Immunolabeling Provides Evidence for Subregions in the Songbird Nucleus Accumbens and Suggests a Context-Dependent Role in Song in Male European Starlings (Sturnus vulgaris)

Abstract

Birdsong is well known for its role in mate attraction during the breeding season. However, many birds, including European starlings (Sturnus vulgaris), also sing outside the breeding season as part of large flocks. Song in a breeding context can be extrinsically rewarded by mate attraction; however, song in nonbreeding flocks, referred to here as gregarious song, results in no obvious extrinsic reward and is proposed to be intrinsically rewarded. The nucleus accumbens (NAC) is a brain region well known to mediate reward and motivation, which suggests it is an ideal candidate to regulate reward associated with gregarious song. The goal of this study was to provide new histochemical information on the songbird NAC and its subregions (rostral pole, core, and shell) and to begin to determine subregion-specific contributions to gregarious song in male starlings. We examined immunolabeling for tyrosine hydroxylase (TH), neurotensin, and enkephalin (ENK) in the NAC. We then examined the extent to which gregarious and sexually motivated song differentially correlated with immunolabeling for the immediate early genes FOS and ZENK in each subdivision of the NAC. We found that TH and ENK labeling within subregions of the starling NAC was generally similar to patterns seen in the core and shell of NACs in mammals and birds. Additionally, we found that gregarious song, but not sexually motivated song, positively correlated with FOS in all NAC subregions. Our observations provide further evidence for distinct subregions within the songbird NAC and suggest the NAC may play an important role in regulating gregarious song in songbirds.

Keywords: Communication; Dopamine; Immediate early genes; Nucleus accumbens; Opioids; Reward; Songbird.

Fig. 1.

Images adapted from the 3D starling atlas (A and C) [De Groof et al., 2016] and illustrations (B and D) showing locations of the rostral pole of the nucleus accumbens (ACR) (panels A and B), and the accumbens core (ACC) and accumbens shell (ACS) (panels C and D) in the left hemisphere of coronal sections. TSM = tractus septomesencephalicus.

Fig. 2.

Photomicrographs of tyrosine hydroxylase (TH) labeling in the rostral pole of the nucleus accumbens (ACR), nucleus accumbens shell (ACS) and nucleus accumbens core (ACC). A. TH labeling in the ACR at 2x magnification. B. 10x magnification of the ACR. C. TH labeling in the ACC and ACS at 2x magnification. D. 10x magnification of the ACS and ACC. Arrows highlight the tractus septomesencephalicus (TSM) and location of Area X for orientation.

Fig. 3.

Percent Area Covered (A) and Optical Density (B) of tyrosine hydroxylase labeling in the rostral pole of the nucleus accumbens (ACR), nucleus accumbens shell (ACS) and nucleus accumbens core (ACC). Error bars represent one within-subject standard error about the mean. *P<0.05

Fig. 4.

Photomicrographs of neurotensin (NT) in the NAC. See Figure 2 for additional details.

Fig. 5.

Photomicrographs of enkephalin (ENK) in the NAC. See Figure 2 for additional details.

Fig. 6.

Percent Area Covered (A) and Optical Density (B) of enkephalin labeling in the rostral pole of the nucleus accumbens (ACR), nucleus accumbens shell (ACS) and nucleus accumbens core (ACC). Error bars represent one within-subject standard error about the mean. No significant differences were found between any of the subregions for either variable.

Fig. 7.

Photomicrographs showing the comparison of FOS in the NAC of low singing (song = 5) (left) and high singing (song = 32) (right) European starlings singing in a gregarious context. Images are centered within the same locations as shown in Figures 2, 4, & 5.

Fig. 8.

Numbers of FOS labeled cells within the rostral pole of the nucleus accumbens (ACR), accumbens core (ACC), and accumbens shell (ACS) correlate positively with gregarious song production. Data shown are the number of FOS-labeled cells within the ACR, ACC, and ACS correlated with total song (point sampling every minute for an hour) for each individual. Each point represents one starling. Individuals singing gregarious song are in the left column and individuals singing sexually-motivated song are in the right column. The inclusion of a regression line indicates a significant correlation (p<0.05).

Fig. 9.

Numbers of ZENK labeled cells within the rostral pole of the nucleus accumbens (ACR) correlates with gregarious song production. Data shown are the number of FOS-expressing cells within the ACR, ACC, and ACS correlated with total song (point sampling every minute for an hour) for each individual. Each point represents one starling. Individuals singing gregarious song are in the left column and individuals singing sexually-motivated song are in the right column. The inclusion of a regression line indicates a significant correlation (p<0.05).