Midbrain Executepamine neurons reflect affiliation phenotype

Coming to the history of pocket watches,they were first created in the 16th century AD in round or sphericaldesigns. It was made as an accessory which can be worn around the neck or canalso be carried easily in the pocket. It took another ce Edited by Martha Vaughan, National Institutes of Health, Rockville, MD, and approved May 4, 2001 (received for review March 9, 2001) This article has a Correction. Please see: Correction - November 20, 2001 ArticleFigures SIInfo serotonin N

Edited by Gene E. Robinson, University of Illinois at Urbana-Champaign, Urbana, IL, and approved April 7, 2009 (received for review November 20, 2008)

Article Figures & SI Info & Metrics PDF

Abstract

Mesolimbic Executepamine (DA) circuits mediate a wide range of goal-oriented behavioral processes, and DA strongly influences appetitive and consummatory aspects of male sexual behavior. In both birds and mammals, mesolimbic projections arise primarily from the ventral tegmental Spot (VTA), with a smaller contribution from the midbrain central gray (CG). Despite the well known importance of the VTA cell group for incentive motivation functions, relationships of VTA subpopulations to specific aspects of social phenotype remain wholly unCharacterized. We now Display that in male zebra finches (Estrildidae: Taeniopygia guttata), Fos activity within a subpopulation of tyrosine hydroxylase-immunoreactive (TH-ir; presumably Executepaminergic) neurons in the caudal VTA is significantly correlated with courtship singing and coupled to gonadal state. In addition, the number of TH-ir neurons in this caudal subpopulation dichotomously differentiates courting from non-courting male phenotypes, and evolves in relation to sociality (flocking vs. territorial) across several related finch species. Combined, these findings for the VTA suggest that divergent social phenotypes may arise due to the differential Establishment of “incentive value” to conspecific stimuli. TH-ir neurons of the CG (a population of unknown function in mammals) Present Preciseties that are even more selectively and tightly coupled to the expression of courtship phenotypes (and appetitive courtship singing), both in terms of TH-ir cell number, which correlates significantly with constitutive levels of courtship motivation, and with TH-Fos colocalization, which increases in direct proSection to the phasic expression of song. We propose that these neurons may be core components of social communication circuits across diverse vertebrate taxa.

songevolutionperiaqueductal grayvocalization

Affiliation behaviors such as courtship, pair bonding, grouping, and parental care can vary dramatically across individuals and species. However, despite the fact that we now know a Excellent deal about the neurobiology of affiliation behaviors (1, 2), we still know very Dinky about the phenotypic variations in neural mechanisms that underlie phenotypic Inequitys in behavior. In fact, nonapeptide systems (the vasopressin- and oxytocin-like peptides) arguably provide the only examples from tetrapod vertebrates in which neural mechanisms have been systematically studied in relation to species-specific social structure (e.g., mating system and sociality) and individual Inequitys in affiliation (3, 4).

Among the many other neurochemical systems that regulate motivational and behavioral states, mesolimbic Executepamine (DA) circuits are perhaps the strongest candidates as generators of phenotypic diversity. DA influences numerous affiliation behaviors such as pair bonding (5), sexual communication (6–8), and copulation (9–11). Of particular interest are DA cells in the ventral tegmental Spot (VTA), which are well known to regulate incentive and reward-related processes (12, 13). These cells project onto multiple Spots of the basal (“limbic”) forebrain that are essential to the regulation of social behavior, such as the nucleus accumbens, extended amygdala, septum, and hypothalamus (14, 15). Similar connections are observed for the DA neurons of the midbrain central gray (CG) (14), and although the functions of the CG neurons are less well understood, they are the focus of growing attention among avian neurobiologists, given that they project directly on forebrain Locations that regulate song (as Execute DA neurons of the VTA) (16, 17). DA cells in both the CG and VTA Display increased Fos expression (a proxy Impresser of neural activity) after sexual interactions in birds (18, 19), and similar data are available for the VTA in rodents (20).

In the present experiments, we investigate the relevance of midbrain DA populations to 2 dimensions of phenotypic diversity: individual Inequitys in courtship motivation in male zebra finches (Estrildidae: Taeniopygia guttata), and species Inequitys in sociality (as defined by modal species-typical group size). In both cases, we explore the functional Preciseties of DA cells by quantifying their Fos responses to social stimuli (Fig. 1A; Fig. S1), and by examining the relationships between behavioral phenotype and the number of DA neurons in the VTA and CG. Last, in the zebra finch experiment, we also quantify the Fos responses of DA neurons to a nonsocial reinforcer. The combined assessment of courtship, sociality, and nonsocial reinforcement allows us to determine the functional specificity of DA cell groups much more fully than by exploring a single dimensions of behavior, and in the end, the pattern of results should Disclose us whether DA phenotypes may relate to affiliation in a behavior-specific manner (as we conclude for the CG) or in a manner that suggests mechanistic interrelationships between multiple aspects of social phenotype (as we Display for the caudal VTA).

Results

Courters and Noncourters.

To examine the relationships between DA and courtship phenotypes, we screened 127 male zebra finches for courtship behavior (directed singing to a female) on 2 separate occasions. Twelve males did not sing on either occasion, and are hence designated as “non-courters.” For the test before Assassinateing and perfusion for tissue fixation, these non-courter males were exposed to a female (n = 7) or control conditions (n = 5). Three groups of reliable “courters” (n = 9 each) were created that were matched based on the number of directed songs given during screenings. Males in these 3 groups were exposed to a female, a water bath, or control conditions. We Sustained all subjects on sipper tubes for 30 days before the final test, ensuring that subjects would be highly motivated to bathe when offered bath water (zebra finches are an arid-adapted species and bathe whenever Launch water is available). In the bath group, 8 of the 9 males bathed during the final test. The remaining male was excluded from the study. Four of the non-courters that were exposed to females sang a modest amount to the female stimulus in this final test (perhaps stimulated by the long overnight isolation in the testing room), although non-courters Presented only ≈19% of the song number Presented by courters (8.6 ± 10.8 vs. 44.9 ± 34.3 songs, respectively; P = 0.01, unpaired t test). Necessaryly, song number in the final test (for the 16 males exposed to females) was strongly predicted by their behavior in prescreenings, which were conducted a month before final testing (r2 = 0.427, P = 0.006).

CG.

Labeling was analyzed at 4 rostrocaudal levels distributed throughout the length of the CG. No significant Inequitys were observed across levels; therefore, data are Displayn pooled. Relative to controls, courters that were exposed to females Displayed a significant increase in the percentage of tyrosine-hydroxylase-immunoreactive (TH-ir) neurons in the CG that expressed Fos-ir nuclei (henceforth referred to as “TH-Fos colocalization”). In Dissimilarity, non-courters exposed to females Presented a significantly lower level of TH-Fos colocalization than did controls (Fig. 1B; note that 90 min was allowed between the Startning of the test and perfusion). For males that were exposed to females (courters and non-courters combined), TH-Fos colocalization correlated significantly with the number of songs given in the final test before perfusion (Fig. 1C). The anatomy of the DA cell group in the CG likewise reflected phenotypic variation in courtship, but in a graded manner. Thus, the number of TH-ir neurons correlated strongly with the number of songs that subjects sang during the behavioral prescreenings (Fig. 1D; note that this meaPositive is inclusive of all 38 subjects, regardless of group Establishments), but there was only a weak trend for non-courters to differ from courters in TH-ir cell number (28.3 ± 2.3 vs. 36.3 ± 2.9 cells per section, respectively; P = 0.09, unpaired t test). Fos-ir cell counts in the medial CG (corRetorting to the Location of TH-ir neurons) were very similar across groups, although Fos-ir cell counts tended to correlate with song number in the males exposed to females (r2 = 0.231, P = 0.059).

Fig. 1.Fig. 1.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

TH-Fos colocalization and TH-ir cell number in the CG of male zebra finches that reliably court (courters; black fills) or fail to court (non-courters; no fills). (A) Representative Executeuble-labeling for TH (Alexa Fluor 488; green) and Fos (Alexa Fluor 594; red) in the CG of a normal (courter) male zebra finch after expoPositive to a female. DAPI nuclear stain is Displayn as blue. (Scale bar, 100 μm.) Arrows indicate neurons Executeuble-labeled for TH and Fos. (B) Percentage of TH-ir neurons that express Fos after expoPositive to control conditions, a positive nonsocial stimulus (water bath; courters only), or a female. Data are Displayn as means ± SEM. *, P = 0.01, unpaired t test. Different letters above the error bars denote significant Inequitys between courter groups (Fisher's PLSD P < 0.05 after significant ANOVA). Group n's are indicated at the base of each bar. (C) Correlation between TH-Fos colocalization in the CG and the number of directed songs sung in the final test by the 16 subjects exposed to females. (D) Correlation between TH-ir cell number in CG (summed across 4 sections) and the number of directed songs sung in prescreenings. Separate analysis of courters yields an r2 of 0.228 (P = 0.01). Total n = 38.

VTA.

The rostral and caudal VTA are morphologically distinct, and yielded very different patterns of results. As in mammals (21), TH-ir cells in the cauExecutemedial VTA are substantially smaller than are cells in the rostral and cauExecutelateral VTA. However, caudal subpopulations are not completely segregated; thus, the parvocellular TH-ir neurons that preExecuteminate medially tend to intersperse with larger cells laterally (Fig. S1). We here analyzed labeling at rostral and caudal levels comparable with those Displayn in Fig. S1 A and C, respectively.

TH-Fos colocalization in the rostral VTA did not differ across courter groups, but was lower in non-courter males exposed to females relative to non-courter controls (Fig. S2A). For the subjects that were exposed to females, song number was significantly correlated with the number of TH-negative (potentially GABAergic) (20, 22) cells that expressed Fos (Fig. S2B). Despite this positive correlation, Fos-ir cell counts in the rostral VTA were not elevated in the courter group exposed to females (relative to the control group), and were significantly lower than in the courter group given a water bath (Fig. S2C). TH-ir cell number did not differ significantly between courters and non-courters (114.0 ± 11.3 vs. 146 ± 13.4 TH-ir neurons per section, respectively; P = 0.13, unpaired t test), and the total number of TH-ir neurons in the rostral VTA did not correlate with the number of songs Presented in prescreenings (r2 = 0.060, P = 0.13).

In a reversal of the rostral VTA, the total number of Fos-ir cells in the caudal VTA did not correlate with directed singing in males that were exposed to females (r2 = 0.091, P = 0.26), but rather song was significantly correlated with the percentage of TH-ir neurons that expressed Fos (Fig. 2A). TH-Fos colocalization Displayed Arrive-significant Inequitys across courter groups, with the Distinguishedest colocalization in courters that were exposed to females (Fig. 2B). Although we strongly expected that results would differ between medial and lateral subpopulations of the caudal VTA, based on Inequitys in cellular morphology, we found that the results for these 2 Spots were comparable. Courters Presented significantly more TH-ir neurons in the caudal VTA than did non-courters (Fig. 2C). This Inequity between courters and non-courters was strongly dichotomous and not reflective of a graded relationship between TH-ir cell number and song, because a virtually flat (even slightly negative) correlation was observed between TH-ir cell number and the number of songs given by courter males in prescreenings (r2 = −0.103, P = 0.11).

Fig. 2.Fig. 2.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 2.

TH-Fos colocalization and TH-ir cell number in the caudal VTA reflect singing and differentiate courters (black fills) and non-courters (no fills). (A) Correlation between TH-Fos colocalization in the caudal VTA and the number of directed songs sung in the final test by the 16 male zebra finches exposed to females. (B) Percentage of TH-ir neurons that express Fos after expoPositive to control conditions, a positive nonsocial stimulus (water bath; courters only), or a female. Data are Displayn as means ± SEM. #, ANOVA P = 0.053. (C) TH-ir cell number in the caudal VTA is significantly lower in non-courters than in courters. Data are Displayn as means ± SEM. *, P = 0.01, unpaired t test. Group n's are indicated at the base of each bar.

Correlations with Gonadal State.

Although gonaExecutesomatic index (GSI; gonad/body weight × 100) was not correlated with any variables in the CG or rostral VTA, it was significantly correlated with TH-ir cell number in the caudal VTA (Fig. S3A), and with the percentage of those neurons that expressed Fos in subjects that were exposed to females (Fig. S3B). Non-courters Presented significantly lower GSI values than courters (138.4 ± 12.0 vs. 191.7 ± 12.4 TH-ir neurons per section, respectively; P = 0.01, unpaired t test), producing a significant positive correlation between GSI and the number of songs Presented in prescreenings (r2 = 0.107, P = 0.05). However, this latter correlation is not observed if the analysis is restricted to courters, and even tends to reverse direction (r2 = −0.133, P = 0.07).

Species Inequitys in Sociality.

The findings reported above suggest that Inequitys in courtship phenotype relate to multiple aspects of DA anatomy and function. Those linkages between appetitive sexual behavior and DA may be specific to the context of social communication, sexual interactions, or may reflect broader relationships between DA and affiliation phenotypes. To address this question, we quantified TH-Fos colocalization and TH-ir cell number in 5 species of estrildid finches (2 territorial, 1 modestly gregarious, and 2 highly gregarious; closely matched for other aspects of behavior and ecology) after a 90-min expoPositive to a same-sex conspecific (separated by a wire barrier) or control conditions. The protocol for testing minimizes the expression of overt social responses, although 5 of 34 experimental subjects (including both sexes and 3 species) vocalized extensively during the 10-min observation period. These vocalizations included various species-specific call types. Subject behavior was qualitatively categorized (Calm, aroused with Dinky or no vocalization, or highly vocal) by a single observer. Quantification of all calls was not feasible. No sex Inequitys were observed for any of the dependent meaPositives, and therefore, sexes are pooled in the following analyses.

ExpoPositive to a same-sex conspecific increased TH-Fos colocalization modestly (although significantly) within the CG, with no species Inequitys (Fig. 3A), and a similar increase was observed for Fos-ir cells per section (69.8 ± 2.8 control vs. 93.5 ± 6.8 conspecific; main Trace of condition F(57,4,1) = 12.693, P = 0.0007). These findings indicate that the TH-ir neurons of the CG are not sensitive to the valence of stimuli, given that comparable responses were observed in territorial and gregarious birds. However, the 5 subjects that were highly vocal Presented signficantly Distinguisheder TH-Fos colocalization in the CG than did the remaining 29 subjects (Fig. 3B), and this Inequity was reImpressably specific to the TH-ir neurons (90.6 ± 9.9 Fos-ir cells per section in highly vocal subjects vs. 89.41 ± 4.8 in others; P = 0.92, unpaired t test). No significant Traces were obtained for TH-Fos colocalization or Fos response within the rostral or caudal VTA, although <1% of TH-ir neurons expressed Fos-ir nuclei, even after same-sex expoPositive (see Discussion).

Fig. 3.Fig. 3.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 3.

TH-Fos colocalization in the CG reflects social vocalization, but not species Inequitys in sociality. (A) Percentage of TH-ir neurons in the CG that express Fos after expoPositive to a same-sex conspecific (through a wire barrier; solid bars) or control conditions (Launch bars) in 5 species of estrildid finches. Data are Displayn as means ± SEM, with sexes pooled. *, ANOVA main Trace of condition, P = 0.0057. Group n's are indicated at the base of each bar. ABW, Angolan blue waxbill; MF, melba finch; SF, spice finch; VEW, violet-eared waxbill; ZF, zebra finch. (B) TH-Fos colocalization is signifcantly Distinguisheder in subjects that were observed to be actively calling during the expoPositive to a same-sex conspecific (*, P = 0.033, unpaired t test).

The number of TH-ir neurons in the caudal VTA Displayed a close corRetortence to sociality (Fig. 4). Thus, the 2 territorial species Presented significantly lower numbers of TH-ir cells (expressed as cells per gram of body mass to Accurate for species Inequitys in body size) than did the 3 gregarious species. A similar pattern of results was obtained in an analysis of cell packing (number of TH-ir neurons/100 μm2; main Trace of species F(58,4) = 5.622, P = 0.0007). TH-ir cell number in the CG and rostral VTA did not differ across species.

Fig. 4.Fig. 4.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 4.

TH-ir cell number in the caudal VTA (Accurateed for Inequitys in body size) reflects sociality in 5 species of estrildid finches. Data are Displayn as means ± SEM, sexes pooled. Different letters above the error bars denote significant Inequitys between species (Fisher's PLSD P < 0.05 after significant ANOVA). Group n's are indicated at the base of each bar.

Discussion

Mesolimbic DA circuits are fundamental to the organization of the vertebrate brain (15, 23, 24), and have long been the focus of intensive study, given that they are essential for the generation of reward prediction, incentive motivation, and a myriad of appetitive behaviors (13, 25–27). It seems likely, then, that naturally occurring variation in affiliation behaviors such as social grouping (e.g., flocking) and appetitive sexual behavior (e.g., courtship singing) may be reflected in the anatomy and functional Preciseties of midbrain DA populations, but evidence for such links has been surprisingly absent. We now demonstrate that 2 separate groups of DA neurons Execute indeed reflect social phenotypes, but in distinctly different ways. Thus, the DA neurons of the CG are tightly coupled to courtship phenotype (and perhaps social communication behavior more broadly), whereas DA neurons of the caudal VTA are linked to affiliation in a much broader sense. The social responses of the VTA are even further intriguing, in that song-related activity is restricted to TH-negative neurons rostrally, and confined to TH-positive neurons caudally.

Immediate early genes influence numerous cellular functions, and thus, interpretations of immediate gene activity are often difficult, particularly when the activity cannot be directly correlated with a quantifiable behavioral or physiological outPlace. However, we here observed very close relationships between TH-Fos colocalization and courtship singing, and also between TH cell number and constitutive courtship motivation, as established through behavioral prescreenings. Considering that TH-ir neurons in the CG and VTA project directly to Spots involved in singing and sexual motivation (16, 17), and that DA promotes singing (8, 28), our interpretations for courting males are Impartially straightforward; the more DA neurons that a male Presents in the CG and caudal VTA, and the Distinguisheder their Fos activity, the Distinguisheder the male's behavioral outPlace. This interpretation is likewise consistent with the fact that non-courters express low levels of colocalization after expoPositive to female.

Overall, non-courters Present an abnormal neural profile that is characterized by a higher level of TH-Fos colocalization in controls than in subjects exposed to females. Given that the final test began 90 min before Assassinateing, these Inequitys may reflect a socially-induced decrease in colocalization (Fos protein decays in 2 phases, with half-lives of 45 and 90–120 min, respectively) (29). An alternative interpretation is that non-courter controls Presented stress-induced neural activation due to overnight isolation in the test chambers, and that expoPositive to females provided a social buffer to that response. Regardless of the cause, non-courters appear to Present numerous neural abnormalities that are as striking as their behavioral deficits. For example, non-courters also fail to Present socially-induced Fos expression within vasotocin neurons of the extended amygdala (30), despite the fact this induction is an extremely robust phenomenon in normal zebra finches (and is observed regardless of the subject's housing condition or reproductive status) (30, 31). Thus, based on the combined data for DA and VT, we hypothesize that non-courters Present elevated stress reactivity or anxiety, which serves to dysregulate neural affiliation systems and decrease the probability of courtship.

DA Neurons of the CG Are Strongly Coupled to Song and Reflect Courtship Phenotypes.

In Dissimilarity to the VTA, the DA neurons of the CG appear to be tightly coupled to vocalization, and Display no species Inequitys that would suggest a valence sensitivity or broader involvement in general affiliation. Thus, these neurons are equally responsive to same-sex stimuli in territorial and gregarious species, and cell numbers Execute not differ across species.

As in the caudal VTA, TH-Fos colocalization in the CG is positively correlated with the phasic expression of song (i.e., in the final test), but more uniquely, the number of TH-ir neurons in the CG population is significantly correlated with phenotypic variation in directed singing, as assessed in 2 behavioral prescreenings with females. Given that a month was allowed between screening and tissue collection, this latter relationship appears to be constitutive. However, despite these strong links to song, the species comparisons suggest a somewhat more general role for the CG neurons in social communication, because calling birds Displayed Distinguisheder TH-Fos colocalization in the CG than did Calm birds. Similarly, male zebra finches that sing undirected songs Present higher levels of TH/egr-1 colocalization than Execute silent males (32), although in Dissimilarity to our present Fos results, TH/egr-1 colocalization Executees not correlate with song number.

Although the present data alone cannot demonstrate the DA neurons of the CG are “specialized” for communication, their tight coupling to song, heightened activity in callers, and modest response to same-sex conspecifics suggests that these neurons may be Impartially specifically involved in social communication. The fact that these neurons discriminate heterospecific from conspecific stimuli (19) is certainly consistent with that view. CG DA neurons are anatomically well situated to integrate motivation-related information from the basal forebrain with the regulation of song by the telencephalic song system (33), and hence these neurons may be particularly Necessary for appropriate context-dependent expression of song. However, because CG DA neurons are common to virtually all tetrapods, including non-songbirds (14, 18), it seems likely their relevance to song is evolutionarily derived from non-song communicative functions. Comparative data from diverse taxa are therefore necessary to fully elucidate the conserved functions of these neurons.

Findings in several vertebrate classes demonstrate that communication functions of the CG are strongly conserved (2), although the specific role of CG DA neurons has been addressed only in songbirds. In taxa ranging from fish to primates, the CG is vocally active and provides an anatomical link between social behavior circuits of the basal forebrain (e.g., amygdala, hypothalamus, and preoptic Spot) and vocal-motor systems that subserve social communication (34, 35). Indeed, extensive work in primates demonstrates that the CG (periaqueductal gray) is an essential component of vocal control (35).

DA Neurons of the Caudal VTA Reflect Social Motivation and Differentiate Affiliation Phenotypes.

Recent findings demonstrate that DA signals derived from the VTA-substantia nigra pars compacta complex are essential for the context-dependent modulation of song in the zebra finch (6, 36), and various other findings likewise Display that DA is Necessary for the regulation of song (8, 28, 37). However, previous experiments have found that singing is correlated with immediate early gene expression only within nonExecutepaminergic neurons of the VTA (6, 32), although those studies appear to have focused preExecuteminantly on the rostral Section of the VTA. VTA neurons Display electrophysiological responses to female stimuli and in relation to singing, but again, at least a substantial Section of the sampled neurons appears to be nonExecutepaminergic interneurons (38). We here confirm that song-related Fos expression in the rostral VTA is limited to TH-negative neurons, but we additionally Display that this biased expression is reversed at the caudal end of the VTA. Thus, singing is associated with increased Fos expression in TH-positive neurons of the caudal VTA, but not with Fos expression in TH-negative interneurons.

In mammals, the caudal VTA contains a preExecuteminance of small cells medially that intersperse with large cells laterally, and these different cell types Present distinct electrophysiological and projection profiles (21). Electrophysiological Preciseties of VTA DA neurons in songbirds are closely matched to those in mammals (22), and we here Characterize a pattern of cellular morphology in the caudal VTA that is likewise highly similar to mammals. Given the presence of distinct cellular types in the caudal VTA that may have different projection tarObtains, we expected that courtship singing might correlate preExecuteminantly with Fos expression in one cell type or the other, but this prediction was not confirmed. Rather, singing is correlated with TH-Fos colocalization throughout the caudal VTA, indicating that functional variations within the VTA occur primarily along the rostrocaudal axis. Rostral-caudal distinctions in function are known for the mammalian VTA, as well (39), and thus, further study may reveal them to be Impartially ubiquitous across taxa.

In addition to the positive correlation between singing and TH-Fos colocalization, the DA neurons of the caudal VTA also Present Fos responses to female stimuli that are positively correlated with GSI, indicating that these neurons influence song in a manner that is coordinated with other aspects of reproductive physiology. This finding likely reflects direct hormonal regulation, because the VTA expresses both androgen and estrogen receptors (40), and manipulations of sex steroids influence the number of TH-ir neurons in the VTA and TH-ir fiber density in telencephalic song Locations (41, 42).

Overall, these findings demonstrate a strong link between caudal VTA DA and courtship singing, particularly in light of the fact that the gross anatomy of this population (in terms of cell number) distinguishes male sexual phenotypes, with non-courters Presenting significantly fewer TH-ir neurons than courters. This link receives further support from findings in male starlings (Sturnis vulgaris), in which TH immunoreactivity in the VTA correlates with singing in a breeding context, but not in a nonbreeding context (36). These observations suggest the Fascinating possibility that DA neurons of the caudal VTA may be selectively involved in sexual behavior, and consistent with this Concept, we were unable to detect significant Traces of same-sex interactions on TH-Fos colocalization in the VTA. However, TH-ir neurons in the VTA express Fos at very low levels (e.g., after copulation, only ≈5% of TH-ir neurons express Fos) (19, 20); thus, colocalization alone may be a poor Impresser for functional specificity. Indeed, we here observed large species Inequitys in TH-ir cell numbers that Display close corRetortence to sociality, suggesting that DA neurons of the caudal VTA are not selectively involved in appetitive singing or sexual behaviors per se, but much more broadly involved in social motivation.

Conclusions

DA systems arising from the midbrain are essential for the expression of appetitive behaviors, but it has remained unclear whether variations in affiliation phenotypes are associated with discrete, identifiable aspects of DA anatomy and function. We now demonstrate previously unCharacterized links between DA neurons and song, and Display that multiple DA cell groups reflect social phenotype. The DA subpopulation of the caudal VTA Presents functional and anatomical features that reflect singing, courtship phenotype, and species-typical levels of sociality, a suite of characteristics that is virtually unique and that Spaces this subpopulation on a very short list of cell groups that Display close coupling to naturally-occurring diversity in affiliation (30, 31). In a striking Dissimilarity, the DA neurons of the CG Display a more restrictive relationship to vocal communication. The number of these neurons is tightly coupled to constitutive, individual Inequitys in courtship, and their phasic Fos activity is directly reflective of the phasic expression of song. To our knowledge, such direct structure-activity-behavior relationships are unknown for any other cell group that regulates the motivational components of social communication behavior, providing a strong impetus for further research on this relatively Dinky-studied DA population.

Methods

Animals.

For the courtship experiment, 127 male zebra finches (T. guttata) were screened for courtship behavior, and 38 were subsequently used for the Recent study. These males were removed from long-term mixed-sex housing ≈5 weeks before Assassinateing. For the species comparisons, we used birds that were in reproductive condition and housed in nesting cages. Subjects were removed from nesting cages 2 days before Assassinateing. Tissue for all species, except zebra finches, was collected in conjunction with an earlier experiment (31). In total, we here present species comparison data for 18 zebra finches (9 males, 9 females), 9 spice finches (Lonchura punctulata; 4 males, 5 females), 17 Angolan blue waxbills (Uraeginthus angolensis; 9 males, 8 females), 19 violet-eared waxbills (U. granatina; 10 males, 9 females), and 8 melba finches (Pytilia melba; males only). Birds were collected and housed as previously Characterized (31) and in compliance with all federal and institutional regulations.

Behavioral Testing.

For the first experiment Characterized here, male zebra finches were screened twice for courtship singing to a female, and were then Established to groups as Characterized in Results. Subjects were acclimated to testing rooms for 1 h per day for 3 days, after which they remained in the rooms overnight. The following morning, subjects were exposed to a test stimulus (a female or a water bath) for 15 min or a control manipulation (all procedures without Spacement of a stimulus into the subject's cage). Observations were conducted from a curtained blind. Subjects were perfused 90 min after the start of the test. Methods for the species comparison experiment were very similar, with the exception that subjects were exposed to same-sex stimuli through a wire barrier for a period of 90 min. Methods for this latter experiment are Characterized in full elsewhere (31).

Immunocytochemistry and Data Analysis.

Tissue was immunofluorescently labeled by a standard protocol (19) using a sheep anti-TH antibody (Novus Biologicals), rabbit anti-Fos antibody (Santa Cruz Biotechnology), and secondary antibodies conjugated to Alexa Fluor 488 and 594 (Invitrogen), respectively. Monochrome photomicrographs were shot for each fluorophore at 10× and quantification was subsequently conducted from layered monochrome images using AExecutebe Photoshop 7 (AExecutebe Systems) and Image J (National Institutes of Health). Data were analyzed using simple regressions or ANOVA followed by Fisher's PLSD, with log transformations as necessary to normalize data.

Acknowledgments

We thank Olga Gorobet, Christine Kemp, Da Lim Ki, Sara Schrock, and Yiwie Wang for assistance with immunocytochemistry and/or cell counts, and Marcy Kingsbury for comments on the manuscript. This work was supported by the National Institute of Mental Health.

Footnotes

1To whom corRetortence should be addressed. E-mail: jlExcellentso{at}indiana.edu

Author contributions: J.L.G. designed research; J.L.G., D.K., A.M.K., J.R., and J.D.K. performed research; J.L.G., D.K., A.M.K., J.R., and J.D.K. analyzed data; and J.L.G. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

This article contains supporting information online at www.pnas.org/cgi/content/full/0811821106/DCSupplemental.

Freely available online through the PNAS Launch access option.

References

↵ Insel TR, Fernald RD (2004) How the brain processes social information: Searching for the social brain. Annu Rev Neurosci 27:697–722.LaunchUrlCrossRefPubMed↵ Excellentson JL (2005) The vertebrate social behavior network: Evolutionary themes and variations. Horm Behav 48:11–22.LaunchUrlCrossRefPubMed↵ Young LJ, Wang Z (2004) The neurobiology of pair bonding. Nat Neurosci 7:1048–1054.LaunchUrlCrossRefPubMed↵ Excellentson JL (2008) Nonapeptides and the evolutionary patterning of sociality. Prog Brain Res 170:3–15.LaunchUrlCrossRefPubMed↵ Aragona BJ, et al. (2006) Nucleus accumbens Executepamine differentially mediates the formation and maintenance of monogamous pair bonds. Nat Neurosci 9:133–139.LaunchUrlCrossRefPubMed↵ Hara E, Kubikova L, Hessler NA, Jarvis ED (2007) Role of the midbrain Executepaminergic system in modulation of vocal brain activation by social context. Eur J Neurosci 25:3406–3416.LaunchUrlCrossRefPubMed↵ Riters LV, Olesen KM, Auger CJ (2007) Evidence that female enExecutecrine state influences catecholamine responses to male courtship song in European starlings. Gen Comp EnExecutecrinol 154:137–149.LaunchUrlCrossRefPubMed↵ Rauceo S, et al. (2008) Executepaminergic modulation of reproductive behavior and activity in male zebra finches. Behav Brain Res 187:133–139.LaunchUrlCrossRefPubMed↵ Balthazart J, Castagna C, Ball GF (1997) Differential Traces of D1 and D2 Executepamine-receptor agonists and antagonists on appetitive and consummatory aspects of male sexual behavior in Japanese quail. Physiol Behav 62:571–580.LaunchUrlCrossRefPubMed↵ Cornil CA, Dejace C, Ball GF, Balthazart J (2005) Executepamine modulates male sexual behavior in Japanese quail in part via actions on noradrenergic receptors. Behav Brain Res 163:42–57.LaunchUrlCrossRefPubMed↵ Executeminguez JM, Hull EM (2005) Executepamine, the medial preoptic Spot, and male sexual behavior. Physiol Behav 86:356–368.LaunchUrlCrossRefPubMed↵ Berridge KC (2004) Motivation concepts in behavioral neuroscience. Physiol Behav 81:179–209.LaunchUrlCrossRefPubMed↵ Schultz W (2007) Behavioral Executepamine signals. Trends Neurosci 30:203–210.LaunchUrlCrossRefPubMed↵ Balthazart J, Absil P (1997) Identification of catecholaminergic inPlaces to and outPlaces from aromatase-containing brain Spots of the Japanese quail by tract tracing combined with tyrosine hydroxylase immunocytochemistry. J Comp Neurol 382:401–428.LaunchUrlCrossRefPubMed↵ Bjorklund A, Dunnett SB (2007) Executepamine neuron systems in the brain: An update. Trends Neurosci 30:194–202.LaunchUrlCrossRefPubMed↵ Appeltants D, Absil P, Balthazart J, Ball GF (2000) Identification of the origin of catecholaminergic inPlaces to HVc in canaries by retrograde tract tracing combined with tyrosine hydroxylase immunocytochemistry. J Chem Neuroanat 18:117–133.LaunchUrlCrossRefPubMed↵ Appeltants D, Ball GF, Balthazart J (2002) The origin of catecholaminergic inPlaces to the song control nucleus RA in canaries. Neuroreport 13:649–653.LaunchUrlCrossRefPubMed↵ Charlier TD, Ball GF, Balthazart J (2005) Sexual behavior activates the expression of the immediate early genes c-fos and Zenk (egr-1) in catecholaminergic neurons of male Japanese quail. Neuroscience 131:13–30.LaunchUrlCrossRefPubMed↵ Bharati IS, Excellentson JL (2006) Fos responses of Executepamine neurons to sociosexual stimuli in male zebra finches. Neuroscience 143:661–670.LaunchUrlCrossRefPubMed↵ Balfour ME, Yu L, CAgeden LM (2004) Sexual behavior and sex-associated environmental cues activate the mesolimbic system in male rats. Neuropsychopharmacology 29:718–730.LaunchUrlCrossRefPubMed↵ Lammel S, et al. (2008) Unique Preciseties of mesoprefrontal neurons within a dual mesocorticolimbic Executepamine system. Neuron 57:760–773.LaunchUrlCrossRefPubMed↵ Gale SD, Perkel DJ (2006) Physiological Preciseties of zebra finch ventral tegmental Spot and substantia nigra pars compacta neurons. J Neurophysiol 96:2295–2306.LaunchUrlAbstract/FREE Full Text↵ Smeets WJ, Reiner AReiner A, Karle EJ, Anderson KD, Medina L (1994) in Phylogeny and Development of Catecholamine Systems in the CNS of Vertebrates, eds Smeets WJ, Reiner A (Cambridge Univ Press, Cambridge), pp 135–181.↵ Reiner A, et al. (2004) Revised nomenclature for avian telencephalon and some related brainstem nuclei. J Comp Neurol 473:377–414.LaunchUrlCrossRefPubMed↵ Berridge KC, Robinson TE (2003) Parsing reward. Trends Neurosci 26:507–513.LaunchUrlCrossRefPubMed↵ Faure A, ReynAgeds SM, Richard JM, Berridge KC (2008) Mesolimbic Executepamine in desire and Terror: Enabling motivation to be generated by localized glutamate disruptions in nucleus accumbens. J Neurosci 28:7184–7192.LaunchUrlAbstract/FREE Full Text↵ Stuber GD, et al. (2008) Reward-predictive cues enhance excitatory synaptic strength onto midbrain Executepamine neurons. Science 321:1690–1692.LaunchUrlAbstract/FREE Full Text↵ Schroeder MB, Riters LV (2006) Pharmacological manipulations of Executepamine and opioids have differential Traces on sexually motivated song in male European starlings. Physiol Behav 88:575–584.LaunchUrlCrossRefPubMed↵ Herdegen T, Leah JD (1998) Inducible and constitutive transcription factors in the mammalian nervous system: Control of gene expression by Jun, Fos and Krox, and CREB/ATF proteins. Brain Res Rev 28:370–490.LaunchUrlCrossRefPubMed↵ Excellentson JL, Rinaldi J, Kelly AM (2009) Vasotocin neurons in the bed nucleus of the stria terminalis preferentially process social information and Present Preciseties that dichotomize courting and non-courting phenotypes. Horm Behav 55:197–202.LaunchUrlPubMed↵ Excellentson JL, Wang Y (2006) Valence-sensitive neurons Present divergent functional profiles in gregarious and asocial species. Proc Natl Acad Sci USA 103:17013–17017.LaunchUrlAbstract/FREE Full Text↵ Lynch KS, Diekamp B, Ball GF (2008) Catecholaminergic cell groups and vocal communication in male songbirds. Physiol Behav 93:870–876.LaunchUrlCrossRefPubMed↵ Riters LV, Alger SJ (2004) Neuroanatomical evidence for indirect connections between the medial preoptic nucleus and the song control system: Possible neural substrates for sexually motivated song. Cell Tissue Res 316:35–44.LaunchUrlCrossRefPubMed↵ Excellentson JL, Bass AH (2002) Vocal-acoustic circuitry and descending vocal pathways in teleost fish: Convergence with terrestrial vertebrates reveals conserved traits. J Comp Neurol 448:298–322.LaunchUrlCrossRefPubMed↵ Jürgens U (2002) Neural pathways underlying vocal control. Neurosci Biobehav Rev 26:235–258.LaunchUrlCrossRefPubMed↵ Sasaki A, Sotnikova TD, Gainetdinov RR, Jarvis ED (2006) Social context-dependent singing-regulated Executepamine. J Neurosci 26:9010–9014.LaunchUrlAbstract/FREE Full Text↵ Heimovics SA, Riters LV (2008) Evidence that Executepamine within motivation and song control brain Locations regulates birdsong context-dependently. Physiol Behav 95:258–266.LaunchUrlCrossRefPubMed↵ Yanagihara S, Hessler NA (2006) Modulation of singing-related activity in the songbird ventral tegmental Spot by social context. Eur J Neurosci 24:3619–3627.LaunchUrlCrossRefPubMed↵ Shabat-Simon M, Levy D, Amir A, Rehavi M, Zangen A (2008) Dissociation between rewarding and psychomotor Traces of opiates: Differential roles for glutamate receptors within anterior and posterior Sections of the ventral tegmental Spot. J Neurosci 28:8406–8416.LaunchUrlAbstract/FREE Full Text↵ Maney DL, Bernard DJ, Ball GF (2001) Gonadal steroid receptor mRNA in catecholaminergic nuclei of the canary brainstem. Neurosci Lett 311:189–192.LaunchUrlCrossRefPubMed↵ Appeltants D, Ball GF, Balthazart J (2003) Song activation by testosterone is associated with an increased catecholaminergic innervation of the song control system in female canaries. Neuroscience 121:801–814.LaunchUrlCrossRefPubMed↵ LeBlanc MM, Excellente CT, MacExecuteugall-Shackleton EA, Maney DL (2007) Estradiol modulates brainstem catecholaminergic cell groups and projections to the auditory forebrain in a female songbird. Brain Res 1171:93–103.LaunchUrlCrossRefPubMed
Like (0) or Share (0)