Differential relationships between D1 and D2 dopamine receptor expression in the medial preoptic nucleus and sexually-motivated song in male European starlings (Sturnus vulgaris).

Converging data in songbirds support a central role for the medial preoptic nucleus (POM) in motivational aspects of vocal production. Recent data suggest that dopamine in the POM plays a complex modulatory role in the production of sexually-motivated song and that an optimal level of dopamine D1 receptor stimulation is required to facilitate singing behavior. To further explore this possibility, we used quantitative real-time PCR to examine relationships between mRNA expression of D1 as well as D2 receptors in the POM (and also the lateral septum and Area X) and sexually-motivated singing behavior in male European starlings. Results showed that both males with the highest and lowest D1 expression in the POM sang significantly less than males with intermediate levels of expression. Furthermore, singing behavior rose linearly in association with increasing levels of D1 expression in POM but dropped abruptly, such that individuals with D1 expression values higher than the mean sang very little. Analysis of birds with low and intermediate levels of D1 expression in POM revealed strong positive correlations between D1 expression and song but negative relationships between D2 receptor expression and song. These findings support prior work suggesting an optimal level of POM D1 receptor stimulation best facilitates sexually-motivated singing behavior. Results also suggest that D2 receptors may work in opposition to D1 receptors in POM to modify vocal production.

Sexually-motivated song is predicted by androgen-and opioid-related gene expression in the medial preoptic nucleus of male European starlings (Sturnus vulgaris).

Across vertebrates, communication conveys information about an individual's motivational state, yet little is known about the neuroendocrine regulation of motivational aspects of communication. For seasonally breeding songbirds, increases in testosterone in spring stimulate high rates of sexually-motivated courtship song, though not all birds sing at high rates. It is generally assumed that testosterone or its metabolites act within the medial preoptic nucleus (POM) to stimulate the motivation to sing. In addition to androgen receptors (ARs) and testosterone, opioid neuropeptides in the POM influence sexually-motivated song production, and it has been proposed that testosterone may in part regulate song by modifying opioid systems. To gain insight into a possible role for androgen-opioid interactions in the regulation of communication we examined associations between sexually-motivated song and relative expression of ARs, mu opioid receptors (muORs), and preproenkephalin (PENK) in the POM (and other regions) of male European starlings using qPCR. Both AR and PENK expression in POM correlated positively with singing behavior, whereas muOR in POM correlated negatively with song. Furthermore, the ratio of PENK/muOR expression correlated negatively with AR expression in POM. Finally, in the ventral tegmental area (VTA), PENK expression correlated negatively with singing behavior. Results support the hypothesis that ARs may alter opioid gene expression in POM to fine-tune singing to reflect a male's motivational state. Data also suggest that bidirectional relationships may exist between opioids and ARs in POM and song, and additionally support a role for opioids in the VTA, independent of AR activity in this region.

Status-appropriate singing behavior, testosterone and androgen receptor immunolabeling in male European starlings (Sturnus vulgaris).

Vocalizations convey information about an individual's motivational, internal, and social status. As circumstances change, individuals respond by adjusting vocal behavior accordingly. In European starlings, a male that acquires a nest site socially dominates other males and dramatically increases courtship song. Although circulating testosterone is associated with social status and vocal production it is possible that steroid receptors fine-tune status-appropriate changes in behavior. Here we explored a possible role for androgen receptors. Male starlings that acquired nest sites produced high rates of courtship song. For a subset of males this occurred even in the absence of elevated circulating testosterone. Immunolabeling for androgen receptors (ARir) was highest in the medial preoptic nucleus (POM) in males with both a nest site and elevated testosterone. For HVC, ARir was higher in dominant males with high testosterone (males that sang longer songs) than dominant males with low testosterone (males that sang shorter songs). ARir in the dorsal medial portion of the nucleus intercollicularis (DM) was elevated in males with high testosterone irrespective of dominance status. Song bout length related positively to ARir in POM, HVC and DM, and testosterone concentrations related positively to ARir in POM and DM. Results suggest that the role of testosterone in vocal behavior differs across brain regions and support the hypothesis that testosterone in POM underlies motivation, testosterone in HVC relates to song quality, and testosterone in DM stimulates vocalizations. Our data also suggest that singing may influence AR independent of testosterone and that alternative androgen-independent pathways regulate status-appropriate singing behavior.

Seasonal and individual variation in singing behavior correlates with α2-noradrenergic receptor density in brain regions implicated in song, sexual, and social behavior.

In seasonally breeding male songbirds, both the function of song and the stimuli that elicit singing behavior change seasonally. The catecholamine norepinephrine (NE) modulates attention and arousal across behavioral states, yet the role of NE in seasonally-appropriate vocal communication has not been well-studied. The present study explored the possibility that seasonal changes in alpha 2-noradrenergic receptors (α(2)-R) within song control regions and brain regions implicated in sexual arousal and social behavior contribute to seasonal changes in song behavior in male European starlings (Sturnus vulgaris). We quantified singing behavior in aviary housed males under spring breeding season conditions and fall conditions. α(2)-R were identified with the selective ligand [(3)H]RX821002 using autoradiographic methods. The densities of α(2)-R in song control regions (HVC and the robust nucleus of the arcopallium [RA]) and the lateral septum (LS) were lower in Spring Condition males. α(2)-R densities in the caudal portion of the medial preoptic nucleus (POM) related negatively to singing behavior. Testosterone concentrations were highest in Spring Condition males and correlated with α(2)-R in LS and POM. Results link persistent seasonal alterations in the structure or function of male song to seasonal changes in NE α(2)-Rs in HVC, RA, and LS. Individual differences in α(2)-R in the POM may in part explain individual differences in song production irrespective of the context in which a male is singing, perhaps through NE modification of male sexual arousal.

D1-like dopamine receptor density in nuclei involved in social behavior correlates with song in a context-dependent fashion in male European starlings.

Research in songbirds shows that singing behavior is regulated by both brain areas involved in vocal behavior as well as those involved in social behavior. Interestingly, the precise role of these regions in song can vary as a function of the social, environmental and breeding context. To date, little is known about the neurotransmitters underlying such context-dependent regulation of song. Dopamine (DA) modulates highly motivated, goal-directed behaviors (including sexually motivated song) and emerging data implicate DA in the context-dependent regulation of singing behavior. This study was performed to begin to examine whether differences in DA receptors may underlie, in part, context-dependent differences in song production. We used autoradiographic procedures to label D1-like and D2-like DA receptors to examine the relationship between DA receptor density and singing behavior in multiple contexts in male European starlings (Sturnus vulgaris). Within a breeding context (when testosterone (T) was high), D1-like receptor density in the medial preoptic nucleus (POM) and midbrain central gray (GCt) negatively correlated with song used to attract a female. Additionally in this context, D1-like receptor density in POM, GCt, medial bed nucleus of the stria terminalis (BSTm), and lateral septum (LS) negatively correlated with song likely used to defend a nest box. In contrast, in a non-breeding context (when T was low), D1-like receptor density in POM and LS positively correlated with song used to maintain social flocks. No relationships were identified between song in any context and D2-like receptor densities. Differences in the brain regions and directional relationships between D1-like receptor binding and song suggest that dopaminergic systems play a region and context-specific role in song. These data also suggest that individual variation in singing behavior may, in part, be explained by individual differences in D1-like receptor density in brain regions implicated in social behavior.

Seasonal variation in androgen-metabolizing enzymes in the diencephalon and telencephalon of the male European starling (Sturnus vulgaris).

In seasonally breeding songbirds, seasonal fluctuations occur in serum testosterone (T) concentrations and reproductive behaviours. Many T-dependent behaviours are regulated by the activity of androgenic and oestrogenic metabolites within specific brain regions. Male European starlings breed in spring when circulating T concentrations peak. T and its metabolites act within portions of the diencephalon to regulate the pituitary-gonadal axis and to activate courtship and copulation. Song in male starlings is critical for mate attraction during the breeding season and is regulated by steroid-sensitive nuclei in the telencephalon and diencephalon. Outside the breeding season, T is undetectable, however, males continue to sing at high levels. This suggests that singing outside of the breeding season might not be T-dependent as it appears to be in the spring. Alternatively, singing when T is low might continue to be regulated by T due to increased sensitivity of the brain to the action of the steroid. This increased sensitivity could be mediated by changes in intracellular T metabolism leading to increased production of active or decreased production of inactive metabolites. To explore the relationship between T-metabolism and reproductive behaviour, we analysed seasonal changes in the activity of four brain T-metabolizing enzymes: aromatase, 17beta-hydroxysteroid dehydrogenase (17beta-HSDH), 5alpha-reductase (all three convert T into active metabolites) and 5beta-reductase (converts T into an inactive metabolite) in the diencephalon and telencephalon. In the anterior and posterior diencephalon, the highest aromatase was observed in spring when this region is critical for courtship and copulation. In the telencephalon, aromatase was highest and 5beta-reductase was lowest throughout the winter months well prior to the reproductive season and these enzymes presumably maximize T-activity within this region. Although these data do not indicate whether the metabolic changes occur specifically within song nuclei, these findings are compatible with the idea that singing in male starlings outside the breeding season may be regulated by steroids despite the presence of low serum T concentrations. Overall, seasonal changes in T-metabolizing enzymes appear to play a significant role in seasonal changes in behaviour and reproductive physiology.

Preoptic aromatase cells project to the mesencephalic central gray in the male Japanese quail (Coturnix japonica).

Previous tract-tracing studies demonstrated the existence of projections from the medial preoptic nucleus (POM) to the mesencephalic central gray (GCt) in quail. GCt contains a significant number of aromatase-immunoreactive (ARO-ir) fibers and punctate structures, but no ARO-ir cells are present in this region. The origin of the ARO-ir fibers of the GCt was investigated here by retrograde tract-tracing combined with immunocytochemistry for aromatase. Following injection of fluorescent microspheres in GCt, retrogradely labeled cells were found in a large number of hypothalamic and mesencephalic areas and in particular within the three main groups of ARO-ir cells located in the POM, the ventromedial nucleus of the hypothalamus, and the bed nucleus striae terminalis. Labeling of these cells for aromatase by immunocytochemistry demonstrated, however, that aromatase-positive retrogradely labeled cells are observed almost exclusively within the POM. Double-labeled cells were abundant in both the rostral and caudal parts of the POM and their number was apparently not affected by the location of the injection site within GCt. At both rostro-caudal levels of the POM, ARO-ir retrogradely labeled cells were, however, more frequent in the lateral than in the medial POM. These data indicate that ARO-ir neurons located in the lateral part of the POM may control the premotor aspects of male copulatory behavior through their projection to GCt and suggest that GCt activity could be affected by estrogens released from the terminals of these ARO-ir neurons.

Seasonal changes in courtship song and the medial preoptic area in male European starlings (Sturnus vulgaris).

In male starlings (Sturnus vulgaris) courtship song plays a critical role in mate attraction. During the breeding season courtship song occurs prior to copulation and appears to reflect male sexual arousal. Outside the breeding season starlings sing, but song appears unrelated to reproduction. The aromatization of testosterone (T), likely within the medial preoptic nucleus (POM), is critical for the expression of male sexual arousal. The present study was performed to determine whether seasonal changes in the POM might relate to seasonal changes in courtship singing behavior in male starlings. T concentrations, the volume of the POM, and aromatase within the POM were examined both during and outside of the breeding season in male starlings. Song was also recorded at these times both with and without a female present. The POM was largest and contained dense aromatase immunostaining only during the spring breeding season, when T concentrations were highest and males responded to a female with an increase in courtship song. Outside the breeding season the volume of the POM was small, T concentrations were low, and males displayed no changes in song expression in response to female conspecifics. Song bout length was positively related to POM volume, and males sang longer songs in spring. Only males with nestboxes in spring responded to a female, and the POM tended to be larger in these males, suggesting that nestbox possession might influence neuroplasticity within the POM. Overall, the findings suggest that T-dependent plasticity and aromatase activity within the POM might regulate courtship singing in a wild songbird.

Lesions to the medial preoptic area affect singing in the male European starling (Sturnus vulgaris).

The aromatization of testosterone (T) in the medial preoptic nucleus (POM) is known to regulate male courtship and sexual behaviors expressed prior to, and in anticipation of, copulation. Singing in male European starlings is used to attract mates prior to physical sexual contact, suggesting that the POM might be involved. The present study was performed to examine the effects of lesions targeting the POM on singing and courtship behavior in reproductively active male starlings. A significant decrease in song output and the gathering of green nest materials was observed in males with lesions to the POM compared to males with damage to brain areas outside of the POM. Lesions did not affect a male's tendency to remain near a female or to occupy a nestbox, suggesting that the effects of POM lesions were specific to courtship behaviors. Behavioral differences were not related to testis mass or volume, and GnRH immunoreactivity was observed within the hypothalamus and median eminence for each male, suggesting that the effects of POM lesions were related specifically to POM involvement in song expression rather than to a disruption of the GnRH axis. These results suggest a general role for the POM in the expression of behaviors related to sexual arousal or anticipation, including song.

Neurochemical evidence for at least two regional subdivisions within the homing pigeon (Columba livia) caudolateral neostriatum.

The distributions of one neurotransmitter, two neurotransmitter-related substances, and five neuropeptides were examined within the homing pigeon caudolateral neostriatum (NCL). All eight neuroactive substances were found within a tyrosine hydroxylase (TH)-dense region that defines the NCL. Overall regional variation in the relative density of these substances suggested at least two neurochemically distinct portions of NCL. Dorsal NCL contained relatively dense staining for TH, choline acetyltransferase, and substance P, whereas vasoactive intestinal polypeptide was more abundant in ventral portions of NCL. Serotonin and cholecystokinin were found to be densest in intermediate portions of NCL. Somatostatin and leucine-enkephalin were homogeneously distributed throughout NCL. The results suggest that NCL may consist of multiple subdivisions. Investigations into the behavioral importance of these regions are necessary to clarify the role of this brain region in avian behavior.

Effects of naloxone on the acquisition and expression of appetitive and consummatory sexual behavior in male Japanese quail.

Previous studies in Japanese quail indicate that central administration of the opioid antagonist naloxone enhances consummatory sexual behavior (CSB). This effect could be related either to a decrease in sexual satiety or to previously documented stimulatory effects of naloxone on GnRH. The present studies were performed to investigate these two possibilities and to explore for the first time opioid involvement in the expression and acquisition of appetitive aspects of sexual behavior (ASB) in castrated, testosterone-treated Japanese quail. Although no effects on either ASB or CSB were observed in response to peripheral naloxone injections, a significant increase in CSB was observed in males receiving central injections of naloxone. Central injections of naloxone had no effect on the acquisition of a social proximity response used to measure ASB. However, compared to controls a greater number of naloxone-treated birds copulated in the test arena on the first day. Overall, these results indicate an inhibitory role for opioids in CSB, and suggest that opioids are differentially involved in different aspects of sexual behavior.

The effects of lesions to the caudolateral neostriatum on sun compass based spatial learning in homing pigeons.

To better define the role of the avian caudolateral neostriatum (NCL) in spatial behavior, we used homing pigeons to explore the effects of NCL lesions on a sun compass based spatial learning task. Although NCL lesioned birds learned the task, they required more sessions to reach criterion than controls. NCL lesioned pigeons were also able to acquire a color discrimination task that was procedurally similar to the sun compass spatial learning task, but they made more errors than controls. Both the deficits observed in sun compass based spatial learning and color discrimination were correlated with the volume of lesion damage to dorsal rather than ventral portions of NCL. Overall, these findings suggest that the role of NCL in homing pigeon navigation from distant unfamiliar locations is not related to a bird's ability to learn stimulus-direction associations using a sun compass. However NCL does appear involved in a pigeon's ability to perform at least some behaviors common to both the color discrimination and the sun compass based spatial learning tasks.

Effects of brain testosterone implants on appetitive and consummatory components of male sexual behavior in Japanese quail.

Aromatization of testosterone (T) into an estrogen is necessary for the activation of consummatory and appetitive sexual behavior in male Japanese quail. T action within the medial preoptic nucleus (POM) is necessary and sufficient to activate consummatory behavior, and some evidence suggests that POM might be involved in the control of appetitive behavior, but other brain regions, such as the bed nucleus of the stria terminalis (BST), an area that contains a dense population of aromatase-immunoreactive neurons, are also likely to be involved. This study was performed to assess the effects of stereotaxic T implants targeting either the POM or the BST on the activation of both components of sexual behavior in castrated male quail. Appetitive sexual behavior was measured by an acquired social proximity response in which a male will approach a window providing visual access to a female after the window has been repeatedly paired with physical access to a female and the possibility to freely interact with her. Rhythmic cloacal sphincter movements that are produced by the male when given visual access to a female were used as another measure of appetitive sexual behavior that does not appear to depend on sexual learning. The experiments confirmed that copulation is necessary for males to develop the social proximity response that is used to measure the appetitive sexual behavior. T implants in the POM activated both components of sexual behavior, suggesting that these components cannot be completely dissociated. In contrast, T implants located within the BST did not affect either component, but because implants in the BST did not activate copulatory behavior, these results do not preclude a role for BST in the expression of a previously acquired appetitive sexual behavior.

Paired-associate learning is unaffected by combined hippocampal and parahippocampal lesions in homing pigeons.

To examine whether the avian hippocampus-parahippocampus (HF) is necessary for nonspatial, paired-associate learning, as has been suggested for rodents, HF-lesioned and control homing pigeons were tested on a visual paired-associate learning task. Both groups learned equally well to discriminate trials that consisted of a stimulus preceded by its paired associate from trials that consisted of a stimulus preceded by stimuli from other paired associates (mispair trials), even when a mispair was experienced for the first time. The groups also learned equally well not to respond to 2 stimuli that were never rewarded. The results demonstrate that HF lesions do not impair nonspatial paired-associate learning in birds, suggesting that the role of HF in nonspatial cognition differs between birds and mammals.

The muscarinic acetylcholine antagonist scopolamine impairs short-distance homing pigeon navigation.

The present study employed intramuscular (i.m.) injections of the acetylcholine (ACh) receptor antagonist scopolamine hydrobromide (0.10 mg/kg) to investigate the possible involvement of ACh in naturally occurring spatial navigation in homing pigeons (Columba livia). Control pigeons receiving injections of saline or scopolamine methylbromide, an ACh antagonist that does not cross the blood-brain barrier, were oriented in a homeward direction when released from a location 8 km from home. In contrast, pigeons injected with scopolamine hydrobromide (0.10 mg/kg, i.m.) were less well oriented and took more time to return home from the same location. These results suggest that homing pigeon navigation is regulated, in part, by central cholinergic mechanisms.

Telencephalic afferents to the caudolateral neostriatum of the pigeon.

The pigeon caudolateral neostriatum (NCL) shares a dopaminergic innervation with mammalian frontal cortical areas and is implicated in the regulation of avian cognitive behavior. Retrograde tracing methods were used to identify forebrain projections to NCL and to suggest a possible role of this area in mediating spatial behavior. NCL receives telencephalic projections from the hyperstriatum accessorium, cells along the border of hyperstriatum dorsale and hyperstriatum ventrale, anterolateral hyperstriatum adjacent to the vallecula, confined cell groups within the anterior neostriatum, and subdivisions of the archistriatum. In addition, labeling of a small number of large cells near the fasciculus prosencephali lateralis was observed at the level of the anterior commissure. In accordance with previous studies, projections of subtelencephalic areas were revealed to originate from the thalamic posterior dorsolateral nucleus and nucleus subrotundus, as well as from the tegmental nucleus pedunculopontinus and locus coeruleus. Forebrain connections of NCL show that somatosensory, visual, and olfactory information can combine in this division of the neostriatum. NCL is therefore suited to participate in a neural circuit that regulates spatial behavior. Moreover, the present study reveals that NCL is reached by a limbic projection from the nucleus taeniae. This projection also suggests similarity between NCL and mammalian frontal cortical areas.

The NMDA-receptor antagonist MK-801 impairs navigational learning in homing pigeons.

The present study employed the N-methyl-D-aspartate (NMDA) receptor antagonist MK-801 to investigate the possible importance of NMDA receptor activation for naturally occurring spatial learning in birds by exploiting the navigational ability of homing pigeons (Columba livia). Control pigeons released from two unfamiliar release sites displayed vanishing bearings that were poorly oriented. However, when released a second time from the same sites they displayed improved homeward orientation. The control birds apparently learned something about the spatial relationships of stimuli at the release sites on the first releases and used that information to orient better when released a second time from the same locations. Experimental pigeons given the NMDA receptor antagonist MK-801 (0.10 mg/kg) initially behaved as controls, orienting poorly when released for the first time from the two sites. In contrast to controls, the experimental birds failed to show significant improvement in orientation when released again from the same sites without MK-801. A second experiment revealed no state-dependent learning. Results of a position/color discrimination task showed that the impairments observed did not generalize to associative learning in an operant chamber, and together with field observations were not a result of sensory or motor drug effects. The data indicate that blocking NMDA receptors can disrupt navigational learning in homing pigeons. As such, the results are consistent with the hypothesis that NMDA receptor activation plays an important role in spatial learning in birds.