Curvilinear relationships between mu-opioid receptor labeling and undirected song in male European starlings (Sturnus vulgaris)

Abstract

Female-directed communication in male songbirds has been reasonably well studied; yet, relatively little is known about communication in other social contexts. Songbirds also produce song that is not clearly directed towards another individual (undirected song) when alone or in flocks. Although the precise functions of undirected song may differ across species, this type of song is considered important for flock maintenance, song learning or practice. Past studies show that undirected song is tightly coupled to analgesia and positive affective state, which are both mediated by opioid activity. Furthermore, labeling for the opioid met-enkephalin in the medial preoptic nucleus (POM) correlates positively with undirected song production. We propose that undirected song is facilitated and maintained by opioid receptor activity in the POM and other brain regions involved in affective state, analgesia, and social behavior. To provide insight into this hypothesis, we used immunohistochemistry to examine relationships between undirected song and mu-opioid receptors in male starlings. Polynomial regression analyses revealed significant inverted-U shaped relationships between measures of undirected song and mu-opioid receptor labeling in the POM, medial bed nucleus of the stria terminalis (BSTm), and periaqueductal gray (PAG). These results suggest that low rates of undirected song may stimulate and/or be maintained by mu-opioid receptor activity; however, it may be that sustained levels of mu-opioid receptor activity associated with high rates of undirected song cause mu-opioid receptor down-regulation. The results indicate that mu-opioid receptor activity in POM, BSTm, and PAG may underlie previous links identified between undirected song, analgesia, and affective state.

Keywords: Birdsong; Motivation; Mu-opioid receptor; Opioids; Reward; Vocal communication.

Figure 1

Evidence for curvilinear relationships between mu-opioid receptor labeling and undirected song rates. An inverted-U shaped curve showing the measure of undirected song on the x-axis and mean mu-opioid receptor total pixel area on the y-axis in A) POM, B) PAG, and C) BSTm. Individual birds are represented by a single black dot. Sample size indicated in the upper right corner of the figures. Presence of the red regression line indicates a significant relationship p<0.05.

Figure 2

Photomicrographs illustrating mu-opioid receptor labeling. Representative images (20X) of mu-opioid receptor punctate labeling within POM in individuals that were classified as a A) low singer, B) intermediate singer, or C) high singer. Abbreviation: V= ventricle. Scale bar in bottom left corner.

Figure 3

Relationships between the area covered by mu-opioid receptor label (mean pixel area) and undirected song. The measure of undirected song is on the x-axis and the mean mu-opioid receptor total pixel area is on the y-axis in A) VTA, B) PVN, C) SL. Individual birds are represented by a single black dot. Sample size indicated in the upper right corner of the figures.

Figure 4

Relationships between the area covered by mu-opioid receptor label (mean pixel area) and bouts of feeding behavior. The total number of bouts of feeding is on the x-axis and mean mu-opioid receptor total pixel area is on the y-axis for A) PAG and B) VTA. Individual birds are represented by a single black dot. Sample size is indicated in the upper right hand corner of the figure. Presence of the red regression line indicates a significant relationship p<0.05.

Figure 5

Non-significant relationships between mu-opioid receptor optical densities and undirected song rates. The measure of undirected song on the x-axis and mean mu-opioid receptor optical density on the y-axis in A) POM, B) PAG, C) BSTm, D) VTA, E) PVN, F) SL. Individual birds are represented by a single black dot. Sample size indicated in the upper right corner of the figures.

Figure 6

Brain schematic indicating approximate areas in measures of mu-opioid receptor labeling was quantified. Abbreviations: A, arcopallium; BSTm, medial bed nucleus of the stria terminalis Cb, cerebellum; CO, optic chiasm; PAG, periaqueductal gray; GLV, nucleus geniculatus lateralis, pars ventralis; ICo, nucleus intercollicularis; SL, lateral septum; NIII, third cranial nerve; N, nidopallium; NC, caudal nidopallium; POM, medial preoptic nucleus; PVN, paraventricular nucleus of the hypothalamus; Rt, nucleus rotundus; VMN, ventromedial nucleus; VTA, ventral tegmental area.