also contribute to this response. Further, it appears that prolactin mediates several target tissue changes which are sex-specific during incubation.

A pigeon crop sac radioreceptor assay was used to measure changes in pituitary prolactin levels in parent ring doves of both sexes on the third day after hatching of their young. After a deprivation of 17 h from the squabs, exposure to a 3-day-old squab for 1 h resulted in a significant decrease in the prolactin content of the pituitary gland as compared with levels obtained in control birds deprived of their young for 18 h. No significant sex differences in prolactin levels were observed in either group. Because exposure to the young also promotes prolactin-induced crop sac growth, it appears probable that the squab-induced decrease in prolactin content of the pituitary gland reflects the release of prolactin into the circulation. Accordingly, the environmental regulation of prolactin secretion in parent ring doves appears similar to that observed in lactating mammals.

Specific binding sites for prolactin have been previously detected and characterized in ring dove brain membranes. In order to map the distribution of these sites, specific binding of 125I-ovine prolactin was examined in slide-mounted sections of 6 male and 6 female ring dove brains by in vitro film autoradiography and densitometry. Analysis of 34 brain regions revealed a sex-specific pattern in specific binding activity. Although no significant sex differences were observed in any individual brain region, a trend in this direction was observed in the preoptic area. Specific binding levels in which the lower limit of the 99% confidence interval was greater than zero were detected in choroid plexus, medial habenula, lateral mesencephalic nucleus, hippocampus, parahippocampal area, preoptic area and 4 hypothalamic sites: paraventricular nucleus, ventromedial nucleus, suprachiasmatic nucleus, and tuberal region. Autoradiographic analysis of specific binding in cerebellum, ventromedial hypothalamus, and hippocampus/parahippocampus yielded relative differences that closely approximated those obtained in binding studies on tissue homogenates from these regions. These results suggest possible sites of prolactin action in altering behavioral state and neuroendocrine function in this species.

Virgin female hamsters were mated and tested once daily for maternal retrieving behavior beginning on days 0, 5, 9, 13, 15, of the 16 day gestation period to determine if responsiveness toward newborn pups changes as pregnancy proceeds. Upon initial exposure to 3 newborn pups, only a small percentage of early-to-mid-pregnant females exhibited maternal retrieving behavior spontaneously. In contrast, over half of the 15 day pregnant females displayed retrieving during the first test. Despite the high frequency of initial pup-directed aggression and cannibalism, maternal retrieval was induced in the majority of the females in all groups by repeated daily exposure to 3 newborn pups. However, no significant differences were observed in the number of pup exposure periods required to induce maternal retrieving in 0, 5, and 9 day pregnant females. It is concluded that the high level of maternal responsiveness observed in the parturient hamster develops somewhat abruptly during late pregnancy. In this respect, the pattern observed in the hamster differs from the more gradual increase in maternal responsiveness reported in mid-to-late-pregnant mice and rats.

The binding of intravenously administered prolactin to choroid plexus and brain tissue was determined radioautographically in the ring dove, a species that exhibits prolactin-induced alterations in brain function. An intense autoradiographic reaction was detected over the epithelial cells of the choroid plexus 5 min after the intravenous injection of 125I-ovine prolactin. A significant reaction was also observed over the infundibulum but no significant uptake of prolactin occurred in other brain areas. The binding of radiolabelled prolactin to infundibulum appeared to be non-specific, since excess unlabelled hormone did not reduce silver grain density. In contrast, 125I-ovine prolactin binding in choroid plexus was significantly reduced by excess unlabelled ovine prolactin or human growth hormone, but not by ovine luteinizing hormone. Specific binding to choroid plexus was also detected in vitro. The lack of significant brain uptake of prolactin in vivo is discussed in relation to recent in vitro evidence for specific binding sites for prolactin in several dove brain regions. Similarities between the binding results obtained in this avian species and those reported previously in mammals suggest that the two vertebrate groups exhibit similar patterns of prolactin interaction with neural target tissues.

Ingestive behavior and body weight were measured in male and female ring doves given twice daily subcutaneous injections of ovine prolactin (7 mg/kg/day) or vehicle and in male doves given daily intracerebroventricular (ICV) injections of ovine prolactin at doses ranging from 0.1 to 2.0 micrograms/day. Changes induced by ICV administration of turkey prolactin, turkey growth hormone, ovine growth hormone, human growth hormone, and vehicle were also examined. Subcutaneous injections of ovine prolactin markedly increased food intake and body weight in both sexes. Similar effects occurred in dose-related fashion in male doves given ICV injections of ovine prolactin. The three growth hormone preparations also increased feeding and body weight significantly, but turkey prolactin was ineffective in this regard. Changes in drinking generally paralleled feeding patterns but were less pronounced and may have been secondary to feeding changes. We conclude that feeding in this species is strongly stimulated by some prolactin and growth hormone preparations. However, the physiological mechanisms underlying these effects remain to be clarified.

Mating-induced inhibition of sexual receptivity was examined in ovariectomized, estrogen (E) treated and estrogen plus progesterone (E + P) treated hamsters given 10 min of exposure to male mounting stimulation alone or to mounts, intromissions, and ejaculations at eight hourly intervals. In E + P treated females, no differential effects of exposure to full mating stimulation vs. mounting stimulation alone were observed. In contrast, females given E treatment alone showed a marked differential response. Fully mated, E-treated females showed more lordosis than E-treated females exposed to mounts alone during the initial test. However, total lordosis duration declined precipitiously in the fully mated group by 2 hr and remained significantly below that in other groups during subsequent tests. Levels of receptivity in E-treated females mounted-only remained relatively constant until 8 hr. These results suggest that P reduces the inhibitory effects of vaginocervical cues received during mating without affecting the response to mounting stimulation alone. In addition, vaginocervical stimulation may initially facilitate lordosis in E-treated females.

Specific binding of 125I-labeled ovine prolactin (125I-oPRL) was detected in crude membrane fractions prepared from ring dove (Streptopelia risoria) liver homogenates. In characterization studies, specific binding was found to depend upon pH, incubation time, incubation temperature, and membrane protein concentration. Competitive inhibition of specifically bound 125I-oPRL was observed with human growth hormone, human and rat prolactin, and dove pituitary extract but not with turkey prolactin, human placental lactogen, and several nonlactogenic hormone preparations. Dove liver membranes showed high affinity (Kd = 3 X 10(-10) M) for binding to oPRL but had relatively low binding capacity (Bmax less than 20 fmol/mg protein). PRL binding activity in pooled liver fractions from breeding doves during early stages of incubation prior to crop sac growth did not differ markedly from that observed in doves sampled at the end of incubation when crop sac weight and serum PRL were elevated. However, binding activity was higher in pooled male liver fractions than in pooled female liver fractions at both reproductive stages. A two- to threefold increase in binding capacity was observed in pooled liver fractions from late-incubating doves following MgCl2-induced binding site desaturation. The MgCl2 treatment did not eliminate the differences in specific binding observed between male and female liver fraction pools, thus suggesting the possibility of sex-specific mechanisms of hepatic PRL binding site regulation in this species. It is concluded that the dove liver possesses specific binding sites for PRL with properties similar, but not identical, to those found in other vertebrate target tissues.

Testicular weights and concentrations of LH in plasma were measured in individually housed adult male ring doves given five daily intracerebroventricular injections of saline-NaHCO3 vehicle (2 microliter), ovine prolactin (0.1, 0.5, 1.0 or 2.0 micrograms/day), turkey prolactin (1.0 micrograms/day), turkey GH (1.0 micrograms/day) or ovine GH (1.0 micrograms/day). Administration of ovine prolactin resulted in a dose-dependent suppression of plasma LH concentration, with values in the two highest dose groups averaging three- to fivefold less than those of vehicle-injected controls. Reductions of similar magnitude were obtained following intracranial administration of turkey, ovine or human GH. Whilst effective in reducing plasma LH, turkey prolactin was less effective than an equivalent dose of ovine prolactin. Testicular regression was observed in all treatment groups which showed a significant decrease in plasma LH concentrations. Because crop sacs remained undeveloped in all treatment groups, it was concluded that these centrally administered hormones acted primarily at the level of the brain or pituitary to exert their suppressive effects. The possibility that prolactin and GH interact with different binding sites to inhibit LH secretion is discussed, together with evidence for a possible role of prolactin and GH in gonadotrophin regulation under normal physiological conditions.

Systemic administration of prolactin (PRL) reportedly maintains readiness to sit on eggs, increases body weights and liver weights, and reduces gonadal and reproductive tract tissue weights in male and female ring doves. To determine if these effects are mediated by PRL-induced changes in the central nervous system, ring doves were isolated from their mates, nests, and eggs on Day 4 of incubation and given twice-daily intracerebroventricular injections of PRL (1 microgram, 0.031 IU) or saline vehicle (2 microliter) during the next 10 days. Food and water consumption were monitored daily during treatment and tests for incubation behavior and observation of organ weight changes were conducted at the end of the period. The incidence of incubation behavior in the PRL-treated group was not significantly different from that in the control group and was significantly lower than that reported in a previous study following subcutaneous PRL injections. However, gonadal and reproductive tract tissue weights were markedly reduced and food and water consumption were significantly elevated in PRL-treated birds. Body weights and liver weights were also increased following PRL administration. In contrast, crop sac weight did not increase in the PRL treated birds, thereby indicating that the effects of icv prolactin treatment were not mediated by increased plasma PRL levels and peripheral target organ stimulation. These results suggest that PRL can act directly on the brain to inhibit gonadotropin release and to stimulate ingestive behavior but central effects of PRL alone are not sufficient to maintain incubation readiness.

Intracranial injections of prolactin (PRL) have been previously shown to elevate food and water intake in ring doves. In an attempt to further characterize these PRL-induced behavioral responses and the time course of PRL action, food and/or water intake were measured as frequent intervals in male doves given a single intracerebroventricular (ICV) injection of ovine PRL (44 pmoles) or vehicle under food deprivation, water deprivation, or nondeprivation conditions. PRL increased food consumption by 35-50% over baseline levels in water deprived and nondeprived doves, although response latencies (10 hr) and durations (greater than 24 hr) were considerably longer than those reported for other orexigenic peptides. Behavioral observations of nondeprived doves further revealed that PRL significantly increased total time spent feeding and average feeding bout duration. In contrast to this pattern, water intake remained unchanged in food deprived doves and was only marginally increased in nondeprived doves following PRL treatment. Collectively, these results suggest that PRL promotes a selective and long-lasting hyperphagia which may in turn augment drinking activity.

Radiolabeled ovine prolactin ([125I]oPRL) was found to bind specifically to crude membrane fractions prepared from ring dove brain homogenates. Specific binding changed with incubation time and temperature but did not fluctuate systematically with pH. Specific binding levels were highest in hypothalamus and forebrain and were lowest in cerebellum and brain stem. A linear increase in specific binding was observed in pooled midbrain and forebrain membrane fractions as membrane protein concentrations increased from 0.2 to 6.0 mg/ml. Scatchard analysis of binding saturation and competitive binding data revealed that dove midbrain/forebrain membranes bound oPRL with high affinity (Kd = 2.1-2.6 X 10(-10) M) but had low binding capacity (Bmax = 2.9-4.8 fmol/mg protein). Unlabeled oPRL and human growth hormone competed most effectively with radiolabeled oPRL for occupation of midbrain/forebrain binding sites. Turkey PRL and human placental lactogen were less effective as competitors but were 8-15 times more effective than ovine or turkey growth hormone, porcine insulin, or ovine luteinizing hormone. Subcutaneous injection of unlabeled oPRL lowered specific binding in liver and midbrain/forebrain by 76 and 39%, respectively, over that observed after saline injection. Prior MgCl2-induced desaturation of membrane binding sites greatly reduced or eliminated these treatment differences. These results suggest the existence of specific, saturable binding sites for PRL in dove brain which conceivably could mediate the reported effects of PRL on behavior and gonadal function in this species.