Abstract: Many songbirds sing in non-reproductive contexts while in flocks. Singing in such gregarious contexts is critical for maintaining and learning songs; however, song is not directed towards other individuals and has no obvious, immediate social consequences. Studies using conditioned place preference (CPP) tests of reward indicate that song production in gregarious contexts correlates positively with a bird’s intrinsic reward state and with opioid markers in the medial preoptic nucleus (mPOA). However, the causal involvement of opioids in gregarious song is unknown. Here we report that the selective mu opioid receptor (MOR) agonist fentanyl dose-dependently facilitates gregarious song and reduces stress/anxiety-related behavior in male and female European starlings. Furthermore, infusion of siRNA targeting MORs specifically in mPOA both suppresses gregarious song and disrupts the positive association between affective state and singing behavior, as revealed using CPP tests of song-associated reward. Results strongly implicate opioids in gregarious song and suggest that endogenous opioids in the mPOA may facilitate song by influencing an individual’s intrinsic reward state.
Discussion
This study is the first to demonstrate a causal role for MOR (mu opioid receptor) in gregarious song in both male and female songbirds. Results also suggest a role for MOR in mPOA in gregarious song and the positive affective state associated with singing behavior. More broadly, this study is consistent with a growing body of research that expands what is known about the role of mPOA to include an important role in non-sexual social behaviors, a role that may generalize to other species and other intrinsically-rewarded social behaviors as detailed below.
MOR agonism reduces stress/anxiety and stimulates gregarious singing behavior
We report that peripheral injections of the selective MOR agonist fentanyl cause dose-dependent increases in gregarious singing behavior in male and female starlings, while at the same time decreasing beak wiping, which is considered a sign of stress or anxiety39,40. The fentanyl did not affect landings, indicating that fentanyl effects on behavior were not caused by gross deficits in motor activity.
Past studies demonstrate that opioids act at MOR to induce both a positive affective state and to reduce signs of anxiety or fear41,42,43,44,45,46. Gregarious singing behavior is facilitated by the presence of flock mates when birds are free from stress and fear (e.g., in the absence of predators) 18,47,48,49. Thus our findings suggest that the presence of flock mates/safety of a flock may naturally lead to opioid release and stimulation of MOR to induce a positive/low anxiety state that is conducive to gregarious singing behavior. This idea is further supported by the observation that in both the female and male studies, control birds sang very little. Given that birds were only selected for inclusion in the study if they sang at high rates consistently during pre-test observations, this suggests that the injection procedure, which involves chasing, catching, and injecting the bird, induced a state of stress/anxiety in the birds that reduced singing behavior. The finding that fentanyl rescued singing behavior is consistent with the idea that natural MOR stimulation may facilitate song by reducing a state of stress or anxiety. It may also be that opioids are released naturally by the act of singing itself to maintain ongoing singing behavior.
It is noteworthy that in past studies the highest dose of fentanyl (0.25 mg/kg), which we report here to stimulate gregarious song, inhibited sexually-motivated song in male starlings50. These opposing findings are consistent with studies of intrinsically-rewarded social behavior and extrinsically-rewarded, mate-directed behavior in mammals. Specifically, stimulation of MOR in rats facilitates gregarious social play behavior (behavior that, similar to gregarious song, has been shown to be intrinsically-rewarded using CPP tests27,31,51,52,53). In contrast, MOR agonists in male rats generally inhibit mate-directed behaviors that can be extrinsically-rewarded by copulation54,55,56,57,58,59,60, as in our past study on sexually-motivated song in male starlings50. The similarities identified in the roles played by opioids in gregarious and sexual behaviors in songbirds and rats, suggest that mechanisms of opioid action on socio-sexual behaviors may be conserved across species. Furthermore, the finding that MOR agonism affected behaviors similarly in males and females suggests that similar mechanisms may underlie this type of singing behavior in both sexes.
MOR downregulation disrupts singing behavior and associated reward
siRNA targeting MOR in mPOA suppressed gregarious song. Specifically, relative to birds infused with negative control sequences, birds infused with siRNA sang significantly less both 24 h and 48 h post infusion. Unlike the peripheral MOR manipulations, this treatment did not significantly impact beak wiping, indicating that MOR acts in other brain regions (such as the periaqueductal gray21) to reduce stress/anxiety needed to facilitate gregarious song. No effects were observed on feeding or drinking behaviors, suggesting that siRNA treatment did not induce non-specific changes in behavior. (Although MOR are involved in feeding behavior61,62, the mPOA has not been identified as a critical site of action for this behavior.)
Because infusion of opioids that stimulate MOR into the mPOA induces reward in rats63, we hypothesized that MOR in mPOA may underlie the reward state associated with gregarious song in songbirds. Consistent with this hypothesis, siRNA targeting MOR in mPOA disrupted the positive correlation observed in controls between singing behavior and a positive affective state, measured using CPP. Specifically, for control birds song rate correlated positively with the amount of time a bird spent in a chamber in which it had been placed previously after singing. Thus these birds demonstrated a song-associated CPP. In contrast, there was no positive relationship between song rate and CPP in siRNA treated birds. We interpret these results as consistent with a role for MOR in mPOA in the positive affective state associated with singing behavior; however, other interpretations are also possible. For example, the lack of correlation in MOR knockdown birds may be interpreted to reflect learning or memory deficits rather than a lack of reward. Although we found no publications implicating MOR in mPOA in learning and memory, such alternative interpretations must be considered.
We interpret our findings cautiously because in this study the sample size for control birds was limited; however, the positive relationship between song and CPP observed in the controls here replicates results of four prior studies from our lab (including one in male zebra finches) that used this method to assess song-associated reward12,22,23, suggesting the correlation in controls is not likely due to chance. Although sample sizes for the sexes were not large in this study, these initial findings do not demonstrate sex differences, suggesting that mechanisms rewarding gregarious song may be similar across males and females, but this must be tested in future studies.
Additional considerations related to siRNA methodology
Our initial validation study demonstrates strong suppression of MOR mRNA in mPOA at 24 h. After using an identical infusion protocol for the behavioral study, we see the same pattern for MOR protein 48 h post-infusion; however, MOR protein was not significantly lower in siRNA treated birds compared to controls. This lack of a significant difference is not unexpected given individual differences in MOR are likely present in individuals prior to treatment and because MOR synthesis may begin to be restored at the time that brains were collected. Thus, given the effectiveness of the method at 24 h we assume that we did successfully knockdown MOR receptors in the mPOA at the time of conditioning in the birds used in the behavioral experiment.
A second caveat is that we do not know how far the siRNA infusions spread. It is likely that infusions spread and downregulated MOR beyond the boundaries of mPOA. Thus, it is possible that effects on behavior are caused by MOR downregulation outside mPOA. Although possible, past studies that show that lesions to mPOA nearly abolish spring song also show that lesions located even slightly outside the boundaries of mPOA have no effect19,64,65. Similarly, studies that demonstrate strong effects of intra-mPOA opioid and dopamine receptor manipulations on singing behavior also show no effects on behavior when cannulae miss mPOA, even by a small margin38,66. Finally, in songbird studies MORs appear to be denser in mPOA than in the immediately surrounding regions67,68. Altogether these past studies suggest that the area immediately surrounding mPOA is not a primary site involved in the regulation of song but do not preclude the possibility that MOR downregulation in sites outside of mPOA may have influenced behavior.
Additional considerations related to the CPP methodology
Our attempt to assess rewarding properties associated with the act of producing song presents unique challenges that require modification of the CPP methodology somewhat from CPP tests used in studies of drug or food reward (reviewed69,70). Unlike food or drugs we cannot administer the “act of singing”. Birds either sing or they do not, which means that we cannot pair the act of singing with one chamber in a CPP apparatus and a lack of singing with another chamber an equal numbers of times and then compare time spent in each chamber to a neutral zone, as is common in studies of food or drug reward. This imbalanced design causes birds to be exposed to the song-paired side of the apparatus more than the non-song-paired side. Thus, it may be that on test day something about the more familiar side of the CPP apparatus (i.e., the previously song-paired side) is more appealing to birds singing higher rates of undirected song. Although we interpret our results cautiously, without a compelling reason to propose that high singers should be more attracted to familiarity than low singers, we suggest that a straightforward interpretation of the results is that gregarious singing behavior is coupled to a positive affective state. Detailed discussion of this and other interpretational issues can be found in12.
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