Social Context-Appropriate Vocal Communication and Opioids in Male European

Social Context-Appropriate Vocal Communication and Opioids in male European

Starlings (Sturnus vulgaris)

Cynthia A. Kelm-Nelson

A dissertation submitted in partial fulfillment of the requirements for the degree of

Doctor of Philosophy

(Zoology)

at the

UNIVERSITY OF WISCONSIN-MADISON

2012

Date of final oral examination: 12/05/2012

The dissertation is approved by the following members of the Final Oral Committee: Lauren V. Riters, Professor, Zoology Catherine J. Auger, Assistant Professor, Psychology Craig W. Berridge, Professor, Psychology Stephen C. Gammie, Professor, Zoology Charles T. Snowdon, Professor, Psychology

©Copyright by Cynthia A. Kelm-Nelson 2012

Acknowledgment There are many people who deserve thanks for their varied contributions to my dissertation. I am grateful to my academic advisor and role model, Lauren Riters, for her acceptance, encouragement, mentorship, and friendship. I would also like to offer my appreciation to the members of my dissertation committee: Dr. Cathy Auger, Dr. Craig Berridge, Dr. Stephen

Gammie and Dr. Charles Snowdon. They have generously given their time and expertise as well as valuable guidance and direction to better my scientific work. It has been a privilege to work with such scientists.

I would also like to thank the members of the Riters’ lab (both past and present): Sarah

Heimovics, Sarah Jane Alger, Ben Pawlisch, Jesse Ellis, and Melissa Cordes. I have been fortunate to interact with a variety of exceptional graduate students. And, many, many thanks to my friend and research collaborator, Sharon Stevenson. There have been many undergraduates who have helped assist the work descript in this dissertation. Those in need of special acknowledgment for technical work include: Griff Gessay and Rachel McCormick.

The animal care staff in Birge Hall is exceptional and I must thank Kate Skogen and Chris Elliot for animal care. I also would like to thank Bill Feeny for his artistry and creation of conference posters, as well as the zoology department staff.

Lastly, I must thank my family. My parents and my brother Pete have always been so loving and supportive throughout my many years of education. And, I must thank my husband Jim for providing love, encouragement, proof-reading and much patience. ii

Table of Contents

Acknowledgment ...... i

Table of Contents ...... ii

Abstract ...... iii

Chapter I: General Introduction ...... 1

Chapter II: Context-Dependent Links between Song Production and Opioid-Mediated Analgesia in Male European Starlings (Sturnus vulgaris) ...... 24

Chapter III: Curvilinear relationships between mu-opioid receptor densities and undirected song in male European starlings (Sturnus vulgaris) ...... 54

Chapter IV: Mu-opioid receptor densities are depleted in regions implicated in agonistic and sexual behavior in male European starlings (Sturnus vulgaris) defending nest sites and courting females ...... 81

Chapter V: Modulation of male song by naloxone in the medial preoptic nucleus ...... 116

Chapter VI: Conclusions and General Discussion ...... 138

iii

Social Context-Appropriate Vocal Communication and Opioids in male European

Starlings (Sturnus vulgaris)

Cynthia A. Kelm-Nelson

Under the supervision of Professor Lauren V. Riters

At the University of Wisconsin-Madison

Abstract Vocal communication is a necessity for successful social relationships and interactions as well as survival and reproductive success. The neural regulation of vocal behavior has been well studied using songbird model systems; yet, less is known about the neurochemical mechanisms regulating context-appropriate vocal behaviors. Endogenous opioid neuropeptides represent likely candidate modulators of vocal behavior as they are involved in a variety of social behaviors, including vocal behavior. The unifying element of this dissertation evaluates the role of mu-opioid receptors in vocal communication produced within distinct social contexts.

Starlings use song to immediately influence a conspecific (directed) and also sing spontaneously at high rates in large flocks (undirected). Past work suggests that opioids play an important role in regulating song but that the role may differ depending upon the social context. In this dissertation I further explored this possibility. Using an indirect assay of opioid-mediated analgesia my first chapter supports the possibility that opioids may facilitate undirected song but inhibit directed song. In order to determine where in the brain opioids may act to influence song in distinct contexts and to further evaluate relationships between receptor densities within individuals, I examined relationships between immunolabeling for the mu-opioid receptor and iv both directed and undirected singing behavior. For undirected song, data showed links between low densities of mu-opioid receptors in the medial preoptic nucleus (and other areas in which mu receptors induce analgesia and regulate vocal communication) and both low and high rates of undirected singing behavior, whereas high receptor densities were associated with intermediate rates of undirected song. In males singing directed song, there was significantly higher mu- opioid immunolabeling in several brain regions, including the medial preoptic nucleus in males singing at low compared to high rates. This suggests that receptors may be acting to inhibit vocal communication in males singing low rates of directed song. This prediction was further supported by a site-specific pharmacology study that demonstrated that blocking receptors in the medial preoptic nucleus in low singing males stimulated directed song. Together, the results of this dissertation provide new insight into where and how opioids regulate context-appropriate vocal communication.

Chapter I: General Introduction For humans and multiple vertebrate species, vocal communication is a necessity for successful social relationships. Multiple mental health disorders in humans, including depression, anxiety, post-traumatic stress disorder and pervasive developmental disorders, are characterized by deficits in social communication, social withdrawal, or inappropriate responses to social signals

[1]. For instance, Autism Spectrum Disorders (ASDs) are a complex class of pervasive developmental brain disorders diagnosed using an assessment of a triad of impairments: social interaction (lack of spontaneous seeking to share with others, failure to develop peer relationships), social communication (delay or lack of development of spoken language), and repetitive patterns of behavior (stereotyped or repetitive motor mannerisms) [2]. These deficits are often times context specific (i.e., communication deficits are seen in specific situations)

[1,3,4] and may lead to social exclusion and / or difficulties maintaining relationships, deficits that persist into adulthood. Communication through the use of vocalizations is essential in both human and non-human animals to convey various forms of information. And, non-human models are an excellent way of studying the neural mechanisms of socially appropriate behavior where the methodology can later be translated to human research and potentially lead to novel clinical treatments and therapeutic (i.e. druggable) targets.

Socially appropriate behavior is critical for successful social interactions within the animal kingdom and may dictate survival and reproductive success. Vocal communication is one way members of the same species convey information to one another, and it is critical that vocal communication be regulated and fine-tuned in response to the appropriate stimuli [5]. For example, in vervet monkeys (Cercopithecus aethiops) different acoustic alarm calls signal 2 predator danger and are specific to whether the danger is above or below the treeline [6].

Accurate signals must be made so the vervet population takes the proper actions (fleeing to the trees, looking above for the threat of eagles or looking down for snakes). Across species, vocal adjustments must be made to match the social context and what is socially appropriate for one individual is not necessarily appropriate for another. An example from human behavior: it may be appropriate to cheer raucously at a sporting event whereas it is inappropriate to do so in the quiet study section of the library.

Songbirds are well known for their ability to regulate and fine-tune vocal behavior in response to specific social stimuli within functionally distinct social contexts [7] and, historically, both the function and neural regulation of vocal communication have been well studied using this model system [8]. However, less is known about the neurochemical mechanisms regulating context- appropriate vocal behavior. Recent evidence suggests that opioid neuropeptides may play an important role in regulating vocal communication so that it is appropriate within a given social context [9,10]. The goal of the present dissertation is to examine further opioid involvement in context-appropriate singing behavior in male European starlings (Sturnus vulgaris).

The function of birdsong differs depending on the context in which song is produced

Songbirds provide a unique system for studying social aspects of vocal communication. As in humans, a primary function of songbird vocalizations is to influence the behavior of other conspecifics (directed song); for example, defending territory boundaries from intruders, repelling competing males or attracting potential mates to nesting sites [7,11]. Some species

(including starlings) also sing high rates of spontaneous song which doesn’t immediately appear 3 to affect behavior of conspecifics. This type of song is common in birds singing in large flocks and has been referred to as undirected [12,13]. In this case the function of song is not well known, but in starlings (and also zebra finches) this type of song appears to be affiliative, produced in large flocks and likely functions in maintaining social contact or song practice

[13,14]. In starlings this song is typical outside breeding season.

Introduction to the starling model system

Similar to other songbirds, starlings sing at specific points during development and within multiple social contexts as adults [11,14,15]. As adults, both male and female starlings sing year round (with the exception of the molting period), but the function of song and the stimuli that elicit male song change seasonally [11,16].

In the spring, as day length begins to increase, circulating gonadal steroid hormone levels (i.e. testosterone; T) are elevated in male starlings [17,18,19]. At this time male song is important for mate attraction and for repelling competitors. For example, males will sing at high rates in response to females and to a lesser extent in response to competing males [20,21,22]. Song in this context is dependent upon the steroid hormone testosterone (T). For example, in male starlings castration abolishes spring song; whereas, systemic T treatment restores song levels in this context [23]. Male song is sexually motivated, where males sing immediately prior to nearly every copulation or upon removal of a female mate [24].

While annual variations in photoperiod and systemic hormone concentrations are necessary for female-directed vocal production in starlings, elevated testosterone and the presence of a female 4 are alone not sufficient to trigger male directed song production in spring. Resource attainment also has direct influences on spring song production [25]. Starlings are semi-colonially hole nesting passerines, and within the spring breeding season individuals either acquire a nest site or do not. Acquisition of a nest cavity, a critical resource, is often times a rate-limiting factor for starling reproduction. Males that acquire nest sites are found to socially dominate other males

[26], and use song to attract a mate to this site [27]. Additionally, the complex song production from these sites functions to defend the territory from other competing males [14,28,29]. It can be considered socially appropriate for male starlings that acquire a nest site sing to at high rates to attract females for mating success. However, it may be considered inappropriate for males to sing at high rates when they fail to acquire a nest site as high rates of song are energetically taxing and may attract the attention of predators [30,31,32].

Male starlings are ideal experimental subjects for the study of complex vocal communication and socially appropriate behavior due to the fact that they sing consistently at high rates within both of these distinct social contexts (breeding and non breeding) in a laboratory setting, much like they would in a natural setting. Little is known about the neural regulation of context-specific song or how social status / resource possession alters the brain to ensure that an individual produces status- or resource-appropriate behavioral responses. Starlings provide a unique model system to address these gaps in knowledge.

Mechanisms regulating directed and undirected song differ

Overall, spring “directed” song and fall “undirected” song rates in male starlings are similar, with the average spring song bout length slightly longer than fall song [33]. Females prefer to 5 mate with a male singing longer songs [29,34,35], thus males adjust song so it is most attractive to females [21]. Female-directed song is used to attract mates, is considered highly sexually- motivated and goal-directed, and may be extrinsically rewarded by successful female attraction and copulation or repulsion of a male competitor [11,22]. Unlike female-directed song, undirected song does not result in an immediate, obvious external rewarding factor (e.g., song does not result in mate attraction or copulation) and it is not clear what motivates males to sing in this context. The factors motivating an individual to sing may differ depending upon the function of song, social context, endocrine state, and social or environmental dynamics. The exact neurological mechanisms that control song in each context are not well known, however evidence suggests that motivational or reward circuitry (reviewed below) may be involved.

The avian song control system controls the production of song

There is little evidence to suggest that the motivation to sing lies within the song control system, a group of cytoarchitecturally distinct forebrain nuclei (Figure 1) that control the production, perception and sensorimotor aspects of song [36,37]. There are two distinct neural pathways that make up the song control system. The anterior forebrain pathway involved in song learning and perception consists of HVC (proper name), Area X, medial nucleus of the dorsal lateral thalamus

(DLM), and lateral magnocellular nucleus of the anterior nidopallium (LMAN). The posterior pathway controls sensorimotor aspects of song and includes projections of HVC to RA to the hypoglossal nucleus and the ventral respiratory group (RAM) which eventually innervates the syrinx and respirator musculature. Research shows that neuronal and gene activity within the area X, LMAN, RA and HVC was higher when males produced undirected compared to directed song [38,39], suggesting that neural differences may contribute to social context-dependent vocal 6 production, but the function of these regions likely relates to structural or motor aspects of singing rather than motivational aspects (discussed next).

Historically, avian neurobiologists have focused on song learning, production and perception.

Beginning with Fernando Nottebohm’s pioneering studies of the song system in the 1970’s, it was discovered that lesions to the canary song control system nuclei produce dramatic effects.

For example, unilateral electrolytic lesions to HVC result in severe song deficits (“silent song”) where the bird exhibits the correct song posture but does not produce vocalizations [36]. Lesions to RA cause song structure deficits whereas Area X lesions result in no deficits to song structure but result in deficits in song learning [40]. The continuation of song production in lesioned birds suggests that the neural system underlying the motivation to sing is still intact.

Neural systems involved in motivation and reward may underlie vocal production

Because vocal communication must be adjusted to reflect an individual’s motivational state, it is likely that brain regions involved in social behavior, reward, and motivation may be involved.

Neural systems outside of the song control system involved in motivation and reward have been conserved across species and are well positioned to influence the song control system nuclei that control the production of song [41,42]. The interconnected nuclei of the “social behavior network”, a group of reciprocally connected, steroid sensitive brain nuclei (Figure 2), represent likely candidates for influencing many social behaviors including sexual behavior, aggression, and vocal communication [43]. 7

Components of the “social behavior network” have been well-studied for their roles in motivation and reward. The incertohypothalamic system (including the medial preoptic area

(abbreviated POM in birds) and the paraventricular nucleus (PVN)) reciprocally connects with mesolimbic nuclei (including ventral tegmental area (VTA), nucleus accumbens (Ac), lateral septum (LS), and medial bed nucleus of the stria terminalis (BSTm)) all of which play an important role in motivation and reward [43,44]. Additionally, these nuclei project (some indirectly) to song control nuclei (Figure 3; [41,42]), providing a route by which they may influence aspects of song production. Neurochemicals, including dopamine and opioid neuropeptides, within the incertohypothalamic system and mesolimbic nuclei have been implicated in motivation and reward [45,46,47,48,49,50]. Specific nuclei within the mesolimbic and incertohypothalamic systems that regulate the motivation or rewarding properties of song are understudied; however distinct patterns of neuronal activity in several regions have been identified in association with directed compared to undirected song, including VTA [51,52,53].

A growing body of data also suggest that POM may play a central role in song production, yet the role of this region differs depending upon whether song is directed or undirected [54,55].

The POM influences social context-appropriate vocal behavior

In songbirds, the POM is well positioned to influence brain areas not only involved in motivation and socio-sexual behavior, but also the song control nuclei through indirect connections (Figure

3) [41,56]. The medial preoptic nucleus is well known for its role in male sexual behavior across vertebrates [56,57,58,59,60] leading to the initial hypothesis that this region would be important for the regulation of female-directed spring song [61]. Several studies support this hypothesis. In 8 male starlings, the volume of POM, concentrations of T and the enzyme aromatase (critical for the metabolism of T) differ seasonally [33]. The volume of POM is the largest and contains the densest aromatase immunostaining in males in spring (when males respond to the introduction of a female with high song rates). In contrast, outside of the breeding season the volume of POM is relatively small and levels of T and aromatase are low [33]. The volume of POM also relates positively to male song bout length. Males that sang the longest songs (occurring in the spring context) had the largest volumes. Interestingly, males that occupied nest sites and sang high rates of song had the largest POM compared to those males that did not obtain a nesting site. This further suggests that individual resource attainment may be linked to activity in the POM.

Lesions to the POM were found to abolish female-directed spring song production in male starlings [61,62,63], and data from Heimovics and Riters (2005) show that the number of c-fos

(immediate early gene, indirect measure of neuronal activity) labeled cells within the POM related positively to the amount of song produced within the breeding season. Males that occupy nest sites also had more c-fos immunolabeling compared to those that do not have nest sites. The number of c-fos labeled cells in the POM of house sparrows (Passer domesticus) also correlated with song produced from a nest site, but not song produced elsewhere [64]. This suggests a central role for the POM in female-directed song production (sexually motivated). Given the role of the POM in male sexual motivation, the results suggest that the POM may provide information about a male’s sexually motivated state to the VTA and song control regions to cause a male with a nest box to sing long complex song that is attractive to females within the breeding season. Interestingly however, the role of the POM in male song is not restricted to sexually-motivated song observed in spring. 9

The POM differentially regulates song production depending on context. Alger and Riters

(2006) demonstrated that bilateral electrolytic lesions of the POM reduce song and nesting behaviors in spring, but increase non-sexually motivated song rates in spring, with a similar trend observed in fall [63]. These findings suggest a stimulatory role for POM in sexually- motivated song, but an inhibitory role for this region in non-sexually-motivated song. This research also suggests that the POM acts with the song control system to regulate song so that it occurs in an appropriate social context, and implicates the POM as an important region for regulating song outside of the breeding context.

Data suggest that distinct patterns of neurochemical activity within POM play a role in adjusting song so it is appropriate given the context; and, various neurochemical markers within POM have been found to be tightly coupled to singing behavior. For example, immediate early gene immunolabeling correlated positively with directed song [51], tyrosine hydroxylase labeling correlated negatively with directed song [65], D1 dopamine receptor densities correlated in opposite directions with undirected and directed song [66], and α2-norepinephrine receptor densities in POM were linked to male starling song when fall and spring song was combined

[67]. In addition to D1 receptors, opioid neuropeptides in POM to date are among the only neurochemicals found to be closely linked to undirected singing behavior [68].

Opioid neuropeptides and context-appropriate vocal behavior

Opioid neuropeptides are involved in a variety of processes including hedonic reward and affective state, motivation, analgesia, sexual, and social interactions [47,69,70]. Multiple studies 10 also implicate opioids in vocal behavior important for the maintenance of social contact

[71,72,73]. Many of these studies support an inhibitory role for opioids in the regulation of directed vocal behavior. For example, normally infant rodents display separation distress in the form of directed vocalizations to their mothers when they are isolated from the mother

[71,74,75]. In general, stimulation of mu-opioid receptors reduces social contact vocalizations,

Administration of naloxone, an opioid receptor antagonist, increases distress vocalization whereas administration of morphine, an opioid agonist, decreases rates of distress vocalizations.

Herman and Panksepp suggest that opiates are possibly replacing endorphins normally released in pups during affiliative interactions with the mother and that separation distress may actually be a form of “endorphin withdrawal”.

In songbirds and other vertebrates, studies implicate mu-opioid neuropeptides in vocal production [9,72,76]; however, the results are contradictory. Data suggest that the effects of opioids on song differ depending upon whether song is directed or undirected. Consistent with the rodent literature, past peripheral pharmacology studies in starlings show that a mu-opioid receptor agonist suppresses directed song production whereas an antagonist leads to an increase in singing behavior [9]. It is possible that successful female-directed male song results in increased opioid activity caused by interactions with the female (much like distress vocalizations discussed above serve to attract a caregiver which results in opioid release). In the absence of opioid activity (as after naloxone treatment) males may sing at higher rates to attract females to restore opioid levels. However, in male zebra finches Khurshid et al. (2010) reported systemic administration of low but not high doses of naloxone decrease directed song rates in high singing males. Interestingly, in the starling study [9,68], males naturally singing at low versus high rates 11 responded differently to the same pharmacological manipulations. Males singing at low rates responded with a larger increase after the opioid blockade. These data suggest that individual differences in receptor densities may be involved in individual differences in the propensity to communicate.

Opioids also influence undirected song but perhaps not in the same way that they affect directed song. In male zebra finches blocking mu-opioid receptors powerfully inhibits undirected song, but does not have the same effect on directed song [77]. This suggests that opioids may stimulate undirected but inhibit directed song, at least based on studies in starlings. Together these past studies suggest that differences may exist in opioid release, opioid receptor densities or binding affinity in association with individual differences in the tendency to sing directed and undirected song.

Where in the brain opioids are acting to influence song is largely understudied. In songbirds opioids (endorphins, enkephalins, dynorphins), their receptors (mu, delta and kappa), and associated enzymes (such as neutral endopeptidase and angiotensin-converting enzyme) are found within the nuclei of the song control system. Specifically, RT-PCR / qRT-PCR analysis of endogenous opioids, met and leu-enkephalin, were analyzed in male zebra finches in LMAN,

Area X, LS, HVC, and RA [78]. Quantitative in vitro autoradiography measures also indicate both mu and delta receptor distribution in vocal control regions [79]. The POM is densely innervated with opioids and their receptors and, as discussed previously, well positioned to influence the song control system [10,41].

Opioid fibers and receptors are found in the POM in multiple species including pigeons, chicks, juncos, zebra finches and starlings [10,80,81,82,83,84], and may be a potential site in which opioids act to regulate context-appropriate behaviors (including vocal communication) as well as general motivation and reward to perform these behaviors. In quail as in rodents, opioids in POM inhibit male sexual behavior and neuronal activity [85]; indicating the POM as a site in which opioids may act to influence sexually motivated male song. Interestingly, in a study of male starlings [10], the opioid met-enkephalin (mENK) in POM did not correlate with song in the breeding context produced in response to a female (directed song). Instead, mENK was solely correlated with song produced outside of the breeding context in the non-breeding condition

(undirected song) only for POM. This suggests that enkephalin opioids in POM are closely linked to undirected song. When paired with data from male zebra finches showing that opioids stimulate undirected song [77], the aforementioned results suggest the possibility that, in contrast to inhibitory effects observed to date on directed song [9,68], opioids in POM may stimulate undirected song.

Dissertation Hypothesis

To date, little is known about the neurobiological mechanisms regulating context-specific vocal behavior. The studies reviewed above indicate that opioids are involved in both directed and undirected song, but that the role may differ. There is a tight linear link between opioid immunolabeling and undirected song production suggesting that opioids may facilitate or simulate song in this context. However, the lack of a linear relationship does not rule out a role for opioids in directed song, for example there are strong effects on song rate due to peripheral opioid administration. 13

The goal of this dissertation is to gain insight into the role that opioids play in regulating socially appropriate vocal communication using a songbird model. To test the hypothesis that the role of opioids in undirected and directed song differs, an indirect test of opioid-mediated analgesia was used to assess the prediction that opioid release occurs in immediate association with undirected, but perhaps not directed song. Additionally, opioid immunomarkers were used to identify brain regions in which the opioids may be acting to influence both directed and undirected song.

Finally, site-specific opioid manipulations were used to determine the central effect of opioids in the POM on directed singing behavior and whether effects differed in individuals naturally singing at low and high rates.

Together, the aims of this dissertation provide new insight into where and how opioids regulate male context-appropriate social vocal behavior. In the future, these data may lead to novel treatments or targets for human communication disorders. 14

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Figures and Figure Legends

Figure 1: A sagittal representation of the avian song control system nuclei. In red, the anterior forebrain pathway for song learning and plasticity, and in black the motor pathway for song production. Abbreviations: Area X (Area X of the medial striatum),

DLM (medial portion of the dorsolateral nucleus of the thalamus), HVC (used as a proper name), LMAN (lateral subdivision of the magnocellular nucleus of the anterior nidopallium), and RA (robust nucleus of the arcopallium). Figure Credits: Nature

Education 2010 [86].

Figure 2: The social behavior network as proposed by Newman (1999). The six limbic areas are interconnected and have been implicated in social behaviors [34].

Figure 3: Starling brain sagittal schematic. Demonstration of projections from the medial preoptic nucleus (POM) to the ventral tegmental area (VTA) and mesencephalic central gray (GCt) and from VTA and GCt to Area X, HVC, and robust nucleus of the arcopallium (RA). Other abbreviations: LMAN, lateral portion of the magnocellular nucleus of the anterior nidopallium; DLM, medial portion of the dorsolateral nucleus of the anterior thalamus; Ram / rVRG, nucleus retroambigualis/rostral ventral respiratory group; nXIIts, tracheosyringial portion of the hypoglossal nucleus [65].

Chapter II: Context-Dependent Links between Song Production and Opioid-Mediated Analgesia in Male European Starlings (Sturnus vulgaris)

Individual manuscript has been previously published in PLoS One.

Kelm-Nelson, C.A., Stevenson, S.A., Riters, L.V. (2012) Context-Dependent Links between Song Production and Opioid-Mediated Analgesia in Male European Starlings (Sturnus vulgaris). PLoS One. 7, e46721.

Abstract

Little is known about the neural mechanisms that ensure appropriate vocal behaviors within specific social contexts. Male songbirds produce spontaneous (undirected) songs as well as female-directed courtship songs. Opioid neuropeptide activity in specific brain regions is rewarding, at least in mammals, and past studies suggest that the opioid met-enkephalin in such areas is more tightly linked to undirected than female-directed song. Recent data using a song- associated place preference paradigm further suggest that production of undirected but not directed song is tightly linked to intrinsic reward. Opioids have analgesic properties. Therefore, if production of undirected song is closely linked to opioid-mediated reward, the production of undirected but not directed song should be associated with analgesia. Consistent with this prediction, in male starlings we identified a positive correlation between analgesia (decreased reactivity to a hot water bath) and undirected song (in non-breeding season condition males in affiliative flocks) but not female-directed song (in breeding season condition males presented with females). When breeding condition males were divided according to social status, a negative correlation was found in subordinate males (i.e. males that failed to acquire a nest box).

These data are consistent with the hypotheses 1) that the production of undirected song is facilitated or maintained by opioids (and / or other neuromodulators that also induce analgesia) and 2) that production of female-directed song is not linked in the same way to release of the same neuromodulators. Results also demonstrate a link between analgesia and song in subordinate individuals lacking a nesting territory within the breeding season. Overall, the findings indicate that distinct neural mechanisms regulate communication in different social contexts and support the working hypothesis that undirected but not directed song is tightly linked to opioid release. 26

Key Words: analgesia; birdsong; opioids; reward; songbird; vocal communication

1. Introduction

Vocal communication plays a critical role in social interactions across vertebrate species, including songbirds [1]. To communicate effectively individuals must adjust vocal production to match particular social contexts, yet little is known about neural mechanisms underlying context- appropriate communication.

In songbirds, opioid neuropeptides are proposed to play a role in male singing behavior that differs depending upon whether song is produced spontaneously (undirected) or is sexually- motivated and directed towards a female (female-directed song). Specifically, immunolabeling density for the opioid met-enkephalin in the medial preoptic nucleus (referred to as POM in birds) correlates positively with undirected but not female-directed singing behavior in male

European starlings (Sturnus vulgaris), with a similar trend observed in the ventral tegmental area

(VTA; p = 0.06) [2]. Additionally, mu-opioid receptor labeling density is lower in both of these regions in male starlings singing high rates compared to those singing low rates of female- directed song [3]. Pharmacological manipulations in male starlings and zebra finches

(Taeniopygia guttata) also indicate that opioids regulate song differently depending on whether it is female-directed or undirected [2,4,5]. In rats, opioid neuropeptides in VTA and the preoptic area are rewarding (e.g., morphine in VTA and enkephalin in the preoptic area [6,7]), and recent data in starlings using a song-associated place preference paradigm suggest production of undirected but not directed song is tightly linked to reward state [8]. Together these studies lead 27 to the hypothesis that undirected song is more tightly linked to immediate opioid release in the

POM and VTA than directed song (reviewed in [9,10]).

Opioids have analgesic properties [11,12], and data indicate that opioid release in both the preoptic area and VTA induces analgesia in rats [13,14]. If production of undirected song is regulated by immediate opioid release in these regions, then production of undirected but not directed song may be associated with analgesia. To test this prediction, flocks of male starlings were observed singing undirected song (males with low testosterone (T) singing in an affiliative non-sexual context) and female-directed song (males with high T singing to females in a breeding season condition). Immediately after the observation period, the latency for each male to remove his foot from a hot water bath was recorded as a measure of analgesia. If undirected but not directed singing behavior is regulated by immediate opioid release, we predicted that measures of undirected but not directed song would correlate positively with the length of time a male maintained a foot in hot water.

2. Materials and Methods

Ethics Statement

Protocols used for bird acquisition, housing, and behavioral testing were in adherence to guidelines approved by the National Institutes of Health Guide for the Care and Use of

Laboratory Animals (DHEW Publication 80-23, Revised 1985, Office of Science and Health

Reports, DRR/NIH, Bethesda, MD 20205). The studies described here were approved by the

University of Wisconsin-Madison Institutional Animal Care and Use Committee (Protocol

Number: L00379-0-08-06).

Animals

In November and December of 2008 and 2009, 81 adult male European starlings (Sturnus vulgaris) and 10 adult females were captured on a single farm west of Madison, WI using baited fly-in traps (Table 1). A Federal Migratory Bird Scientific Collecting permit is not required for

European starlings as they are not covered under the Migratory Bird Treaty Act. After capture, birds were housed indoors in the University of Wisconsin-Madison Department of Zoology animal facilities in single sex cages (91cm × 47cm × 47cm) on photoperiods similar to the outdoor natural light cycle. Food (Purina Mills Start and Grow Sunfresh Recipe, 61S3-IGH-G) and tap water were always provided ad libitum. Each animal was assigned a number as well as a colored leg band for identification.

Analgesia Test

The analgesia test described here is similar to an opioid-sensitive analgesia test used in past work on Japanese quail (Coturnix japonica) [15] and house sparrows (Passer domesticus) [16]. A

250ml beaker was filled with tap water and placed on a hot plate. The water was stirred and the temperature was measured continuously using a digital thermometer with a resolution of 0.1°C

(Traceable Thermometer, -50°C to 150°C). Each subject was held in one hand and the foot up to the ankle joint was quickly lowered into the water bath. The head was covered with a hood to reduce visual distraction. Foot withdrawal latency (i.e., analgesia) was measured as the time for the bird to remove its foot from the water bath in a temperature established in Experiment 1.

Times were recorded using a stopwatch with a resolution of 0.01s (ThermoScientific: Cimarec).

The maximum time allowed for testing was 20s; thereafter the foot was manually removed. The 29 ambient air temperature of the room was within the range of 20-26°C. After the test, the foot was submerged in room temperature water and the animal returned to its home cage.

Experiment 1: Latency to Withdrawal Curve

Nine (Table 1) birds were housed in groups of three on 8 hours of light (L): 16 hours of darkness

(D) in single sex cages and were used to identify the temperature that generated a measurable analgesia response. The foot withdrawal latency for each bird was tested at water temperatures ranging from 40°C to 60°C in increments of 2.5°C [16]. The order of water temperatures tested was randomly selected for each bird, to avoid confounding group and day effects. At least one full day was used as a recovery period between testing (testing did not exceed three times in a single week). Testing was performed in the light phase between 8:30 and 11:30. The total procedure time, from capture to returning a bird to its standard home cage, lasted less than five minutes per trial. Each male was tested a total of nine times.

Experiment 2: Test to confirm the thermal test is opioid sensitive

In this experiment we examined whether peripheral injections of deionized water (vehicle control), naloxone (opioid antagonist, 20 mg/kg dissolved in deionized water), or fentanyl

(opioid agonist, 0.25 mg/kg dissolved in deionized water) modify the foot withdrawal response to hot water.

Males, n=15 (Table 1), were housed on artificial photoperiods of 18L: 6D for a length of six weeks, a photoperiod which induces refractoriness, a physiological state observed naturally in starlings in late summer / early fall in which the level of circulating reproductive hormones is 30 basal [17]. This photoperiod was selected to mitigate any possible effects of steroid hormones on analgesia. Animals were housed in groups of 5 in single sex cages (91cm × 47cm × 47cm). Each male was injected with 0.05mL (subcutaneous injection, inguinal leg fold) of the appropriate dose of the drug (5 birds per drug group), and placed in a covered holding cage (birds were visually but not acoustically isolated). Thirty minutes after injection, analgesia was measured in each male using the hot water test described above. Time of testing after injection was designed to fit within the half-life of the drug provided by the manufacturer. The latency to withdrawal was averaged in each group.

Experiment 3: Tests of Song-associated Analgesia

Breeding Season Condition

Photoperiod and hormone manipulations were used to place males into a physiological state characteristic of the natural spring breeding season. Specifically, birds were placed on photoperiods of 18L: 6D for 6 weeks, followed by 6L: 18D for an additional 6 weeks. Exposure of male starlings to this regime of long followed by short photoperiods induces a physiological state referred to as photosensitivity, a condition in which males respond to increasing day length

(characteristic of the spring breeding season) and testosterone with increases in the production of sexually-motivated behaviors, including courtship song [3,17,18]. Birds were moved into single- sex indoor aviaries (3.5m × 2.25m × 2m) on photoperiods of 11L: 13D, a photoperiod under which male starlings respond to testosterone treatment with increases in female-directed singing behavior (e.g.,[3,19]). Birds were randomly assigned to groups ranging from two to five.

Aviaries contained nest boxes, multiple perches, food and water. Aviaries were visually isolated.

Each breeding season condition male, n=35 (Table 1), received two silastic subcutaneous implants of T (two, 14 mm in length of i.d., 1.47 mm; o.d. 1.96 mm; Dow Corning, Midland, MI

USA, packed with 10-mm crystalline testosterone proprionate, Sigma-Aldrich, St. Louis, MO

USA). Each stimulus female, n=10, received two silastic implants of 17β-estradiol (two, 17 mm in length packed with 13 mm 17β-estradiol, Sigma-Aldrich), to enhance female sexual interest.

Hormone implants were surgically placed above the left breast muscle two weeks prior to behavioral testing as described in [3]. Past results show that the T manipulation in males elevates serum T concentrations to those observed within the breeding season [20].

Immediately prior to a single behavioral observation period, a novel stimulus female and nest material (green grass clippings and leaves) were introduced into the aviary. Male singing behavior was observed for 20 minutes between 10:00 and 14:30 hours. Starling song consists of four components: introductory whistles, complex phrases, click series and high frequency phrases [21]. An observer located behind a one-way mirror recorded the number of times a starling produced any component of song. A song was considered new if separated by at least 2 seconds. Components were summed to create a measure of Total Song. In addition, gathering nest material and the number of nest box entries was also recorded.

Immediately after the 20 min observation period a second experimenter entered the room and rapidly captured the subject. The right foot of the subject was placed in a water bath containing water at 55⁰ +/- 0.3⁰, a temperature established in experiment one, and the Latency to

Withdrawal the foot was recorded.

In order to determine whether the analgesia measure can be explained by behaviors other than song, for a subset of animals (n=20; Table 1), additional measures of behavior were collected, specifically feeding and drinking. A distinct bout of behavior was defined as an event separated from the next event by at least 2 seconds. Additionally, after the testing period, each male was checked to confirm the presence of hormone implants and a blood sample was taken for T analysis. A single sample of 200μL of blood was collected via venipuncture of the ulnar vein of

19 males. Plasma T was measured using a competitive immunoassay (EIA; Cayman Chemical,

Ann Arbor, MI, USA, Catalog No. 582701) in accordance to manufacturer’s directions and as reported in [3].

Non-breeding Season Condition

Males, n=22 (Table 1), in the non-breeding season condition were placed on photoperiods of

18L: 6D for six weeks followed by a photoperiod of 6L: 18D for the remainder of the study. As described above, this photoperiod regime induces photosensitivity. Under natural conditions male starlings become photosensitive in the fall non-breeding season, and as long as day length is relatively short (throughout the fall and winter non-breeding season), testosterone remains low

[17,22]. Males were housed in groups of four in aviaries set up as described for the breeding season condition males. Birds were observed behaviorally and tested for withdrawal response as described above. However, a female and nest material were not introduced into the aviary as these are not biologically relevant stimuli for male starlings in a non-breeding condition [23].

Measures of eating and drinking were also recorded. T was not measured in males in this condition because based on past studies the circulating levels of T are below the detectable level of the assay (e.g. [24]). 33

Statistical Analysis

All data were analyzed using Statistica 6.0 software (Stat Soft Inc., Tulsa, OK). Levene’s test for homogeneity of variance and Lilliefors test for normality were used to test the assumptions required for the use of parametric statistics. Outliers identified in residual analysis plots were removed if they fell outside two times the standard deviation of the mean. This resulted in the removal of a single breeding season condition male from the song-associated analgesia study

(latency to withdraw foot = 19.42 sec, total song=1) and one male from the pharmacology study

(latency to retract foot = 20 sec after each manipulation). (These outliers are not included in sample sizes described above or in Table 1).

Peripheral Pharmacology

A one-way analysis of variance (ANOVA) was used to examine differences in the foot withdrawal response across pharmacological treatments. Post-hoc analyses of significant

ANOVA results were performed using Fisher's LSD tests.

Song-associated Analgesia

Pearson correlations were used to evaluate relationships between latency to withdrawal the foot and total song in the breeding and non-breeding season conditions. The relationship between the analgesia response and additional measures (i.e., feeding and drinking, plasma T) were also analyzed using Pearson correlations for the subset of breeding season condition birds for which these measures were collected.

Within the breeding season condition some males acquired nesting territories (nest boxes); others did not. In starlings nest box owners sing high rates of song in response to the introduction of a female (female-directed song) and socially dominate other males [23,25]. Males without boxes sing, but do not increase their rates in the presence of a female and are socially subordinate to other males [23,25]. Based on these behavioral differences we also analyzed data in males with and without nest boxes separately. Nest box owners were defined as males entering and exiting the same nest box at least 2 times during the observation period.

In all conditions, birds that did not sing during the behavioral observation period were dropped from analysis (non-breeding season condition n = 6, breeding season condition n = 9 (1 nest box owner, 8 non-owners)). (Birds that did not sing are not included in sample sizes described above or in Table 1).

3. Results

Latency to withdrawal

The mean latency +/- the standard error at each temperature (40-60⁰C) was plotted to establish a temperature latency curve (Figure 1). Similar to Japanese quail [15] and house sparrows [16], the latency curve had a sigmoid shape with a “non-response plateau” at low temperatures, a “high response plateau” at high temperatures and a slope. The temperature of 55⁰C +/- 0.3⁰C was selected as it fell within the slope portion of the curve and generated measureable responses.

Peripheral Pharmacological Manipulations 35

A one-way ANOVA indicated that drug treatments significantly altered the analgesia response

(F(2, 11) =8.85, n = 14; p=0.005; Figure 2). Fisher LSD post hoc tests revealed significant differences between naloxone and control treatments (p=0.021) and naloxone and fentanyl treatments (p=0.002). There was no significant difference between fentanyl and control treatments (p=0.14).

Comparisons of Breeding and Non-Breeding Season Condition Birds

The average latency to withdrawal in breeding season and non-breeding season condition males was calculated. There were no significant differences between the two groups

(mean of non-breeding=9.54, SD=6.83; mean of breeding=7.43, SD=6.05; t38=1.02, p>0.10; Figure 3A). There was also no difference when breeding condition males were analyzed based on nest box status (mean of non-owners= 5.93, SD=3.071; mean of owners=8.27, SD=7.17; t23=0.92, p>0.10; Figure 3A). The average bouts of total song were also calculated in each condition. There was a significant difference in total singing behavior between the breeding season and non-breeding season (mean of non- breeding=7.0, SD=2.67; mean of breeding=18.96, SD=14.14; t38=3.23, p=0.0026l;

Figure 3B). There was also a difference between owners and non-owners (mean of non- owners=5.67; SD=3.04; mean of owners=26.43, SD=12.2; t23=4.97, p=0.000050; Figure

3B). There was no difference between non-breeding season and non-owners (t22=1.124, p=0.27; Figure 3B); however, there was a significant difference between non-breeding season and owners (t29=6.023, p=0.000001; Figure 3B).

Non-Breeding Season Condition

A significant positive correlation was identified between total song production and analgesia

(n=15, r=0.59, p=0.017; Figure 4A). There was no correlation between analgesia and the measure of feeding and drinking (n=15, r=-0.37, p=0.16).

Breeding Season Condition

Correlation analyses revealed no significant relationship between total song and analgesia in the breeding season condition males (n=25, r=-0.16, p>0.10; Figure 4B). However, when nest box owners (entering box mean=3.4, SD=5.55; gathering nest material mean=0.9, SD=1.80) and non- owners (no nest box directed behaviors) were analyzed separately, there was a significant negative correlation between the variables in non-owners (n=9, r=-0.845, p=0.0041; Figure 5A) but not owners (n=16, r=-0.42, p=0.11; Figure 5B).

No significant correlations were identified between analgesia and feeding and drinking (n=20, r=0.36, p=0.11), in the 10 nest box owners and 10 non-owners for which these measures were collected. Furthermore, there was no correlation between T and analgesia (n=19, r=-0.24, p>0.10). Additionally, T did not differ between nest box owners and non-owners (mean nest box owners=3391.80 pg/mL, SD=324.82, mean non-owners=3339.48 pg/mL, SD=362.87; t17=0.33, p>0.10).

4. Discussion

In starlings, opioids within the POM and possibly VTA have been linked closely to undirected but not directed song production [2]. Opioid release in these regions also leads to analgesia, at 37 least in rats [13,14]; therefore, if production of undirected song is linked to opioid release in these areas, then production of undirected, but not directed song should be associated with analgesia. The present study supports this hypothesis and is the first to demonstrate a tight link between analgesia measures and production of undirected but not female-directed male song.

Furthermore, in subordinate breeding season condition male starlings that did not defend a nest site (non-owners), analgesia and song were negatively correlated, suggesting a possible distinct role for opioids in singing behavior in this context as well.

Injections of the opioid antagonist naloxone significantly decreased analgesia compared to control injections, whereas the opioid agonist fentanyl increased analgesia compared to the naloxone treated animals. Fentanyl in the present study did not statistically increase analgesia relative to control injection, which may reflect a ceiling effect; however, our results overall are consistent with past studies showing the analgesia test used here to be opioid-dependent [15,16].

The production of undirected song correlated positively with analgesia

Few studies have examined opioids and undirected song, but in those that have, research suggests that opioids stimulate song in this context. For example, in zebra finches undirected song is inhibited by opioid antagonist injections [4]. Consistent with these findings, in male starlings the densities of immunolabeled met-enkephalin fibers in POM correlate positively with undirected song [2]. In addition to mediating analgesic responses, opioids (e.g., met-enkephalin and morphine) in both the preoptic area and VTA respectively is rewarding, at least in mammals

[6,7]. Undirected song is not associated with any form of obvious, immediate, external reward

(e.g., it does not immediately attract a mate). Our working hypothesis is that undirected song is 38 triggered and maintained by intrinsic-reward induced by release of rewarding neurochemicals such as opioids [9,10]. Recently, the rewarding properties of producing directed and undirected song were evaluated in male starlings and zebra finches using a conditioned place preference paradigm [8]. Males of both species were found to develop a strong preference for a place associated with the act of producing undirected (but not directed) song, thereby linking the production of undirected song to a positive affective state (i.e., a reward state). Together, the analgesia results reported here along with the place preference data indicate that opioid release may underlie reward associated with undirected vocal communication. Additional research using

POM and VTA site-specific pharmacological manipulations and measures of analgesia and reward are needed to evaluate this hypothesis.

Female-directed song did not relate to analgesia

Although it is possible that some of the songs produced by males in the breeding season condition were undirected, our assumption based on past studies of males with elevated T (e.g.

[23]) is that a larger proportion of the songs produced by breeding season condition males tested in the presence of a female are directed than undirected; and certainly these males produce more directed songs than non-breeding season condition birds (who based on past literature are unlikely to sing any female-directed songs) [23]. We found no correlation between the measure of analgesia and female-directed song production in dominant breeding season condition males with nesting sites, suggesting that opioid release is not linked to the production of female- directed song in the same way as undirected song. This idea is supported by past data showing that densities of immunolabeled met-enkephalin fibers in POM did not correlate linearly with directed song in male starlings [2]. Furthermore, males that occupy a nest site have a lower 39 density of mu-opioid receptors in POM and VTA compared to males without nest sites [3].

Additionally, enkephalin opioids in the avian POM have been found to suppress male sexual behavior [26]. Thus, a reduction in opioid activity in males with nest sites should serve to facilitate sexually-motivated male behaviors, including production of courtship song. This idea is supported by data showing that peripheral administration of the opioid receptor antagonist naloxone facilitated female-directed song in male starlings [5]. In the VTA, mu-opioid receptor immunolabeling is negatively correlated with measures of total song in breeding condition starlings [3], perhaps reflecting an inhibitory role for opioids in VTA in male song in this context. In contrast to undirected song, female-directed song can result in immediate mate attraction and copulation. Thus, our working hypothesis is that directed song is primarily externally-reinforced by neurochemicals (including opioids) released upon successful mate attraction and copulation [9,10] rather than in close association with the act of song production.

It is also possible that opioids are released during female-directed song at low levels that are not detectable using an analgesia measure such as the latency to withdrawal from a hot water bath.

Therefore, additional direct measures of opioid release such as microdialysis measures should be investigated in future studies.

Song correlated negatively with analgesia in subordinate breeding season condition males

Here, we found a negative correlation between the measure of analgesia and singing behavior in subordinate breeding season condition males that failed to acquire nest boxes. During the breeding season, males that do not obtain nest sites sing, but they do not increase singing behavior in response to females [23]. This type of song may be a form of directed song that is 40 suppressed in males that fail to acquire a nesting location. This idea is supported by the observation that when a nest box owner is removed from an aviary it is common for a male without a nest box to rapidly (within hours) take over the box and initiate high rates of female- directed song (personal observation). Endogenous opioids (e.g. enkephalins and endorphins) inhibit socio-sexual behaviors [27,28]; and in starlings pharmacological manipulations indicate that mu-opioid receptor stimulation inhibits female-directed song [5] (but see [4] for an exception). Male starlings without nest sites have significantly higher densities of mu-opioid receptors in the POM and VTA and other areas than males with nest sites [3]. As reviewed above, enkephalin opioids in the avian POM inhibit male sexual behavior [26]. Thus, heightened tissue sensitivity to opioids (reflected in higher receptor densities) and heightened release

(reflected in the analgesia response reported here) suggest that opioids may be acting to suppress courtship song in males without a nesting site. The possibility that opioid release (and associated analgesia) serves to suppress sexual behavior in contexts in which it may not be appropriate (e.g., for a male without a nest site) is consistent with a past study in mice in which a reduction in female sexual responses to potential mates infected with parasites was associated with opioid- mediated analgesia [29].

Analgesia did not differ categorically across conditions

Although males in spring condition with nest boxes sang at much higher rates than males in spring condition without nest boxes or fall-condition males, mean analgesia responses did not differ categorically across breeding season conditions. We do not believe that the lack of categorical differences in analgesia responses rule out our interpretations of the correlational data

(that opioid release is differentially linked to communication in distinct contexts). The lack of 41 categorical differences may reflect the fact that opioids are involved in multiple processes in addition to singing, that also differ across birds in the three conditions (e.g., feeding, stress, thermoregulation, reproductive physiology (reviewed in [11,28,30])). Furthermore, we expect that differences in tissue sensitivity to opioids or differences in receptor subtype distributions in males in the three conditions also may explain why a high song rate would not always be associated with high analgesia (e.g., in spring-condition birds with nest boxes) and why a low song rate would not always be linked to low analgesia (e.g., in fall-condition birds). This idea is supported by data in male dark-eyed juncos showing that mu and kappa opioid receptor densities differed seasonally in the POM and VTA [31], and data in starlings showing that mu receptor densities were greater in spring condition males with next boxes compared to those without nest boxes [3]. These findings suggest that even though birds in spring condition with a nest box sing more than birds in other conditions, opioid-mediated analgesia may not differ because the densities of opioid receptors in brain regions mediating this response differ in males across conditions. These factors may in part explain why analgesia responses do not differ categorically across groups.

Opioids and Steroid Hormone Interactions

Steroid hormones are known to alter neural reward systems and shape behavior so that it is appropriate for an individual within a particular context. For example, in female rats proceptive behaviors alter steroid hormone levels so that copulation is rewarding [32]; pregnancy hormones are known to influence neural reward systems so that interactions with offspring are rewarding at birth [33]; and reward associated with feeding behaviors is rapidly adjusted by nutrient-induced hormone actions on reward circuitry [34]. In the present study seasonal and social status-related 42 differences in T activity may have contributed to changes in opioid reward systems so that males sing a song appropriate for the season and an individual’s social status. T strongly influences the motivation to sing in male starlings [23] and, in rodents, enkephalin opioids have been found to be affected by T, including within the preoptic area and VTA [35,36,37]. Data also indicate that mu-opioid receptor densities shift seasonally in POM and VTA in association with testis volume

[31]. It is thus possible that T differences in the present study contributed to the differential links identified between analgesia and singing behavior. Here, we found no differences in the overall analgesia responses for males in either hormonal condition. In a subset of our data, we analyzed circulating T concentrations in breeding condition males but did not see any significant relationships between T concentrations and analgesia. Previous studies indicate that T has inconsistent effects on analgesia [38,39], including in birds using the same test employed in the present study [16,40]. Thus, the impact of steroid hormones on opioid activity, singing, and analgesia is at present unclear.

Future Directions and Broader Impacts

While this study focused on opioids and analgesia, there are several other neurotransmitter systems that affect analgesia and may contribute to the effects observed here, including GABA, endocannabinoids, and substance P [41,42,43]. Furthermore, there are several brain regions in addition to the preoptic area and VTA that regulate analgesia and contain opioid receptors, including the periaqueductal gray [44] the nucleus accumbens [45]. and the anterior hypothalamus [13,46]. Finally, while the majority of research has focused on the mu-opioid receptor, future research should target multiple opioid receptor subtypes such as kappa and delta. 43

The mechanisms underlying the links between singing and analgesia reported here must be identified in future work.

Human data also link undirected vocal behaviors to analgesia and opioid release. For example, swearing that was not directed toward another individual increased pain tolerance compared to tolerance in individuals that did not swear [47]. Furthermore, relaxed social laughter in humans that is considered important for group bonding (similar to undirected song in overwintering starling flocks) was associated with feelings of well-being as well as analgesia [48]. Thus, the link between analgesia and undirected vocal behavior identified here appears to extend beyond songbirds. This link may have implications for the use of vocal production in humans to promote positive affect and to reduce responses to painful stimuli in a clinical or hospital setting.

Chapter Acknowledgments

The data presented in this paper are based upon work supported by grants R01 MH080225 to

LVR. We acknowledge Kate Skogen and Chris Elliot for animal care taking, Griffin Gessay,

Rachel McCormick and Dr. Ben Pawlisch for helping to collect pilot data.

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Figures and Figure Legends

Figure 1: Temperature latency curve. The curve showing mean (+/- standard error) latency to withdrawal the foot at increasing water temperatures in male starlings. The temperature of

55.5⁰C (indicated with an asterisk) was selected for all experiments as it fell within the slope of the curve and generated a measurable response in all subjects.

Figure 2: The foot withdrawal response is opioid sensitive. Mean latency to withdrawal and standard error in males receiving peripheral injections of diH20 (control; black bar); 20.0 mg/kg naloxone (NAL; gray bar); 0.25 mg/kg fentanyl (FEN; white bar). Individuals are represented by a single circle in each condition. Sample sizes are indicated in the bottom of each bar. Brackets indicate the results of the Fisher post hoc contrasts.

Figure 3: The average latency to withdrawal the foot did not differ across groups. The average bouts of Total Song in breeding nest box owners were significantly different from non-owners and non-breeding condition. A. Latency to withdrawal the foot in non-breeding condition (black bar) and breeding condition (nest box non-owners and owners, gray bars) +SEM. B. Average bouts of total song in each condition +SEM. Statistical significance is represented by lines and

*** (p<.00001). Sample sizes are indicated in the bottom of each bar.

Figure 4: Analgesia responses correlate with song production in non-breeding season condition males. Data shown are the latency to withdrawal the foot (in seconds) versus the total song produced by each individual. Each point represents one individual. A. Individuals in the non- breeding season condition. B. Breeding season condition individuals. Sample sizes are noted in the bottom right corner. Presence of the regression line indicates a significant correlation

(p<0.05).

Figure 5: Analgesia responses within the breeding season condition correlate with song production in males without nest boxes. Data from Fig. 4B have been replotted to illustrate relationships between the latency to withdrawal the foot (in seconds) versus the total song produced by each individual for A. Breeding season condition individuals that did not occupy a nest site and B. Breeding season males that occupied and defended nest sites. Sample sizes are noted in the bottom right corner. Presence of the regression line indicates a significant correlation (p<0.05).

Tables

Table 1: Final sample sizes for each experiment Experiment Number of Animals 1. Latency to withdrawal curve 9 2. Test to confirm thermal test is opioid sensitive 15 3a. Song Associated Analgesia: Breeding Condition 35 3b. Analysis of behaviors, T and analgesia* 20 4. Song Associated Analgesia: Non-Breeding Condition 22 *subset of the same birds used in 3a.

Chapter III: Curvilinear relationships between mu-opioid receptor densities and undirected song in male European starlings (Sturnus vulgaris)

Individual manuscript is currently under review at Brain Research

Kelm-Nelson, C.A., Riters, L.V. (2012) Curvilinear relationships between mu-opioid receptor densities and undirected song in male European starlings (Sturnus vulgaris). Under Review.

Abstract

The neural basis of female-directed communication in males has been reasonably well studied; yet, relatively little is known about communication in other important social contexts. In songbirds, data show that undirected song produced spontaneously in non-sexual, affiliative social contexts is tightly coupled to analgesia and positive affective state, which are both known to be mediated by opioid activity, at least in mammals. Consistent with this idea, labeling for the opioid met-enkephalin in the medial preoptic nucleus (POM) correlates positively with undirected song production. We propose that undirected singing behavior is facilitated and maintained by mu-opioid receptor activity in the POM and possibly 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 several brain regions in male starlings. Polynomial regression analysis revealed significant inverted-U shaped relationships between measures of undirected song and mu-opioid receptor labeling densities 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. Overall the results suggest that opioid activity in POM, BSTm, and PAG may underlie previous links identified between undirected song, analgesia, and affective state. 56

Key Words: opioids; vocal communication; reward; birdsong; motivation; mu-opioid receptor

1. Introduction

Across the animal kingdom, vocal communication is critical for successful social interactions.

Although female-directed courtship vocalizations produced by males have been relatively well- studied, less is known about communication in other social contexts. In some songbirds, song is observed at high rates in large affiliative flocks [1,2,3] where it has been proposed to play a role in song learning or maintenance [2,4,5]. This type of song is not directed towards a particular individual and it appears to be ignored by nearby conspecifics, and has thus been termed

“undirected song” [3,6,7,8]. Additionally, undirected song does not result in an immediate, obvious external rewarding factor (e.g., song does not result in mate attraction and copulation).

Instead, it has been hypothesized that undirected communication may be facilitated and maintained by intrinsic reward and/or positive affective state, including song-associated opioid- mediated reward [9].

Multiple lines of evidence link opioids to undirected birdsong. First, positive correlations were identified between labeling for the opioid protein met-enkephalin (mENK) in the medial preoptic nucleus (POM) and undirected song produced by male European starlings (Sturnus vulgaris)

(with a similar trend identified for the ventral tegmental area (VTA); p=0.06) [10]. Opioids in the medial preoptic area induce both analgesia and reward/positive affect, at least in rats

[11,12,13], and recent data link undirected song in male starlings to both opioid-mediated analgesia and positive affect. Specifically, analgesia measures correlated positively with male starling undirected song rates [14], and both male starlings and zebra finches singing high rates 57 of undirected song developed a conditioned place preference for a chamber previously paired with undirected singing behavior [15]. These results link undirected song to a positive (or at least a less negative) affective state, which we hypothesize may, in part, be mediated by opioids [9].

The present study was designed to further examine links between opioids and undirected song in male starlings. During fall and winter months (i.e., the non-breeding season) when testosterone concentrations are low male starlings do not sing to attract females or defend nesting territories

[1]. However, they sing at high rates as part of affiliative overwintering flocks [1,2]. Song in these flocks is proposed to function primarily to maintain flock cohesion [4] and can be considered a form of undirected, affiliative communication. We used immunohistochemistry to examine links between undirected song in male starlings with low testosterone singing in flocks and mu-opioid receptor labeling. If previously reported links between undirected song, analgesia and reward are mediated by opioids, then we predict that undirected song production will be linked to mu-opioid receptor densities in regions in which opioids have been found to induce analgesia and / or reward, which include the POM, the ventral tegmental area (VTA), periaqueductal gray (PAG), bed nucleus of the stria teminalis (BSTm), lateral septum (SL) and periventricular nucleus (PVN) (analgesia: [11,12,16,17,18] and reward: [13,19,20,21,22,23].

2. Materials and Methods

Animals and Protocols

Eighteen male starlings were captured on a single farm in Madison, WI using baited fly-in traps.

After capture, males were housed indoors in stainless steel, single sex cages (91cm x 47cm x

47cm) within the University of Wisconsin’s Department of Zoology indoor animal facilities. 58

Food (Purina Mills Start and Grow Sunfresh Recipe, 61S3-IGH-G) and water were provided ad libitum. For identification, each animal was assigned a numbered as well as a colored leg band.

Protocols used for bird acquisition, housing, and behavioral testing were in adherence to guidelines approved by the National Institutes of Health Guide for the Care and Use of

Laboratory Animals (DHEW Publication 80-23, Revised 1985, Office of Science and Health

Reports, DRR/NIH, Bethesda, MD 20205) and approved by the University of Wisconsin-

Madison Institutional Animal Care and Use Committee.

Light Cycle and Housing

Animals were randomly assigned to one of five social outdoor aviaries (3 or 4 birds per aviary;

2.13m x 2.4m x 1.98m) and allowed to habituate prior to the beginning of behavioral testing. The natural light cycle during the experiment, occurring in fall (early October), was approximately

11hr: 40 min light, decreasing daylight by 3 minutes each day.

Over five consecutive days, each aviary was observed for 20 min by a single observer concealed by dark green and brown camouflage netting and blinds. Each aviary contained four nest boxes and branches for perching. Food and water were provided ad libitum. Each aviary was visually, but not acoustically isolated from the others by the use of camouflage blinds. For seven days prior to the first day of behavioral observations an observer sat in front of each aviary (behind a blind) to allow males to habituate to her presence.

Behavioral Observations

The testing order of the aviaries was randomized, with observations performed between 9:00 and

13:00 each day. Measures of starling song included bouts of full song (four distinct components including introductory whistles, complex phrases, click-series, and high frequency phrases [2]), bouts of fragments (at least 2 components of song), and bouts of introductory whistles. These measures were summed to create a measure of Total Song. Additionally, bouts of feeding, drinking, preening and beak wiping were recorded. A distinct bout was defined as an event separated by at least 2 seconds.

T Assay, Tissue Collection, and Immunohistochemistry

Immediately after the last observation, all males in a group were rapidly decapitated. The testes were measured to ensure that they were in a regressed stage, as is typical of the non-breeding season [55]. Additionally, a terminal trunk blood sample was taken immediately after sacrifice to confirm fall typical low or undetectable levels of the hormone T. Plasma T was measured with a commercial grade competitive assay (EIA; Cayman Chemical, Ann Arbor, MI, USA, Catalog

No. 582701) as described previously in Kelm et al. 2011, and by manufacturer’s instructions.

Brains were removed by dissection, fixed in 5% acrolein overnight, rinsed, cryoprotected in 30% sucrose for 3 days and frozen at -80⁰C. Using a cryostat, brains were cut in the coronal plane in three, 40μm series and stored in anti-freeze until processing. Series one was used for mu-opioid receptor labeling discussed here.

Brain tissue was processed using immunohistochemistry to identify mu-opioid receptor protein.

The primary antibody was an anti- mu-opioid receptor antibody made in rabbit (Abcam, ab10275, 1:5000). The secondary antibody was biotinylated goat anti-rabbit (Vector

Laboratories, 1:1000). Briefly, sections were rinsed in phosphate buffered saline (PBS) for

30 min, incubated in 0.5% sodium borohydride solution for 15 min, rinsed in PBS for 20 min, incubated in 0.5% hydrogen peroxide solution for 10 min, rinsed in PBS for 20 min, incubated in

20% normal goat serum solution for 1 h, and then incubated in 2% NGS primary solution overnight at room temperature. Sections were then rinsed in PBS-T for 30 min and incubated in

2% NGS biotinylated secondary solution for 90 min at room temperature. Sections were then rinsed in PBS-T for 30 min, incubated in AB solution (Vectastain Elite ABC, Vector

Laboratories) for 1 h, rinsed in PBS-T for 30 min, and the avidin–biotin complex was visualized using 3,3′-Diaminobenzidine (DAB) tablets (Sigma Aldrich, St. Louis, MO, USA). Sections were float mounted onto gel-coated slides, dehydrated in a series of alcohols, and cover slipped.

Antibody specified was verified using preadsorption, omitting the primary and via Western blot analysis as reported in Kelm et al. (2011).

Quantification of Brain Regions

A Spot Camera (Diagnostic Instruments, Inc.) connecting a microscope to a computer was used to acquire images of immunolabeled brain regions. Using METAVUE (Fryer Company, Inc.,

Huntley, IL, USA) software, the mean total pixel area covered by mu-opioid receptor labeling was quantified from three serial sections for each bird in POM, PAG, BSTm, VTA, PVN, and

SL. The locations of these nuclei were based on Heimovics and Riters (2007) (Figure 4). The portion of PAG measured is the area recently suggested as similar to the ventral PAG column in 61 mammals [56], which is the portion that regulates analgesia [57]. The total pixel area provided a measure of the approximate area covered by labeled receptors (pixels highlighted fibers using a computer generated threshold) within boxes or ovals centered in each region (Table 3). In cases of tissue damage, labeling was quantified either on a fourth section or the individual was dropped from analysis for affected brain areas.

Statistical Analysis

Data were analyzed using Statistica 6.0 software (StatSoft®, Inc., Tulsa, OK, USA). Lilliefors test for normality and Levene’s test for homogeneity of variance were used to test the assumptions required for the use of parametric statistics. When variables did not meet assumptions they were transformed. Specifically, to improve homogeneity of variance the variable was square root transformed and to improve normality the variable was log transformed.

Specific statistical tests are described in the results section.

3. Results

Behavior and testosterone concentrations

Eighteen males were used for the behavioral analysis. Male testosterone concentrations were in the range typical of the non-breeding season. T concentrations were below detectable levels for all but four males (for males with detectable levels n=4, mean=144.85 pg/mL, SD=207.65). For reference, the average T measure for breeding males is approximately 2930.00 pg/mL [24].

Furthermore, individuals did not show behaviors indicative of birds during the breeding season

(i.e., they did not collect nest material, wing wave, or displace other males from nesting sites).

Taken together, these results indicate that the hormone and photoperiod states were similar to 62 wild birds in the non-breeding season when male starlings sing high rates of undirected but not female-directed song.

Mu-opioid Receptor Labeling and Total Song

We first ran linear regression analyses to examine relationships between the mu-opioid receptor immunolabeling average total pixel area and Total Song. In all cases linear regression results were not significant and the data did not fit a linear model (p>0.50; Table 1). Therefore, we ran nonlinear, higher-order curve polynomial regression analyses. Specifically, in separate polynomial regression analyses the average total pixel area for a brain region was entered as the dependent variable and the Total Song was entered as the predictor variable. Statistical outliers identified in residual analysis plots were removed if they fell outside two times the standard deviation of the mean. This resulted in the removal of one animal for POM (total song=25, mean total pixel area POM=1177.83); one for BSTm and SL (total song=44, mean total pixel area

BSTm=34662.8, mean total pixel area SL=12701.8, respectively), and an additional animal for

BSTm (total song= 3, mean total pixel area=23484.0). Additionally, due to tissue damage during processing, measures were lacking for some individuals which explain differing sample sizes.

Descriptively, densities of mu-opioid receptors and Total Song exhibited an inverted U-shaped pattern where mu-opioid receptor densities in POM, PAG and BSTm were low in both the lowest and highest singers. Results of the polynomial regression analyses revealed significant curvilinear relationships between mu-opioid receptor density in POM, PAG, and BSTm and

Total Song (Table 2; Figure 1, A-C). There were no significant polynomial relationships between 63 measures of mu-opioid receptor densities in VTA, PVN, or SL and Total Song (Table 2; Figure

2, A-C).

Mu-opioid Receptor Labeling and General Behaviors

General behaviors (feeding, drinking, and beak wiping) and mu receptor labeling in each brain region did not display curvilinear tendencies. Therefore, to determine whether mu-opioid labeling could be explained statistically by behavioral variables in addition to Total Song, we investigated the relationship of immunolabeling and non-song behaviors (bouts of feeding, drinking, preening and beak wiping) using linear multiple regression analysis. Specifically, multiple regression analyses were run using the brain region as the dependent variable and the behaviors as the independent variables. Both backward and forward analyses were performed, and in most cases were identical. For VTA analysis of both forward and backward resulted in the same (though not identical) significant effects. Results of the forward analysis are provided because this model best explained the data based on the highest adjusted R2, lowest standard error, and the best residual plots.

For measures of the total pixel area covered by mu-opioid receptor labeling, results of the multiple regression analyses revealed bouts of feeding to contribute significantly (negatively) to variance in total pixel area in both PAG (n=16, adj. R2=0.33, feeding beta=-0.74, SE of

2 beta=18491.93, t13=5.14, p=0.03; Figure 3A) and VTA (n=15, adj. R =0.301, feeding beta=-0.59,

SE of beta=7794.5, t13=4.59, p=0.02; Figure 3B). No variables contributed to variance in POM,

BSTm, VTA, PVN or SL.

4. Discussion

In the present study we found links between affiliative, undirected singing behavior and mu- opioid receptor labeling in three regions in which opioids are implicated in analgesia and / or reward (as reviewed in the introduction), POM, PAG and BSTm. Specifically, we report a positive association between mu-opioid receptor labeling in each of these regions in birds singing low to medium rates of undirected song. However, there appeared to be a point, after which the highest rates of song were associated with low densities of receptor labeling, resulting in an inverted-U shaped relationship.

Interpretation of the inverted-U function

We suggest that relatively low rates of undirected song facilitate (and/or are facilitated by) relatively low levels of mu-opioid receptor activity in POM, PAG, and BSTm. Given that mu- opioid receptors down-regulate in response to sustained occupation by enkephalin [25,26], it is possible that high levels of opioid release in these regions are associated with high levels of song production which cause mu-opioid receptor down-regulation. Consistent with this possibility, and in contrast to the curvilinear relationships identified for mu receptors, in a past study immunolabeling for mENK (an endogenous ligand for mu and delta receptors) in POM related positively to undirected singing behavior even in the birds singing at the highest rates [10]. This past study, along with the present results, suggests that when mENK protein is highest (at least in

POM) mu-opioid receptor labeling is lowest, consistent with the possibility that sustained song- associated opioid release results in receptor down-regulation. This possibility must be examined in future work.

Mu-opioid receptors in POM, BSTm, and PAG may modify affect and vocal behavior

The POM, BSTm and PAG are components of a reciprocally connected [27] opioid-rich

[10,28,29,30,31,32] neural circuit that has been implicated in affiliative social behavior (e.g.,

[33,34]) in birds and mammals [35,36]. In male canaries, cell groups send projections from the

PAG to regions controlling song production [37], and labeling for the immediate early gene egr-

1 (often referred to as ZENK in songbird studies) is increased in the PAG in male zebra finches that sing undirected song compared to silent males [38]. In birds and mammals, the PAG is proposed to gather and integrate affective information from other brain regions, which it then relays to vocal production (motor) areas so that an animal emits a vocal signal reflective of its emotional state [38,39,40,41]. In cats, enkephalin opioids in the PAG suppressed negative, non- affiliative vocalizations [42], and in monkeys opioid antagonists increased vocal behavior linked to aversive states [43], suggesting a role for opioids in PAG in the inhibition of negative forms of vocal production. Studies in mammals also show that projections from the preoptic area and

BSTm to PAG promote positive vocal behaviors [44,45]. Together these data suggest that projections from the POM and BSTm to the PAG may promote positive forms of vocal behavior, including undirected singing.

The link reported here between the POM and undirected singing in starlings is consistent with the results of an increasing number of studies [10,46,47]. Bilateral electrolytic lesions to the

POM that suppressed sexually-motivated song increased undirected singing behavior [47], and met-enkephalin in this region was linked tightly to affiliative song [10]. Injections of enkephalin opioids directly into the POM of male quail suppressed neuronal activity [48]. This suggests that increased mu-opioid receptor activity in POM may act to suppress neuronal activity to facilitate 66 undirected singing behavior. This remains to be tested using future site-specific pharmacological manipulations.

The present data are also consistent with past work implicating the BSTm in undirected singing behavior. In male starlings immunolabeling for ZENK in BSTm correlated positively with undirected singing behavior [49]. Although the role of opioids in BSTm in vocal production has not been well studied, met-enkephalin in BSTm in cats suppressed negative forms of vocal production [50,51]. Recently in songbirds a population of BSTm neurons (vasotocin-producing) was found to increase activity in response to positively-valenced social stimuli and to reduce activity in response to negatively-valenced social stimuli [52,53]. These effects were not observed in response to a positive nonsocial stimulus, suggesting that the BSTm responds selectively to positively-valenced social stimuli.

No significant relationships were found between undirected song and mu-opioid receptors in VTA, PVN and SL

Past data in starlings singing undirected song showed a trend for a linear relationship between undirected singing and mENK protein immunolabeling in VTA (p=0.06) [10]. Here, we report no significant relationship between mu receptor immunolabeling in VTA and song. We also report no significant relationships between mu receptor labeling and undirected song in PVN or

SL. Although opioids in each of these regions have been found to induce reward and / or analgesia as reviewed in the introduction, the present results do not implicate mu receptors in these regions in undirected singing behavior in starlings; however additional study is warranted.

Mu-opioid receptors in PAG and VTA negatively correlated with feeding

Although non-specific behaviors generally did not correlate with measures of mu receptors in any of the brain regions examined, linear negative correlations were found between mu labeling in PAG and VTA and feeding behavior. Past studies implicate opioids in the VTA and PAG in feeding behavior [54], thus these findings may reflect a role for mu receptors in PAG and VTA in feeding. Alternatively it may be that birds engaging in other opioid-mediated behaviors such as undirected singing spend less time feeding, explaining the negative correlation between feeding and mu receptors in these regions.

Conclusions

Based on the present findings and past data, we suggest that in response to positive social and environmental conditions (e.g., in the presence of non-threatening conspecifics, an unlimited food supply, and shelter) opioid release in BSTm and POM induces a positive affective state which is relayed to the PAG. The PAG may integrate this information and transmit it to vocal control regions so that a bird produces undirected song when it is appropriate to do so. Opioids in

POM, BSTm, and PAG have been found to induce a positive affective state (at least in rodents), and projections from the preoptic area and BSTm to the PAG in mammals have been found to facilitate positive forms of vocal behavior. Based on these findings, we propose that the link between positive affective state and affiliative singing behavior identified in past work in starlings and zebra finches [14,15] may be regulated in part by song-associated opioid release in

POM, BSTm, and PAG. This prediction must be examined in future work.

Chapter Acknowledgments

Support/Grant Numbers: R01 MH080225 to LVR and the Zoology Department Bunde Fund

(2012) to CKN. Additionally, thank you to Kate Skogen and Chris Elliott for animal care, Bill

Feeny for assistance with the illustrations, and Dr. Ben Pawlisch and Sharon Stevenson for help with tissue processing. 69

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Figures and Figure Legends

Figure 1: Evidence for curvilinear relationships between mu-opioid receptor densities and undirected song rates. An inverted-U curve showing the measure of undirected song on the x- axis and average 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 and statistics indicated in the upper right corner of the figures. Presence of the red regression line indicates a significant relationship p>0.05.

Figure 2: Relationships between average mu-opioid pixel area and undirected song. The measure of undirected song is on the x-axis and the average 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 and statistics indicated in the upper right corner of the figures.

Figure 3: Relationships between average mu-opioid receptor pixel area and bouts of feeding behavior. The total number of bouts of feeding is on the x-axis and average 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 and statistics are indicated in the upper right hand corner of the figure.

Figure 4: Brain schematic indicating approximate areas in which pixel area of mu-opioid receptor 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. 78

Tables

Table 1: Non-Significant Linear Regression Results of Mu-Opioid Receptor Label and Total Song Standard Error of Brain Region F R2 Adj. R2 p-value (df) the Estimate POM 0.0000139(1, 15) 0.00000093 -0.0667 0.7702 0.997 PAG 0.0159(1, 14) 0.0011 -0.070 23381.10 0.904 BSTm 0.163 (1, 13) 0.0124 -0.0636 0.625 0.693 VTA 0.439 (1, 13) 0.033 -0.040 9515.55 0.520 PVN 0.294 (1, 16) 0.018 -0.043 1.349 0.594 SL 0.309 (1, 14) 0.022 -0.048 2735.632 0.587

Table 2: Polynomial Regression Results of Mu-Opioid Receptor Label and Total Song 2 2 Brain Region N F(df) R Adj. R p-value POM 17 4.98(1,14) 0.416 0.33 0.023 PAG 15 4.92(1, 12) 0.45 0.36 0.028 BSTm 14 12.26(1,12) 0.67 0.61 0.0012 VTA 15 1.55(1,12) 0.205 0.07 0.250 PVN 16 1.49(1,15) 0.17 0.05 0.250 SL 16 1.31(1,13) 0.17 0.04 0.300

Table 3: Box Measurements Nucleus Shape Size (mm) BSTm Rectangle Area= 0.67 x 0.37 CG Circle Diameter= 0.60 POM Rectangle Area= 0.43 x 0.44 PVN Rectangle Area= 0.22 x 0.42 SL Rectangle Area= 0.36 x 0.54 VTA Rectangle Area= 0.38 x 0.53

Chapter IV: Mu-opioid receptor densities are depleted in regions implicated in agonistic and sexual behavior in male European starlings (Sturnus vulgaris) defending nest sites and courting females

Individual manuscript has been previously published in Behavioural Brain Research.

Kelm, C.A., Forbes-Lorman, R.M., Auger, C.J., Riters, L.V. (2011) Mu-opioid receptor densities are depleted in regions implicated in agonistic and sexual behavior in male European starlings (Sturnus vulgaris) defending nest sites and courting females. Behav Brain Res. 219, 15-22. 82

Abstract

Social status and resource availability can strongly influence individual behavioral responses to conspecifics. In European starlings, males that acquire nest sites sing in response to females and dominate other males. Males without nest sites sing, but not to females, and they do not interact agonistically with other males. Little is known about the neural regulation of status- or resource- appropriate behavioral responses to conspecifics. Opioid neuropeptides are implicated in birdsong and agonistic behavior, suggesting that opioids may underlie differences in the production of these behaviors in males with and without nest sites. Here, we examined densities of immunolabeled mu-opioid receptors in groups of male starlings. Males that defended nest boxes dominated other males and sang at higher rates when presented with a female than males without nest boxes, independent of testosterone concentrations. Multiple regression analyses showed nest box ownership (not agonistic behavior or singing) predicted the optical density of receptor labeling in the medial bed nucleus of stria terminalis, paraventricular nucleus, ventral tegmental area and the medial preoptic nucleus. Compared to males without nest boxes, males with nest boxes had lower densities of immunolabeled mu-opioid receptors in these regions.

Singing additionally predicted the area covered by labeling in the ventral tegmental area. The results suggest that elevated opioid activity in these regions suppresses courtship and agonistic behavioral responses to conspecifics in males without nest boxes. The findings are consistent with a dynamic role for opioid receptors in adjusting social behavior so that it is appropriate given the resources available to an individual.

Keywords: birdsong; motivation; vocal communication; socially appropriate behavior; dominance; territoriality

1. Introduction

Across vertebrates both social status and the resources available to an individual can strongly influence the way in which an individual responds to conspecifics. For example, in male

European starlings (Sturnus vulgaris), the acquisition and defense of a nest site (or nest box in aviary studies) is a crucial first step for initiating mating behavior [1]. Male starlings with nest boxes sing high rates of song in response to females [2]. In contrast, male starlings that do not successfully obtain nest boxes often sing, but not in response to a female [2]. Males with nest boxes additionally displace other males from food sources and perches more often than males without nest boxes, suggesting nest box owners socially dominate males without boxes [3]. Thus, the distinct behavioral responses to conspecifics observed in males with and without nest boxes are established early in the breeding season through competition over limited available nest sites.

Little is known about how social status or resource possession alters the brain to ensure that an individual produces status- or resource-appropriate behavioral responses to conspecifics. In the present study we begin to explore a possible role for opioids in social behavior associated with nest box possession in flocks of male starlings.

Data implicate opioids in the regulation of birdsong and suggest that the effects of opioids on song differ depending upon whether song is directed towards a female (as in nest box owners) or undirected (as in males without nest boxes). In male starlings with nest boxes, opioids acting at the mu-opioid receptor inhibit courtship song produced in response to a female ([4,5], but see 84

Khurshid et al., (2009) for different effects in zebra finches). Furthermore, male starlings that sang at naturally high or low rates responded differently to opioid receptor pharmacological treatments. For example, treatment with the opioid receptor antagonist naloxone increased song production in low singers, but did not have a dramatic effect on high singers [6]. This suggests differences in mu-opioid receptor density or binding affinity may underlie individual differences in males singing at high and low rates in response to a female (i.e., in males with and without nest boxes respectively).

Studies using immunolabeling for immediate early genes (IEGs) identify differences in neuronal activity in males with compared to males without nest boxes. Specifically, males with nest boxes had higher numbers of IEG labeled cells in regions implicated in both agonistic behavior and birdsong, including the medial preoptic nucleus (POM; see Table 1 for abbreviations), medial bed nucleus of the stria terminalis (BSTm), ventral tegmental area (VTA), and mesencephalic central gray (CG) [7,8,9,10,11]. Each of these regions contains opioids or opioid receptors

[5,12,13,14,15]. In male starlings, immunolabeling for the opioid met-enkephalin in the POM and VTA correlated with male song production, however for POM this correlation existed only for song that was not produced in response to a female (with a similar trend observed for VTA)

[5]. These results link opioids in the POM and VTA specifically to the type of song produced by males without nest boxes. The results of opioid-receptor pharmacological manipulations in male zebra finches also support the idea that opioids more strongly influence undirected song compared to female-directed song [15].

Altogether, the results of pharmacology studies and met-enkephalin immunolabeling suggest that the effects of opioids in part acting within POM and VTA may differ in males singing at high rates in response to a female (i.e., males with nest boxes) compared to those singing low rates of undirected song (i.e., males without nest boxes). To test this, we examined individual differences in densities of immunolabeled mu-opioid receptors in flocks of male starlings in aviaries with nest boxes. If the inhibitory role of opioids in female-directed song in starlings is mediated by opioids in POM and VTA, then we predict that mu-opioid receptor densities will be lower in these and possibly other regions in males that successfully acquire nest boxes compared to those that fail to obtain a nest box.

2. Material and Methods

Animals

In January of 2008, 19 adult male and 10 adult female starlings were captured on a single farm west of Madison, Wisconsin using baited fly-in traps. After capture, birds were immediately housed indoors in stainless steel, single sex cages (91 cm×47 cm×47 cm) within the University of Wisconsin’s Department of Zoology indoor animal facilities. Food (Purina Mills Start and

Grow Sunfresh Recipe, 61S3-IGH-G) and water were provided ad libitum. Each animal was assigned a numbered and colored leg band for identification. All procedures and protocols were in adherence with the National Institutes of Health’s Guide for the Care and Use of Laboratory

Animals and in accordance with the University of Wisconsin-Madison Research Animal

Resource Committee (RARC). All procedures were approved by the University of Wisconsin

Institutional Animal Care and Use Committee. 86

Prior to study initiation, a hackle feather was removed from the breast of each bird and the length of iridescence was measured using calibrated calipers. Previous research [16] shows that males with hackle feathers ranging from 11-15mm are older than one year of age. Based on hackle feather length, all birds in the present study were estimated to be over one year of age at the start of the experiment.

Light cycle and hormone manipulations

Prior to study initiation, birds were housed indoors and placed on artificial photoperiods of 18L:

6D for six weeks, followed by a photoperiod of 6L: 18D for an additional six weeks. These photoperiod manipulations induce photosensitivity, a condition in which when given T implants male starlings with nest boxes display high rates of courtship song and behaviors in response to a female [17,18]. Each male test subject received two subcutaneous implants of testosterone (T)

(as detailed below). Each stimulus female also received two subcutaneous implants of 17β- estradiol, to maximize female sexual interest in males in the laboratory.

Hormones were implanted seventeen days prior to the first day of behavioral testing. All birds were lightly anesthetized using isoflurane gas anesthesia and a small pocket-like incision was made in the skin over the breast muscle. The incision site was sutured closed (Ethilon nylon suture, 13mm, 3.8c, #698G). Males received two, 14-mm lengths of silastic tubing (i.d., 1.47- mm; o.d. 1.96-mm; Dow Corning, Midland, MI) packed for 10-mm with crystalline testosterone proprionate (Sigma-Aldrich, St. Louis, MO). Females received two, 17-mm lengths of silastic tubing packed for 13-mm with 17β-estradiol (Sigma-Aldrich). After recovering on a heated pad, 87 male test subjects were placed in outdoor aviaries on natural day length and allowed to acclimate to study conditions. Female stimulus birds were returned to their home cages.

Housing and acclimation

Animals were randomly assigned to one of five outdoor aviaries (4 birds per aviary; 2.13m x

2.4m x 1.98m per aviary) and allowed to habituate prior to the beginning of behavioral testing.

Each aviary contained four nest boxes and branches for perching. Food and water were provided ad libitum. Each aviary was visually, but not acoustically isolated from the others by the use of camouflage blinds. Each day during the week prior to the first day of behavioral observations, green nest materials (green grass clippings and leaves) and a novel female were introduced into each aviary to allow males to habituate to the study procedures as well as the presence of an observer. During this time the observer additionally, practiced observing each male starling.

Starlings display high site fidelity and behavioral data can reliably be collected from multiple birds in a single aviary simultaneously [17,19,20].

Behavioral observations and study procedures

Over five consecutive days, each aviary was provided with nest material and one stimulus female

(a novel female each day) was released into the aviary. Five minutes after the introduction of the female, each aviary was observed for 20min by a single observer concealed by dark green camouflage. The order of observations across aviaries was randomized each day, with observations performed between 8:00 and 13:00. Starling song is complex and consists of at least four distinct components including introductory whistles, complex phrases, click series, and high-frequency phrases [21]. During each observation period the total number of complete song 88 bouts (i.e., bouts containing each of the 4 components) was recorded. A distinct bout was defined as an event separated from the next event by at least two seconds. Furthermore, nest box directed behaviors were recorded, including the number of times males entered and exited nest boxes, collected nesting material and looked in the nest box. Additionally, measures of agonistic behavior (including chasing and displacements from perches or food dishes) as well as bouts of wing waving, feeding, and drinking were also recorded.

Tissue collection and processing

Immediately after the last twenty-minute observation period, all males in a group were sacrificed via rapid decapitation. Each male was checked to confirm the presence of hormone implants.

Brains were removed by dissection, fixed and agitated in a 5% acrolein solution overnight, rinsed and placed in a 30% sucrose cryoprotectant for 4 days, rapidly frozen with crushed dry ice, and stored at − 80 °C until sectioning. Using a cryostat, brains were cut in the coronal plane in three,

40 μm series (each series contained every third section) and stored in anti-freeze cryoprotectant

(PBS, polyvinylpyrrolidone, sucrose and ethylene glycol mixture) until processing. Storage in a cyroprotectant allows for the long term storage of free floating sections to reduce the loss of antigenicity [22]. Series one was used for mu-opioid receptor immunohistochemistry described here.

Blood sampling and serum analysis

As part of a separate study not reported here, a single sample of no more than 200uL of blood was collected via venipuncture of the ulnar vein of each male once during the five days of 89 testing. A terminal trunk blood sample was taken immediately after sacrifice for use in the present study.

To insure that T concentrations were elevated by the implants, T plasma metabolites in the terminal blood sample were measured with a commercial grade competitive assay immunoassay

(EIA; Cayman Chemical, Ann Arbor, MI, USA, Catalog No. 582701). Samples were run in duplicate, using the manufacture’s protocol at a dilution of 1:8 (determined in pilot studies as the optimal concentration) in buffer solution and visualized at 405nm with a BioTek 800 plate reader

(#7331000, ELv800™, BioTek Instruments, Inc. Winooski, VT, USA). Sensitivities of the commercial EIA according to the manufacture’s specifications indicate a limit of detection: 80%

B/B0: 6 pg/ml and sensitivity: 50% B/B0: 32 pg/ml. The assay is specific (cross reactivity to 5 α-

DHT is 27.4%, and to 17- β -estradiol <.01 %). All samples were run within a single assay. The intraassay CV was 12.39%.

Antibody specificity tests

Brain tissue was processed using immunohistochemistry for the mu-opioid receptor. Antibody specificity was verified using multiple techniques. First, a preadsorption test (tissue was incubated in mu-opioid receptor antibody (Abcam, ab10275) with the mu-opioid receptor peptide

(1:50; Abcam, ab46988)) revealed no labeling; additionally, in pilot studies that omitted the primary antibody no labeling was detected. Secondly, a Western blot analysis was run to confirm specificity. Snap frozen dissected brain blocks from the hypothalamus were homogenized after adding 300 μL ice-cold lysis buffer consisting of 50 mM Tris-HCl, 1% Na-deoxycholate, 0.25%

Nonidet P-40, 150 mM NaCl, 1 mM EDTA, protease inhibitor cocktail (P-8340, as directed; 90

Sigma, St. Louis, Missouri, USA) and phosphatase inhibitor cocktail (P-2850, as directed,

Sigma, St. Louis, Missouri, USA). In order to remove cellular debris and nuclei, the samples were centrifuged at 14000 r.p.m. for 10min at 4˚C. The supernatant was collected and protein concentration determined by a BCA protein assay (Cat# 23225, Thermo Fisher Scientific,

Rockford, IL, USA). Fifteen micrograms of total protein from each animal were gel electrophoresed using a precast SDS-PAGE 4–20% Tris Glycine gel (Cat# 58645, Cambrex Bio

Science Rockland, Inc., Rockland, Maine, USA) and transferred to a polyvinyl difluoride membrane (Immobilon-P; Cat# IPVH20200 Millipore, Bedford, Massachusetts, USA).

Membranes were blocked for one hour in 0.1 M TBS containing 5% nonfat dry milk with constant agitation at room temperature. Membranes were then incubated overnight at 4˚C with agitation in TBS containing 0.05% Tween-20 (TBS-T) and 2% nonfat dry milk with primary antibody (for mu-opioid receptor, Ab10275 [Abcam, 1:5000]) overnight at 4°C. The next day, the membranes were given three quick washes and then four five minute washes in TBS-T.

Following washes, membranes were incubated in a goat anti-rabbit horseradish peroxidase-linked secondary antibody (Cat# 7074, Cell Signaling Technology, Inc., Beverly,

Massachusetts, USA, 1:3000) and horseradish peroxidase conjugated anti-biotin antibody (Cat#

7075, Cell Signaling Technology, Inc., Beverly, Massachusetts, USA, 1:5000) for 30min at room temperature with agitation and then washed three times for 5min each with TBS-T and twice for

5min each with TBS. Immunoreactive bands were detected using a chemiluminisense kit (Ca#

7003, Cell Signaling Technology, Inc., Beverly, Massachusetts, USA) and exposed to X-ray film

(Cat #178 8207, Eastman Kodak Co., Rochester, New York, USA).

Western immunoblot indicated a dark band at 48kDa, the molecular weight for the mu-opioid receptor protein [14,23{Khurshid, 2009 #59]]. Two additional bands were observed at approximately 50 and 160 kDa. These bands were lighter and may represent splice variants

[24,25] or dimeric forms of the mu-opioid receptor due to glycosylation [26].

Immunohistochemistry

Immunohistochemistry on tissue from all males (n = 19) was run in a single batch and background labeling was observed to be similar across all sections. Sections were rinsed in phosphate buffered saline (PBS) for 30 min, incubated in 0.5% sodium borohydride solution for

15 min, rinsed in PBS for 20 min, incubated in 0.5% hydrogen peroxide solution for 10 min, rinsed in PBS for 20 min, incubated in 20% normal goat serum (NGS (made in PBS with 0.2% triton (PBS-T))) solution for 1 h, and then incubated in 2% NGS (made in PBS-T) primary solution overnight at room temperature (rabbit anti-mu-opioid receptor at 1:5000 (Abcam, ab10275)). Sections were then rinsed in PBS-T for 30 min and incubated in 2% NGS (made in

PBS-T) biotinylated secondary solution for 90 min at room temperature (goat anti-rabbit at

1:1000 (Vector Laboratories, Burlingame, CA)). Sections were then rinsed in PBS-T for 30 min, incubated in AB solution (Vectastain Elite ABC, Vector Laboratories) for 1 h, rinsed in PBS-T for 30 min, and the avidin–biotin complex was visualized using 3,3′-Diaminobenzidine (DAB) tablets (Sigma Aldrich, St. Louis, MO, USA). Sections were float mounted onto gel-coated slides, dehydrated in a series of alcohols, and cover slipped.

Quantification

Using a Spot Camera (Diagnostic Instruments, Inc., Sterling Heights, MI, USA) connecting a

Nikon microscope to a computer to acquire images of brain sections and using METAVUE

(Fryer Company, Inc., Huntley, IL, USA) software, mean optical density and pixel area for mu- opioid receptor labeling were quantified bilaterally on three serial sections for each bird in regions implicated in reward, agonistic, and sexual behavior (POM, VTA, CG, LS, and BSTm).

The locations of these nuclei were based on Heimovics and Riters (2007). Additionally, we took bilateral measures of labeling in the paraventricular nucleus (PVN) of the hypothalamus from three serial sections containing the anterior commissure. This region was included because it was rich in mu-opioid receptor labeling, and past work implicates PVN in sociosexual behaviors

[27]. Labeling was not observed at high levels in areas in the nidopallium or arcopallium, thus measurements were not made in song control regions.

For measures of optical density and pixel area, a single computer generated threshold selected material that a blind observer agreed represented labeling. A separate threshold was generated for each brain region of interest. The METAVUE autoscale function was used to calculate the correct exposure of each image as a percentage of the total range of light, which further reduced the variation in background among individuals. Optical density is a measure of how much light is transmitted through the tissue, providing an index of labeling density. Pixel area (pixels highlighting fibers using the computer generated threshold) provides a measure of the approximate area covered by labeled receptors. Measures in POM, VTA, CG, PVN, LS, and

BSTm were made within boxes or ovals (Figure 1; Table 2) centered within each region of interest, and measures were generated by the METAVUE software in each section in both 93 hemispheres for each bird. In cases of tissue damage, labeling was quantified either on a fourth section or the individual was dropped from analysis for affected brain areas.

Statistical analysis

Data were analyzed using Statistica 6.0 software (Stat Soft Inc., Tulsa, OK). Males were defined as either nest box owners or non-owners. Specifically, if a male was observed entering and exiting the same nest box(es) multiple times (more than 5 times) on more than one day, he was designated as a nest box owner. Birds that did not display any aspect of song (intro whistles, fragments, or full song) during the behavioral observation period were dropped from analysis

(n=3, non-owners).

A multivariate analysis of variance (MANOVA) was run with total song, nest box directed behaviors, total agonistic behavior, and the sum of feeding plus drinking behavior entered as repeated measures dependent variables and nest box ownership as a categorical between subjects independent variable (males with nest boxes, n=7; males without nest boxes, n=9). Separate

MANOVAs were run with mu-opioid receptor optical density and pixel area in POM, VTA, CG,

BSTm, LS, and PVN entered as repeated measures dependent variables and nest box ownership as a categorical between subjects independent variable. For the MANOVAs on mu-opioid receptors, a mean substitution (mean of all other animals in the group) was used to replace missing data. Specifically, data were substituted for: one male with no box for the POM, one male with a nest box for LS, and three males with and one without a box for BSTm. Because four replacements were required for BSTm (tissue folded or tore from the ventricle in this area) the MANOVA was run with and without this region included. The results were similar; 94 therefore, the BSTm is included in the analysis. Finally, because mu-opioid receptors differed in males with and without nest boxes we ran multiple regression analyses to determine what best explained variance in mu-opioid receptor optical density and pixel area. Specifically, in separate multiple regression analyses a brain region was entered as the dependent variable and total number of songs, total number of agonistic interactions, and whether or not a male possessed a nest box were entered as predictor variables. Multiple regression analyses were run on the original data set for each region with no mean replacement. Influential statistical outliers were removed from analyses, resulting in removal of one data point from BSTm pixel area measures.

For multiple regression analyses backward and forward analyses were performed. In all cases except for BSTm results were identical. For BSTm backward regression resulted in the same

(though not identical) significant effects. Results of the backward analysis are provided because this model best explained the data based on the highest adjusted R2, lowest standard error, and the best residual plots.

3. Results

Behavior

Sixteen males were used for behavioral analysis. Seven males obtained one or more nest boxes and nine failed to acquire a nest box. The results of a MANOVA revealed an overall effect for nest box ownership (F1, 14 = 45.59, p = 0.000009). Fischer’s post hoc analyses indicated that compared to males without nest boxes, nest box owners responded to the introduction of a female with significantly higher rates of total song (p = 0.0005; Figure 2A), displayed significantly more nest box directed behaviors (p = 0.000009; Figure 2B), and higher levels of agonistic behavior (the sum of all chases and displacements; p = 0.011; Figure 2C). Bouts of 95 wing waving were only performed by nest box owners. Feeding and drinking behavior were not significantly different between males with or without nest boxes (p>0.05).

Mu-opioid receptor labeling and nest box ownership

The results of a MANOVA revealed an overall effect for nest box ownership on both the optical density (F1,14 = 8.68, p = 0.01) and pixel area covered by labeled receptors (F1,14 = 8.26, p =

0.011). Specifically, mu-opioid receptor labeling area and density were significantly lower in males with compared to males without nest boxes (optical density shown in Figure 3; pixel area means in Table 3).

Mu-opioid receptor labeling and behavior

Results of multiple regression analyses revealed nest box ownership (and not total songs or agonistic interactions) to contribute significantly to variance in mu-opioid receptor optical density in BSTm, PVN, POM, and VTA (Figure 3 and 4; BSTm: adj R2 = 0.28, N = 12, nest box

2 beta = 0.587, sd of beta = 0.26, t10 = 2.29, p = 0.045; PVN: adj R = 0.24, N = 16, nest box beta =

2 0.541, sd of beta = 0.22, t14 = 2.40, p = 0.031; POM: adj R = 0.40, N = 15, nest box beta =

2 0.668, sd of beta = 0.21, t13 = 3.24, p = 0.006; VTA: adj R = 0.27, N = 16, nest box beta =

0.566, sd of beta = 0.22, t14 = 2.56, p = 0.022). No variables contributed significantly to variance in LS or CG.

For measures of the pixel area covered by mu-opioid receptor labeling, results of multiple regression analyses revealed nest box ownership (and not total songs or agonistic interactions) to contribute significantly to variance in mu-opioid receptor pixel area in BSTm and POM (BSTm: 96

2 2 adj R = 0.39, N = 11, nest box beta = 0.670, sd of beta = 0.25, t9 = 2.71, p = 0.024; POM: adj R

= 0.26, N = 15, nest box beta = 0.557, sd of beta = 0.23, t13 = 2.71, p = 0.031; results of analyses of pixel area were similar to optical density and are thus not presented graphically but appear in

Table 3). For VTA pixel area the total number of songs (rather than nest box ownership or agonistic behavior) contributed significantly (negatively) to variance in mu-opioid receptor pixel

2 area (VTA: adj R = 0.24, N = 16, song beta = -0.540, sd of beta = 0.22, t14 = 2.40, p = 0.031;

Table 3; Figure 5). No variables were found to contribute significantly to variance in pixel area in LS, CG or PVN.

Testosterone

An independent t test revealed no significant differences in T concentrations between males with or without nest boxes (with nest box: Mean= 0.08pg/ml, sd=0.04, without nest box: Mean=0.10 pg/ml, sd =0.07, p> 0.05).

4. Discussion

Consistent with past studies, males with and without nest boxes differed dramatically with respect to their behavioral responses to conspecifics [3,4,7,28]. Males with nest boxes sang at high rates and accompanied song with wing waves (a courtship display); whereas, males without nest boxes sang at low rates and did not wing wave. Males with nest boxes also chased and displaced other males from perches or feeding sites more frequently than males without nest boxes indicating that they were socially dominant.

Mu-opioid receptor densities were lower in POM and VTA in males with compared to without nest boxes

As predicted based on the extreme differences in singing behavior in males with and without nest boxes, as well as past studies implicating opioids in the POM and VTA in male song [5], nest box ownership significantly explained variance in mu-opioid receptor labeling in both POM and

VTA. Specifically, male starlings with nest boxes had lower densities of mu-opioid receptors in these regions than males without nest boxes. This finding is similar to a study in rats in which social defeat (similar to a failure to acquire a nest box) resulted in increased mu-opioid receptor mRNA in VTA [29,30]. In addition to finding mu-opioid receptor optical density to be lower in

VTA in males with nest boxes, multiple regression analysis also revealed song to best explain variance in mu-opioid receptor pixel area. This latter finding reflected the fact that males without nest boxes sang at the lowest rates in response to a female and had the greatest area covered by mu-opioid receptor labeling.

In starlings, pharmacological manipulations demonstrate an inhibitory role for opioids in female- directed song [4]. Furthermore, opioids in the avian POM inhibit neuronal firing [31], as well as suppress agonistic, and sexual behaviors in Japanese quail [32]. Given the inhibitory role of opioids in POM, the high densities of mu-opioid receptors in this region in males without nest boxes may make these regions more sensitive to opioid input thereby inhibiting inappropriate social responses to conspecifics. In contrast, the low opioid receptor density in males with nest boxes may render the POM less sensitive to opioids, thereby disinhibiting behavioral responses to female and male conspecifics. 98

In songbirds, opioids in VTA also act at mu-opioid receptors to inhibit neuronal firing [33]; however unlike opioids in the POM, in rat studies opioids in VTA increase sexually-motivated behavior [34]. This effect is mediated by opioid inhibition of GABA receptors, which causes a disinhibition of activity in VTA dopamine neurons [35,36]. However, in a recent study elevated firing rates in VTA neurons and dopamine release were found in association with social defeat in male rats [37]. Thus, the finding that males that failed to acquire nest boxes have elevated densities of mu-opioid receptors in VTA may reflect a role for opioid stimulation of dopamine release associated with social defeat.

Previously, in male starlings immunolabeling density for the opioid met-enkephalin in POM correlated positively with undirected but not female-directed song (with a similar trend observed for VTA; p = 0.06) [5]. The limited range of singing observed in males without nest boxes in the present study prevented us from running a similar analysis; however, the present finding that males without nest boxes (i.e., males that sing undirected but not directed song) have elevated densities of mu-opioid receptors in POM and VTA is consistent with the met-enkephalin study and with a study in male zebra finches showing opioid blockade to more potently suppress undirected as opposed to directed song [15]. We have previously proposed that the production of undirected song is intrinsically motivated and reinforced by opioid release in POM and VTA [6].

The present data showing opioids to be elevated in the group of males singing only undirected song are consistent with this hypothesis.

Here, we report a negative correlation between the area covered by mu-opioid receptor pixel area in VTA and song when males with and without nest boxes were combined. This may reflect an 99 inhibitory role for mu receptors in VTA in song in response to a female in males without nest boxes. However it is somewhat in contrast with past data showing labeling for the opioid met- enkephalin to relate positively to male song production when males singing to attract a female in spring and males singing undirected songs in fall were combined [5]. Interpretation of these differences is difficult because met-enkephalin labeling can reflect stored or released enkephalin and because of the dynamic relationships that exist between receptors and peptides (e.g., receptors can be up-regulated in the presence of low levels of opioid release). Thus, the reason that song relates positively to met-enkephalin labeling but negatively to mu-opioid receptor labeling must be examined in future work using direct measures of opioid release or opioid receptor manipulations in the VTA.

Possible roles for opioids in BSTm and PVN in males with and without nest boxes

The present results also suggest two novel regions in which opioids may regulate song and agonistic interactions, the BSTm and PVN. BSTm is implicated in sexual and agonistic behaviors in birds as in mammals [11,38,39,40,41,42]. Immediate early gene responses in

BSTm differ in male starlings with and without nest boxes and depending on whether males are singing to attract a female or singing undirected song [7,9]. Across vertebrates, the PVN has been implicated in agonistic behaviors and responses to stressors, including social stressors such as opposite sex conspecific intruders [43,44,45,46,47]. Little is known about the role of opioids in BSTm or PVN in social behavior, however opioids in BSTm can inhibit neuronal firing [48] and male sexual behavior in rodents [49]. Therefore, the elevated mu-opioid receptor activity in

BSTm in males without nest boxes may serve to inhibit sexual behavior. Opioids in the PVN have been found to modify physiological responses to stress in rodents [50]. Accordingly, the 100 differences observed in mu-opioid receptors in PVN in the present study may reflect opioid regulation of differential stress responses to social challenges in males with and without nest boxes.

What do differences in receptor densities reflect?

Reductions in mu-opioid receptor density measures may reflect a decrease in receptor numbers or reduced opioid receptor activation. When bound and activated mu-opioid receptors internalize, which is reflected as an increase in optical density [51,52,53,54]. Thus, opioid receptors may be more active in males without nest boxes in POM, VTA, BSTm and PVN. In

POM and BSTm the area (pixel area) covered by labeled receptors was also reduced in nest box owners. The differences in pixel area may reflect differences in the sensitivity of a region to opioids; with the larger the area covered by receptors indicating a possible greater sensitivity of the region to opioids.

Interestingly, only nest box ownership (and not agonistic or singing behavior) explained variance in mu receptor measures in POM, PVN, and BSTm. This suggests owning a nest box rather than exhibiting associated behaviors either causes a reduction in opioid receptors in these areas, or conversely that only males with lower densities of mu receptors are motivated to own a nest box.

Whether the differences in mu-opioid receptor densities are the cause or consequence of acquiring a nest box is not clear from the present study. This issue must be resolved with studies in which males are assigned individually to aviaries with or without nest boxes or by examining effects of site specific pharmacological manipulations of opioid receptors on behavior of males with and without nest boxes. 101

Testosterone and differences in males with and without nest boxes

T is linked to nest box ownership [2,3,28], as well as song in response to a female [2,3,20]. In the present study males received implants of T, and serum concentrations of T did not differ in males with or without nest boxes. This suggests that differences in behavior and mu-opioid receptors observed in males with and without nest boxes were not determined by differences in serum T, but perhaps by resource possession alone or by the production of behaviors associated with nest box ownership. However, a recent study in territorial California mice (Peromyscus californicus) demonstrates that winning an agonistic interaction (which can be considered similar to “winning” a nest box in starlings) results in increased expression of androgen receptors in

BSTm and VTA [55]. Thus, it may be that acquisition of a nest box results in increased sensitivity of neural tissue to androgens which then modifies mu-opioid receptors and behavior

(independent of serum T concentrations). This possibility must be addressed in future studies.

Concluding remarks

The results of the present study suggest that the acquisition of a nest box may cause a reduction in opioid receptor activity within the BSTm, PVN, POM, and VTA of male starlings to promote agonistic responses to males and sexually motivated vocal responses to females. The results are consistent with past studies suggesting a context-dependent role for opioids in birdsong.

Moreover, they suggest an important, testosterone-independent, role for opioids in modifying social behavior so that it is appropriate given the resources available to an individual.

102

Chapter Acknowledgements

The data presented in this paper are based upon work supported by grants from R01 MH080225 to LVR. We gratefully acknowledge Kate Skogen, and Chris Elliot for animal care taking;

Sharon Stevenson for help with tissue processing and Bill Feeny for help with illustrations.

103

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Figures and Figure Legends

Figure 1: Brain nuclei measurement locations. Boxes indicate approximate areas in which optical density and pixel area of mu-opioid receptor labeling was quantified. Abbreviations: A, arcopallium; BSTm, medial bed nucleus of the stria terminalis; Cb, cerebellum; AC, anterior commissure; CO, optic chiasm; CG, medial central gray; ICo, nucleus intercollicularis; NIII, third cranial nerve; N, nidopallium; NC, caudal nidopallium; POM, medial preoptic nucleus;

PVN, paraventricular nucleus of the hypothalamus; Rt, nucleus rotundus; LS, lateral septum,

VMN, ventromedial nucleus; VTA, ventral tegmental area.

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Figure 2: Difference in behaviors between males with and without nest boxes. Mean number of behaviors in nest box owners, in black boxes, and non-owners in white for (A) Song, (B) Nest box specific behaviors (looking in the nest box, entering the nest box, collecting nest material), and (C) measures of agonistic behavior (chasing and displacements of conspecific males).

Asterisk indicates significant differences between nest box owners and non-owners (p < 0.05).

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Figure 3: Differences in mu-opioid receptor labeling in brain regions in males with and without nest boxes. Mean optical density of mu-opioid receptor labeling in (A) VTA, (B) POM, (C)

BSTm, (D) PVN. Black bars represent males with nest boxes; white bars, males without nest boxes. Asterisk indicates a significant contribution of nest box ownership to mu-opioid receptor labeling density based on multiple regression analysis (p < 0.05).

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Figure 4: Photomicrographs illustrating mu-opioid receptor labeling. Representative images include POM (top) and PVN (bottom) in males without (left) and with (right) nest boxes. Scale bar at the bottom right box is approximately .005mm.

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Figure 5: Scatterplot showing the relationship between pixel area measurements in VTA and song. Each point represents one individual, darkened circles represent males with nest boxes, open circles represent males without nest boxes. Presence of the regression line indicates a significant relationship (p<0.05).

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Tables

Table 1: List of Abbreviations Abbreviation Full Name BSTm Bed nucleus of the stria terminalis, medial part CG Midbrain central gray LS Lateral septum

POM Medial preoptic nucleus PVN Paraventricular nucleus T Testosterone VTA Ventral tegmental area

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Table 2: Box measurements Nucleus Shape Size (mm) BSTm Rectangle Area= 0.67 x 0.37 CG Oval Diameter= 0.60 POM Rectangle Area= 0.43 x 0.44 PVN Rectangle Area= 0.22 x 0.42 LS Rectangle Area= 0.36 x 0.54 VTA Rectangle Area= 0.38 x 0.53 115

Table 3: Pixel Area Measurements (without mean data substitution) Brain Region Nest box Owners Pixel Area Mean +/- Nest box Non-Owners Pixel Area owners SEM Non- Mean +/- SEM owners VTA* 7 9103.66 +/- 3206.408 9 28010.14 +/- 6502.517 POM** 7 4976.16 +/- 1366.120 8 11528.34 +/- 2227.134 BSTm** 4 4859.77 +/- 1415.464 8 16397.77 +/- 5684.518 PVN 7 18342.13 +/- 4061.620 9 19848.47 +/- 2138.746 LS 6 7429.17 +/- 1527.734 9 12099.39 +/- 2271.638 CG 7 17085.27 +/- 5289.357 9 26433.04 +/- 5474.350

* indicates a significant contribution of singing behavior to mu-opioid receptor labeling density based on multiple regression analysis (p < 0.05). ** indicates a significant contribution of nest box ownership to mu-opioid receptor labeling density based on multiple regression analysis (p < 0.05).

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Chapter V: Modulation of male song by naloxone in the medial preoptic nucleus

Individual manuscript is under re-review at Behavioral Neuroscience.

Kelm-Nelson, C.A., Stevenson, S.A., Cordes, M.A., Riters, L.V., 2012. Modulation of male song by naloxone in the medial preoptic nucleus Behavioral Neuroscience. Under Re-review.

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Abstract

Studies in songbirds implicate opioid neuropeptides in singing behavior, however past results are contradictory. In starlings, the effect of opioid manipulations on sexually-motivated courtship song differed in birds naturally singing at low compared to high rates, and mu-opioid receptors were denser in several regions, including the medial preoptic nucleus (POM) in low singing males. In the present study we found that low singing male starlings also had significantly higher enkephalin (ENK) immunolabeling densities in the POM than high singers. We then blocked opioid receptor activity in the POM with naloxone injections and found that this increased both song rate and song bout length in low singers, suggesting that high densities of mu receptors and

ENK in the POM actively suppress song in these males. In contrast to its effects on low singers, naloxone in the POM of high singers dose dependently decreased song rate and tended to reduce song bout length. This suggests that at least some level of opioid activity in POM is necessary for song production. Our results are the first to demonstrate that direct administration of opioids into the POM influences sexually-motivated song and that effects differ depending on an individual’s initial rate of song and associated density of ENK. We suggest that differential effects seen in past studies of opioids and song may in part be explained by differences in the natural song rate of subjects and accompanying differences in enkephalin activity and neural substrate sensitivity to opioids in POM.

Key Words: opioids; vocal communication; birdsong; motivation; medial preoptic area 118

1. Introduction

Little is known about the neurochemical mechanisms regulating differences observed across individuals in the motivation to communicate. In songbirds and other vertebrates studies implicate opioid neuropeptides in vocal production [1,2,3]; however, the results are contradictory. For example, in male European starlings peripheral treatment with the opioid receptor antagonist naloxone increased sexually-motivated song production [1]; whereas in male zebra finches, naloxone treatment reduced the number of female-directed songs [4].

Interestingly, responses to opioid receptor manipulations in male starlings appeared to depend on whether males were singing naturally at low or high rates [5]. Specifically, the mu receptor agonist fentanyl uniformly abolished song in all high singers. In contrast, low singers did not respond uniformly to fentanyl, with some increasing and others decreasing song production.

Naloxone uniformly increased song production in low singers, but resulted in a less uniform response in high singers [5]. This suggested that individual differences may exist in opioid receptor densities or binding affinity in individuals communicating at low or high rates; a hypothesis supported by a study showing that densities of immunolabeled mu-opioid receptors are higher in low compared to high singing males [6].

In birds as well as mammals, endogenous opioids, including enkephalin opioids, in the POM inhibit male sexual behavior [7,8,9]. This suggests that enkephalin activity may also be elevated in the POM of males singing low compared to high rates of sexually-motivated courtship song.

High densities of mu receptors have been found in the POM of low singing males and may act to suppress sexually-motivated song. If true, mu-opioid receptor blockade in POM should facilitate song in these males. In contrast, the lower densities of mu receptors in POM in high singing 119 males may facilitate sexually motivated song by reducing region sensitivity to opioids. Thus, mu receptor blockade in high singing males may act to further increase singing behavior. However, it may be that at least some level of mu receptor activity is needed to maintain song even in high singing males. Here, we tested these predictions.

2. Materials and Methods

All procedures and protocols are in adherence with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (DHEW Publication 80-23, Revised 1985, Office of

Science and Health Reports, DRR/NIH, Bethesda, MD 20205) and approved by the University of

Wisconsin-Madison Institutional Animal Care and Use Committee.

Experiment 1:Song and enkephalin (ENK) labeling

Animals and housing

Twenty adult male European starlings (Sturnus vulgaris) and five females were trapped on the same farm in Madison, WI (2008-2010). Birds were assigned leg bands for identification and were randomly assigned to single-sex indoor communal aviaries (47cm H x 47cm W x 91cm L) containing multiple perches and nest boxes.

Light cycle and hormone manipulations

Manipulations of photoperiod and testosterone implants were used to place males into a breeding-like physiological state as in [6,10,11]. Two weeks prior to study initiation each male received testosterone implants as in Kelm et al. (2011), and each stimulus female received two 120 subcutaneous implants of 17β-estradiol as in Riters, Oleson and Auger (2007) to facilitate sexually motivated behaviors the laboratory.

Habituation

After two weeks, an observer screened aviaries to identify singing males. Nest material

(consisting of green grass, leaves, and straw) and a stimulus female were introduced into male aviaries. Only males observed to sing in response to a female during screening were used in the study. We have found that when housed in pairs males tend to sing at much higher rates to females than when housed singly (unpublished observation). Thus, selected males were placed in pairs in new aviaries equipped with a one-way observation mirror (same dimensions as the community rooms). Pairs were housed with one nest box, multiple perches, food and water.

Behavioral testing

Each aviary was provided with nest material and a novel stimulus female. During a single 20 min observation period between 0900 and 1300, the total number of full song bouts (i.e., bouts containing each of the 4 components of starling song [12,13]) and the mean song bout length

(total seconds of full song/number of songs) were measured in all males by a single observer.

Furthermore, nest box directed behaviors were recorded, including the sum of times males entered nest boxes, collected nesting material, looked into the nest box and landed on the perch of the nest box. Measures of agonistic behavior (chasing and displacements from perches or food dishes) and general behavior (bouts of feeding or beak wiping) were also recorded.

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ENK immunolabeling

Approximately 90 min after the start of the observation period all males, n=20, were sacrificed via rapid decapitation. This is a window in which labeling for the ENK protein was found to increase in association with singing in area X (a song control region) in male zebra finches [14].

After decapitation, the presence of the hormone implants was confirmed. Brains were fixed in a

5% acrolein solution cryoprotected, frozen and sectioned in the coronal plane in three, 40 μm series (each series contained every third section) and stored in anti-freeze until processing as detailed in Kelm et al. (2011). Series one was used for ENK labeling. Immunohistochemistry on tissue from all males was run in a single batch following a prior protocol (Kelm et al., 2011), except that the primary antibody was rabbit anti-ENK at 1:2500 (Immunostar, #20065, previously manufactured by IncStar, Sillwater, MN) and secondary antibody was goat anti-rabbit at 1:2500 (Vector Laboratories, Burlingame, CA, USA). To ensure antibody specificity, a pilot study was run where primary antibody was omitted, and tissue failed to show labeling. This antibody has also been validated in starling brain tissue using preadsoption tests [15], and in male zebra finches specificity was validated using Western immunoblots [14]. In a more recent paper, pretreatment with leu-enkephalin also partially blocked immunoreactive staining [16], therefore we refer to immunolabeling generally as enkephalin (ENK).

Using a Spot Camera (Diagnostic Instruments, Inc., Sterling Heights, MI, USA) connecting a

Nikon microscope to a computer, images of brain sections were analyzed using METAVUE

(Fryer Company, Inc., Huntley, IL, USA) software. Specifically, the average total pixel area covered by ENK receptor labeling was quantified bilaterally using methods detailed in Kelm et 122 al. (2011) on three serial sections for each bird within a box centered in the POM (Area= 0.496 x

0.440; Fig. I).

Experiment 2: POM cannula and pharmacological manipulations

For this study, males were captured, housed, and hormonally treated following an identical protocol to that used for the birds in Experiment 1, except that in each aviary one focal male per pair, n=22 out of 44 individuals, was randomly selected to receive a cannula guide targeting the

POM.

POM cannula

Males were deeply anesthetized with isofluorane/oxygen gas and stereotaxically fitted with a permanent unilateral, 26 gauge cannula guide (#C315G/SPC, Plastics One, Inc., Roanoke, VA).

The stereotaxic apparatus (Kopf Instruments 995) was set with the beak approximately 45° below the plane of the ear bars. Ear bars were placed in the most rostral (anterior) ear position possible. The point between the ear-bars was used as the anterior-posterior zero point. A small incision was made through the skin, exposing the skull, and the cannula guide tip lowered to the skull at the center of the mid vein. The mid vein can be seen quite clearly through the skull immediately upon exposure. Both the lateral and vertical zeros were established at this position.

The anterior-posterior target coordinate was placed 0.8mm posterior to the stereotaxic zero. The lateral target was placed 0.6mm from skull zero, either to the right or left hemisphere (birds were randomly assigned to receive a cannula in either the right or left hemisphere). The apparatus was adjusted to a 2⁰ angle to the right or left of center so that the cannula was implanted obliquely. A hole was drilled and the vertical target was lowered -5.0mm from skull zero. Each cannula was 123 secured to the skull with screws using dental cement (clear Ortho-Jet powder combined with acrylic liquid; Lang Dental Manufacturing Company, Inc. Wheeling, IL). Birds were allowed three days to recover from surgery, confirmed by presence of singing behavior in response to a novel female conspecific. The day before the observation period, a mock injection was performed to familiarize males to the injection procedure.

Pharmacological manipulations and testing

On each test day, males were anesthetized with isofluorane/oxygen gas and either vehicle or a dose of the general opioid receptor antagonist (Naloxone (Sigma, #N165, molecular weight=

327.27 g/mol) low dose = 0.20 nmol (.065ug in 0.03uL), intermediate dose = 2.0 nmol (0.65ug in

0.03uL), high dose = 20.0 nmol (6.54ug in 0.03uL), dissolved in 0.9% saline (Ricca Chemical,

#721016), which served as vehicle) was infused (using a Latin squares design).

The dose was infused using a 33-gauge cannula that was attached to a Hamilton syringe, with

PE50 tubing (Plastics One). The volume and rate of the infusion was controlled by an 11 Plus pump (Harvard Apparatus; Holliston, MA). The cannula extends 2mm below the cannula guide when inserted. The infusion rate was set at 0.100 µl/min until 0.30 µl of drug was infused.

Infusions were verified by following movement of an air bubble in the tubing, and the injection cannula remained in place for 180 sec following each injection. The cannula was left within the guide for five min to allow the drug to diffuse out from the cannula tip. Males were allowed fifteen min to recover in the aviary before observations began. Behavioral observations of the experimental male began 15 min after injection and followed protocols identical to that described for Experiment 1. The observer was blind to the treatment condition of the focal male. 124

Cannula verification, histological confirmation of injection sites

After behavioral testing on the last day, males were infused with Chicago Blue 6B (Fisher

Scientific) while under anesthesia and then rapidly decapitated. The brains were dissected out of the skull, frozen immediately with dry ice, and stored at -80⁰C. The brains were sectioned at 50

µm using a cryostat and thaw mounted on to gel-coated slides. Alternate sections were either coverslipped without stain (to avoid washing away the blue dye) or Nissl stained, dehydrated in a series of alcohols, and coverslipped (to highlight the POM and other landmarks). Cannula placement was determined using a microscope based on perforations within POM and the location of blue dye by 2 independent observers blind to the behavior of the males during the study.

3. Results

Experiment 1: Song and enkephalin (ENK) labeling

Data were analyzed using Statistica 6.0 software (Stat Soft Inc., Tulsa, OK). The assumptions of parametric statistics were tested using Lilliefors tests for normality and Levene’s test for homogeneity of variance. In cases in which assumptions were not met, data were square root transformed.

Twenty males were used in the analysis. Males were categorized as either high or low singers based on a median split of full song bouts on the testing day (mean low singers=0.10, SD=0.32, mean high singers=7.44, SD=4.53). A Student’s t-test revealed that ENK labeling density in 125

POM was significantly lower in males that sang at high rates compared to males that sang at low rates (Figure I A-D). Immunolabeling did not correlate with other behavioral measures (p>0.05).

Experiment 2: POM cannula and pharmacological manipulations

Results of analyses of males with cannulae targeting POM

Cannulae were centered in the POM in 16 males (Figure II A-D). A residual plot analysis was performed and one male for whom data were greater than 2 standard deviations of the mean for full song production on the saline administration day (full song= 12) was removed from the analysis. Animals used in analysis were defined as either low or high singers (male categorization) based on their full song production the day of the vehicle (saline) injection

(median for birds combined=2.50, mean=3.150, SD=3.498; mean for high singers=6.62,

SD=2.67; mean for low singers=0.38, SD=0.74).

Mean Song Bout Length

For the measure of mean song bout length there was a significant interaction between dose of drug and male categorization (F(3,42)=3.29, p=0.03). There was no significant main effect of male categorization (F(1,14)=2.48, p=0.14) or dose (F(3,42)=0.77, p=0.52). Post hoc contrasts examining the effects of doses of naloxone on the mean song bout length in low singers demonstrated a significant increase in song bout length at the high dose compared to saline (p=0.008; Figure II,

E). Additionally, there was a trend for a decrease in mean song bout length in high singers after administration of the high dose of naloxone compared to saline vehicle (p=0.06).

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Full Song Production

Repeated measures ANOVA showed significant main effects of male categorization (F(1,14) =

9.50, p=0.0081) and there was a significant interaction between dose of drug and male categorization (F(3,42) = 4.18, p=0.011). There was no significant main effect of dose (F(3,42)=0.40, p=0.75). Post hoc contrasts demonstrated that there were significantly more full song bouts produced after a low dose of naloxone compared to saline vehicle administration for males singing at low rates (low dose p=0.03, trend for the high dose p=0.09; Figure II, F). There were no significant effects at the medium or high doses for males singing at low rates. In contrast, naloxone decreased the number of full song bouts produced by high singers in a dose-dependent linear fashion. Specifically, there were fewer full songs produced after low, medium and high doses of naloxone, compared to saline, for males singing at high rates (trend for the low dose p=0.0572, medium dose p=0.019, high dose p=0.014; Figure II, F).

Results of analysis of negative control animals

Four males had cannulae that were positioned outside the POM (Figure IIA, C) and were analyzed here as controls (Song Bout Length: mean saline=20.4, SD= 24.10, mean low=9.87,

SD=19.75, mean medium=41.92, SD=8.40, mean high=19.57, SD=23.28; Full Song Bouts: mean saline=1.75 SD, 2.06, mean low=2.25, SD=4.5, mean medium=5.75, SD=4.11, mean high=2.25,

SD=2.25). Repeated measures ANOVA demonstrated no significant effect of dose on mean song bout length (F(3,9)=2.25, p=0.14) or full song bouts (F(3,9)=2.34, p=0.15). One of these animals fell in the range of high singers and 3 fell in the range of low singers. When separated according to low and high still there were still no consistent effects or trends for drug treatments.

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Nest Box Directed Behaviors, Agonistic Behaviors and General Behaviors

For nest box directed behaviors there was a significant main effect for male categorization

(F(1,14)=6.55, p=0.022), in which high singers performed more nest box directed behaviors than low singers. But, there was not a significant main effect for dose (F(3,42)=1.20, p=0.32) or interaction between male categorization and dose (F(3,42)=2.04, p=0.12). No significant main effects or interactions were identified for agonistic behaviors, feeding or beak wiping. There were also no significant findings in the negative control birds (cannula misses).

4. Discussion

The POM plays a central role in facilitating sexually-motivated male song [17,18,19]. We found that low singing males had substantially higher densities of ENK labeling in the POM compared to high singing males, a finding consistent with past data showing that low singing males had higher densities of mu-opioid receptors in the POM [6]. Enkephalin opioids in the POM suppress male sexual behavior in Japanese quail [7], thus it may be that high levels of opioid activity in the POM of low singers suppress sexually-motivated song production. This prediction was strongly supported by the present data showing that blocking opioid receptors in POM with naloxone caused low singing males to sing at higher rates and to sing longer songs (a feature of song found to be attractive to female starlings [13,20]). These findings suggest that under natural conditions high mu-opioid receptor activity and ENK release may suppress sexually motivated singing behavior to match environmental conditions, for example in males that do not have access to nesting sites [6] or have reduced fitness (i.e. high parasitic load), as seen in rodents

[21].

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The relatively lower densities of ENK labeling and mu-opioid receptor labeling observed in high singing males suggest that low levels of mu-opioid receptor activity may be necessary to facilitate singing behavior and that targeted opioid receptor blockade in the POM would further increase sexually-motivated singing behavior in high singing males. In contrast to this prediction, we found that naloxone reduced song rate in males that sang at high rates. A possible explanation for this finding relates to the well-known inverted-U shaped relationship between opioids and behavior [22,23,24,25,26]. Specifically, opioid-dependent behaviors tend to be observed at high rates when opioids are released at intermediate levels, and to be produced at low rates when opioid levels are extremely low or extremely high. It is possible in the present study that the further reduction by naloxone of already low opioid activity in the POM (based on the measures of ENK and mu receptors) of high singers reduced opioid activity to an extreme low level resulting in song suppression. In contrast, the same treatment in low singers may have lowered extremely high opioid levels down to an intermediate range resulting in song facilitation.

Another contrasting interpretation is that in high singers song is linked to elevated use of ENK.

Immunolabeling measures do not reveal whether ENK is stored or released and changes in ENK protein induced by singing can be observed within 1-2 hours [14]. It is thus possible that high singing is linked to high ENK use (resulting in protein depletion in POM) and the high ENK release results in receptor down regulation (reflected in lower densities of mu receptors in these males) [6]. The finding that naloxone in the POM suppressed singing in high singers is consistent with this hypothesis. Future studies are needed to explore further the possibility that opioids differentially modify singing behavior in low and high singers. 129

Although naloxone differentially affected singing behavior in low compared to high singers, drug treatment did not affect nest box directed, agonistic, feeding, or beak wiping behaviors.

This indicates that deficits are specific to singing behavior and not likely caused by general naloxone-induced impairments of behavior. Furthermore, effects on song were only observed in males in which the cannula was centered in the POM, indicating that these effects are specific to opioid receptors in the POM.

Relationship to past studies on opioids and sexually-motivated song

The present data may provide insight into previous conflicting results in studies using peripheral manipulations of opioid receptors. Specifically, naloxone significantly increased sexually- motivated singing behavior in one study in male starlings [27], decreased sexually-motivated song in male zebra finches [4], and in another study in starlings had no significant effect on song

[1]. In the zebra finch study [4], males were screened and the study included only the very highest singing males. The finding that naloxone suppressed singing behavior in high singers in this study is thus consistent with the present data showing naloxone to suppress singing only in high singing males. When data from the low and high singing male starlings from the Riters et al., 2005 study were examined separately, naloxone was found to uniformly increase sexually- motivated song in low singers, but to less uniformly affect song production in high singing males

[28]. This past finding is thus consistent with the present data showing naloxone to increase singing only in low singing males.

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The relationships between ENK labeling and singing behavior reported here also differ somewhat from a past study in male starlings in which ENK immunolabeling density did not differ in the low compared to high singers [27]. The results of the two studies cannot be compared directly as the earlier study focused on a correlated but different measure of song (i.e., the proportion of minutes at which a male sang). However, it is possible that the birds in the past study produced more uniformly high rates of song in response to a female given that they were housed under more natural conditions in outdoor aviaries in flocks without cannula. Thus, it may be that a more restricted range of singing masked a significant relationship between song production and ENK fiber density. Although this possibility must be tested, both our past and present data indicate that in future studies of opioids and behavior it is necessary to consider the range of singing behavior in a study population.

Future directions and concluding remarks

Overall, our results are consistent with past studies suggesting that opioid neuropeptides influence an individual’s motivation to communicate. Several additional brain regions have been implicated in motivation, reward, and song, including the ventral tegmental area and periaqueductal gray [6]. Furthermore, the songbird POM is rich in mu, kappa, and delta opioid receptors [29]. Future studies examining additional opioid sensitive brain regions as well as specific opioid receptor subtypes is necessary to elucidate further the role of opioids in vocal communication. 131

Chapter Acknowledgments

We gratefully acknowledge support from the National Institutes of Health (R01 MH080225) to

LVR, the Bunde Fund Zoology Department research grant (2011) to CKN, Kate Skogen and

Chris Elliott for animal care, and Bill Feeny for assistance with illustrations. 132

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13. Eens M, Pinxten R, Verheyen RF (1990) On the function of singing and wing- waving in the European Starling Sturnus vulgaris. Bird Study 37: 48-52.

14. Wada K, Howard JT, McConnell P, Whitney O, Lints T, et al. (2006) A molecular neuroethological approach for identifying and characterizing a cascade of behaviorally regulated genes. P Natl Acad Sci 103: 15212-15217.

15. Ball GF, Absil P, Balthazart J (1995) Assessment of volumetric sex differences in the song control nuclei HVC and RA in zebra finches by immunocytochemistry for methionine enkephalin and vasoactive intestinal polypeptide. Brain Res 699: 83- 96.

16. Stevenson TJ, Calabrese MD, Ball GF (2012) Variation in enkephalin immunoreactivity in the social behavior network and song control system of male European starlings (Sturnus vulgaris) is dependent on breeding state and gonadal condition. J Chem Neuroanat 43 (2): 87-95.

17. Alger SJ, Riters LV (2006) Lesions to the Medial Preoptic Nucleus Differentially Affect Singing and Nest Box-Directed Behaviors Within and Outside of the Breeding Season in European Starlings (Sturnus vulgaris). Behav Neurosci 120: 1326-1336.

18. Alger SJ, Maasch SN, Riters LV (2009) Lesions to the medial preoptic nucleus affect immediate early gene immunolabeling in brain regions involved in song control and social behavior in male European starlings. Eur J Neurosci 29: 970-982.

19. Riters LV, Ball GF (1999) Lesions to the Medial Preoptic Area Affect Singing in the Male European Starling (Sturnus vulgaris). Horm Behav 36: 276-286.

20. Eens M, Pinxten R, Verheyen RF (1991) Male Song as a Cue for Mate Choice in the European Starling. Behaviour 116: 210-238.

21. Kavaliers M, Colwell DD, Choleris E (1998) Analgesic responses of male mice exposed to the odors of parasitized females: Effects of male sexual experience and infection status. Behav Neurosci 112: 1001-1011.

22. Lukas SE, Brady JV, Griffiths RR (1986) Comparison of opioid self-injection and disruption of schedule-controlled performance in the baboon. J Pharmacol and Exp Ther 238: 924-931.

23. Van Ree JM, Gerrits MAFM, Vanderschuren LJMJ (1999) Opioids, Reward and Addiction: An Encounter of Biology, Psychology, and Medicine. Pharmacol R 51: 341-396.

24. Harrigan SE, Downs DA (1978) Self-administration of heroin, acetylmethadol, morphine, and methadone in rhesus monkeys. Life Sci 22: 619-623. 134

25. Martin TJ, Kim SA, Harris LS, Smith JE (1997) Potent reinforcing effects of dihydroetorphine in rats. Eur J Pharmacol 324: 141-145.

26. Katz JL, Cooper JMLSJ (1989) Drugs as reinforcers: Pharmacological and behavioural factors. The neuropharmacological basis of reward. New York, NY, US: Clarendon Press/Oxford University Press. pp. 164-213.

27. Riters LV, Schroeder MB, Auger CJ, Eens M, Pinxten R, et al. (2005) Evidence for opioid involvement in the regulation of song production in male European starlings (Sturnus vulgaris). Behav Neurosci 119: 245-255.

28. Riters LV (2012) The role of motivation and reward neural systems in vocal communication in songbirds. Front Neuroendocrin 33: 194-209.

29. Woods JK, Deviche P, Corbitt C (2010) Opioid receptor densities analyzed across seasons in the POM and VTA of the dark-eyed junco, Junco hyemalis. J Chem Neuroanat 40: 123-129.

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Figures and Figure Legends

Figure I: Differences in ENK immunolabeling in POM in males that sang low (black bar) and high (white bar) rates of female-directed song. A) Mean square root of the total pixel area +SEM labeled by ENK in POM. Brackets indicate significant result of a Student’s t-test. Sample size is indicated at the bottom of each bar. B) Photomicrograph showing ENK labeling (dense uniform fiber label with sparsely labeled cell bodies) in a coronal brain section with the boundaries of

POM indicated by white arrows. The boxed area indicates the approximate region in which ENK was measured. Photomicrographs illustrating mu-opioid receptor labeling in POM in C) low and

D) high singing males at a higher magnification (within the white box in panel B). White boxes indicate approximate area measured in POM at 20X. Abbreviations: v=ventricle.

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Figure II: Schematic of cannula placement and effects of naloxone on singing behaviors. Black and gray circles represent areas in which the cannula tip ended within the boundaries of POM, with gray circles located in a portion of POM located approximately 50microns rostral to the black circles and open circles indicating cannulae placed outside the boundaries of POM. A)

Intermediate (i) POM; nhit=10, nmiss=2. B) Photomicrograph of tip of cannula in iPOM taken at

2X; tissue injected with Chicago Blue dye. Dashed ovals represent approximate boundaries of

POM. C) Caudal (c) POM; nhit=7, nmiss=2. D) Photomicrograph of tip of cannula in cPOM. Scale 137 bar at the lower right corner is 2µm. Abbreviations (avianbrain.org): POM, medial preoptic nucleus; TSM, septopallio-mesencephalic tract; SL, lateral septum; SM, medial septum; M, mesopallium; N, nidopallium; GLV, nucleus geniculatus lateralis pars ventralis; CO, optic chiasm; PP, lateral striatum; Rt, nucleus rotundus; LHy, region lateralis hypothalami; BST, bed nucleus of the stria terminalis; Hp, hippocampus; CoA, anterior commissure; PVN, paraventricular nucleus of the hypothalamus; VMN, ventromedial nucleus of the hypothalamus.

The square root of E) mean song bout length (+SEM) and F) the number of full song bouts after treatment with vehicle (saline), a low (0.20 nmol), medium (2.0 nmol), and high (20.0 nmol) dose of naloxone, respectively, in male starlings grouped based on a median split into low (black bars, n=8) and high (white bars, n=8) singers. The same birds were tested under each drug condition in a repeated measures design. Brackets indicate significant p values resulting from

Fisher’s post-hoc analyses.

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Chapter VI: Conclusions and General Discussion Effective social communication requires the motivation to communicate and the production of signals that are appropriate within a given social context. The studies described in this dissertation offer support for the hypothesis that opioids are involved in the regulation of singing

/ vocal behavior and that differences in individual song rates may be related to available opioid receptors, quantity of opioid ligand, specific binding affinities and the social context in which the vocalizations are produced. Starlings sing consistently at high rates within functionally distinct social contexts. These high rates of birdsong suggest that individuals are motivated to sing and that the act of singing itself may be rewarding [1]. As discussed throughout this dissertation, starlings use song to immediately influence a conspecific (directed singing) and also sing at high rates in large affiliative flocks (undirected song). Evidence from this dissertation advances the field in a positive direction offering support for a context-dependent role for opioids in vocal production.

Dissertation summary

A. Song-associated analgesia

Using an indirect measure of central opioid release, the data from Chapter II examined links between opioids and directed and undirected song using an opioid-mediated test of analgesia.

Results suggest that undirected, but not directed song, is associated with opioid-mediated analgesia, linking, albeit indirectly, affiliative song linearly to opioid release. Specifically, affiliative song was positively correlated with analgesia. Interestingly, in males singing low rates of directed song there was a negative correlation between song and analgesia. In contrast, there was no relationship between song and analgesia in males singing high rates of female-directed song. These findings suggest that opioids may facilitate affiliative song but inhibit female- 139 directed song and that individual differences in communication may be due to differences in opioid release, receptor binding affinity or densities. This study served as the basis for the next two studies, to investigate where in the brain opioids may act to regulate song behaviors in each social context.

B. Immunohistochemical analysis of mu-opioid receptors and singing behavior

i. Undirected song

Mu-opioids appear to facilitate production of undirected song, for example blocking opioid receptors decreases undirected singing [2]. Labeling for met-enkephalin in the medial preoptic nucleus (POM) correlated positively with song production [3], suggest this region as at least one site in which opioids may act to influence undirected singing behavior. In Chapter III, I found additional links between undirected singing behavior and another opioid marker, mu-opioid receptor labeling, in the POM and additionally the PAG and BSTm (all regions in which mu receptors induce analgesia). Specifically, mu receptor immunolabeling was low in both low and high singing males, whereas high receptor densities were associated with intermediate rates of song. These relationships resulted in the formation of an inverted-U. These results are consistent with the common the inverted-U shaped relationships seen between opioids and behavior in past studies [4,5,6]. I hypothesize that receptor up-and down-regulation acts to ensure that song is produced at a biologically relevant rate. I also propose that undirected singing behavior may be facilitated and maintained by mu-opioid receptor activity in the POM, BSTm and PAG.

Additionally, these data point to a potential affiliative-vocal communication circuitry. As discussed in chapter III, neural connections between POM, BSTm and PAG may regulate vocal communication so that it is reflective of an animal’s internal state. 140

ii. Directed song

The results of the analgesia study suggested that opioids may inhibit directed song in socially subordinate males (i.e., males without nest boxes). Social status and resource availability can strongly influence individual behavioral responses to conspecifics. Densities of immunolabeled mu-opioid receptors in groups of male starlings that either defended nest boxes or did not were analyzed. Male starlings that sang high rates of sexually-motivated directed song in response to the introduction of a female and defended a nest box had relatively low opioid densities in VTA,

PVN, BST, and POM. In contrast, males without nest boxes that sang low rates of song had high densities in the same brain regions. These results are consistent with multiple pharmacology studies which demonstrate an inhibitory role for opioids in vocal production in male starlings that do not have access to breeding resources [7,8,9] and fit within the analgesia framework

(males without nest boxes had significant negative correlations to opioid-mediated analgesia).

C. Site-specific manipulation of opioids and directed singing

In starlings, the effect of opioid manipulations in POM on sexually-motivated directed song differed in birds naturally singing at low compared to high rates [10]. As chapter IV demonstrated, mu-opioid receptors are denser in several regions, including the POM in low singing males. Chapter V data showed that low singing male starlings also have significantly higher met-enkephalin (ligand) immunolabeling densities in the POM than high singers.

Furthermore, in this chapter I found that blocking opioid receptor activity directly in the POM with naloxone injections increased both song rate and song bout length in low singers, suggesting that the high density of mu receptors and mENK within POM may actively suppress 141 song in these males. In contrast to its effects on low singers, naloxone in the POM of high singers dose-dependently decreased song rate and tended to reduce song bout length. This finding suggests that at least some level of opioid activity in POM may be necessary for song production in high singers. The results from Chapter V are the first to demonstrate that direct administration of opioids into the POM influences sexually-motivated (directed) song and that effects differ depending on an individual’s initial rate of song and associated density of mENK.

Conclusions

Opioids are central to a variety of behaviors including cardiovascular regulation [11], thermoregulation [12], analgesia [13], feeding [14], gastrointestinal function [15], stress responses [16], social [7] and sexual behaviors [17] and much more [18]. Opioids are also strongly implicated in brain reward processes (reviewed in [19]). One possibility, based on the results of my study, combined with past research reviewed below, is that opioids play a role in reward associated with singing behavior that differs depending upon whether communication is directed or undirected [1,20,21].

A. A role for reward in vocal communication: behavioral evidence

Starlings sing at high rates within and outside of the breeding season, this suggests not only are they highly motivated to sing but that the act of singing itself may be rewarding. Motivational states are closely linked to reward, thus if a behavior is rewarding the individual is more likely to continue the behavior. However, there are obvious differences in the social environment 142 mediating directed (breeding) and undirected (non-breeding) song, and it can be further hypothesized that the factors rewarding song within these contexts differ.

The primary function of directed singing is mate attraction; this song is highly sexually motivated. For example, male nest box owners will increase song rates in response to both the introduction and removal of a potential mate, yet they cease song immediately after copulation

[22,23]. This suggests that the presence or absence of a female is critical to song rate. Because directed singing functions to attract mates and can lead to copulation, it may be that directed song is externally rewarded through the behavioral response of the receiver (i.e., through copulation). Thus, opioid release induced by copulation may reward female-directed singing behavior (leading to satiety and a temporary inhibition of singing behavior).

Unlike directed singing, undirected song does not appear to be reinforced through any external means as it does not elicit a clear response from conspecifics. While the function of undirected song may be related to group cohesion or song practice [23,24], the exact mechanisms for what motivates high rates of this type of song are not clear. One hypothesis is that undirected song is induced by or results in an intrinsic reward. That is, it may be that undirected song is facilitated by a positive affective state (possibly induced by opioid release associated with group membership or hearing others sing) and / or that the act of song production itself induces a positive affective state (through opioid release).

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The possibility that undirected but not directed singing behavior is linked to a positive affective state was supported by a study using a conditioned place preference test. Specifically, Riters and

Stevenson (2012) found that male starlings and zebra finches singing high rates of undirected song developed a preference for a chamber associated with singing behavior. Interestingly, males singing high rates of directed song did not exhibit a place preference. CPP studies provide powerful information for elucidating aspects of behavior that are rewarding [25]. This study is thus the first to suggest that undirected song production is linked to reward / a positive affective state [26].

The results of the CPP study are also consistent with the relationship between song and analgesia

(Chapter II). High rates of undirected song were associated with analgesia, which I interpret as being facilitated or simulated by opioids. However, there is no relationship between analgesia and high rates of directed song. Both CPP and my analgesia measure suggest that opioid release may contribute to the rewarding aspects of undirected vocal communication.

Although the CPP study did not link directed song to a positive affective state, none of these males were allowed the opportunity to copulate with a female. I previously suggested that copulation may be the rewarding stimuli in these males, and I hypothesize that if allowed to copulate, these males would show place preference to the side paired with copulation. There is some evidence to support the hypothesis that song contributes to reward in this context; for example male starlings that sang directed song but did not successfully attract a female showed a 144 place aversion in the CPP test [26]. This further supports the overarching hypothesis that song in this context may be reinforced by the behavioral response of a female.

B. The role of opioids and song-associated reward

My data show high singing nest box owning males have low densities of labeling for both mu- opioid receptors and endogenous ligand. Opioids are known to inhibit sexual behavior

(especially when administered centrally at higher doses [29]), and it is proposed that sexual satiety (and an inhibition of sexual behavior) is regulated by opioid release [30,31]. For example, beta-endorphin infused into the hypothalamus, preoptic area and bed nucleus of the stria terminalis virtually abolishes all sexual activity in male rats [32]. Therefore, I hypothesize that opioid activity may be lower in high singers to prevent an inhibition of sexually-motivated singing behavior and that it is possible that a surge of opioids immediately after copulation

(should a male’s song successfully attract a female) would result in satiety and an inhibition of singing. This idea is supported by evidence in male rats, in which both morphine injections and intromissions / ejaculation induce the same reward value using a CPP model [33]. Consistent with the opioid-induced reward of copulation, naloxone infused into the preoptic area blocks

CPP results [34].

Past data in male zebra finches [2] as well as data from chapter II and III suggest that opioids may facilitate undirected song production. Furthermore, the CPP data suggest that opioid release is linked to a positive affective state. Low rates of undirected song may facilitate (and / or are facilitated by) relatively low levels of mu-opioid receptor activity in POM, PAG, and BSTm.

Given that in-vitro mu-opioid receptors down-regulate in response to sustained occupation by 145 enkephalin [35,36], it is possible that high levels of opioid release in these regions is associated with high levels of song production which cause mu-opioid receptor down-regulation.

Consistent with this possibility, and in contrast to the curvilinear relationships identified for mu receptors, past research using immunolabeling for mENK in POM related positively to undirected singing behavior even in the birds singing at the highest rates [3]. This past study, along with the present results presented in Chapter III, suggest that when mENK protein is highest (at least in POM) mu-opioid receptor labeling is lowest, consistent with the possibility that sustained song-associated opioid release results in receptor down-regulation.

Inverted U-shaped functions for opioids and behavior are commonly observed in opioid

(morphine) self-administration studies in rats and monkeys [4,5,6]. These U-shaped relationships suggest that at low levels opioid receptor stimulation induces reward but not satiety [37], thus an animal is willing to respond behaviorally to self-administer opioids. However, at higher doses opioids lead to a higher level of reward resulting in satiety and a temporary reduction in the motivational drive for the animal to respond behaviorally for opioid injection [38]. Data from

Chapters II and III can be interpreted within this drug-of-abuse framework. Specifically, the lower mu-opioid receptor densities associated with high rates of undirected song may also lead to

“tolerance” causing birds to sing at higher rates (in a sense “self-administering” opioids) to receive the higher levels of opioid release needed to stimulate the fewer numbers of available opioid receptors (to induce reward or positive affective state).

Chapter II and III data indicate that opioids are stimulating or facilitating undirected song production and it may be that opioid release induces a rewarding neural state. Starlings sing high 146 rates of undirected song in large social affiliative flocks, however when housed singly they do not sing (personal observation). Positive affective state has been associated with extroversion and gregariousness [39]. Additionally, multiple studies have suggested that social exclusion (or isolation) is a painful experience. For example, in humans, the aversive emotional state of social pain is similar to the unpleasant sensation associated with physical pain [40]. As an evolutionary adaptation, social and physical pain brain processing centers may overlap [41]. Opioid substrates may play an important role in the modulation of both pain and pleasure [42] and there are multiple lines of research that suggest commonalities between the neural circuitry of pain and pleasure. For example, human subjects were tested using an assay to cold pain sensitivity after being randomly assigned to groups with either social support or no support. Those receiving vocal social support reported less pain from the task [43]. In humans, several studies link undirected vocal behavior to analgesia and opioid release [44,45]. The cold pain sensitivity test has also been used to evaluate human undirected communication (laughing and swearing), with significant relationships between these types of vocal communication and increased pain tolerance. The inference that opioids are involved in both social and physical modulation of pain can be translated to human ASD conditions, where often (but not always) there are deficits in social communication and individuals also have increased physical sensitivity to stimuli [46].

C. Mu-opioid receptor dynamics: Dual role of opioids

The interesting factor in regards to opioid modulation of song is that opioids appear to have dual actions; they both stimulate song in one context and block / reduce song in other contexts. This is not uncommon, as opioids are known to underlie reward associated with hedonic feeding [47] 147 and sexual behavior [30], yet opioids stimulate feeding behavior [48,49] but tend to inhibit sexual behavior [17].

The mu-opioid receptor is part of a class of seven transmembrane G-protein coupled receptors where activation of the receptor is regulated by desensitization as well as internalization (down regulation) which in turn modulates the number and activity of receptors present on the plasma membrane [50,51]. Internalization is a rapid process whereas desensitization is a slower process both of which have been implicated in tolerance and dependence [31,52]. Future studies should investigate the time course for mu-opioid receptor activation and internalization as well as receptor desensitization in regards to song production.

Opioid ligands are promiscuous as they will bind to multiple receptor subtypes; however, they show preference for their primary receptor [53]. Mu receptors bind endorphins as well as enkephalins and are implicated in positive affective state (as discussed within this dissertation).

Delta receptors have been found to reduce anxiety [54,55] , and kappa receptors are implicated in negative affective state and anxiety behaviors [56]. Therefore, it is possible that mu, delta, and kappa opioid receptors work together to modulate vocal communication so that it occurs in response to the individual’s affective state. In songbirds, receptor subtypes have been found to change seasonally, at least within the POM and VTA [57]. Shifts in subtype proportion may lead to seasonal differences in song rate; therefore future studies should investigate multiple receptors and their relationship to vocal communication.

D. Additional neurochemicals linked to vocal communication and reward 148

There are numerous other neurochemicals that may be involved in vocal communication. The neurotransmitter dopamine acting within both the mesolimbic and incertohypothalamic systems is a likely candidate for influencing vocal behaviors. Dopamine primes the neural system underlying anticipatory behaviors as well as reward seeking [58,59]

Dopamine: opioid interactions may also regulate starling song. Both mu and delta opioid receptors in the VTA indirectly trigger dopamine release as well as neuron firing rates (through the inhibition of GABA) [60,61]. If opioids are acting to either trigger or block dopamine release, this could lead to changes in song rate within and between contexts. High rates of song production before copulation may also be due dopamine release. Baseline levels of opioids may be necessary to stimulate the mesolimbic dopamine system, for example, in male rats beta- endorphin injections into the VTA stimulate sexual motivation by stimulating the dopamine system [62].

Additionally, arginine vasotocin (AVT), which has been implicated in the activation of courtship and aggressive behaviors in songbirds, has been shown to increase song and other vocal behaviors [63]. For example, vasotocin-producing neurons in the BSTm increase activity and firing rates in response to positive social stimuli and reduce their actively in response to negative stimuli. Whether or not opioids are directly interacting with these neurons to regulate communication rates are unknown and should be investigated using electrophysiology in the future. While AVT may be implicated in nest box defense, future studies should also target AVT in sexually motivating, directed singing.

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It is worth noting that within the spring breeding context, the steroid hormone testosterone is elevated in males. Throughout all experimental studies outlined in this dissertation, all males

(both owners and non-owners) were T-implanted, and no significant differences were found between owners and non-owners. T has rewarding properties in itself. For example, systemic administration of a pulse of T has been shown to induce a place preference suggesting that T has rewarding or positive affective properties [64]. Furthermore, the act of obtaining a territory has been associated with an increase in androgen receptors [65], thus acquisition of a nesting site may cause an up-regulation of androgen receptors an increased sensitivity to testosterone. It is unknown whether copulation induces a T pulse in starlings. The rewarding properties of T should be considered in future studies.

Future Directions

Future studies should examine opioids, vocal communication and analgesia centrally within the aforementioned social brain regions. The combination of central opioid pharmacological manipulations, vocal behavior and analgesia yield powerful tools for elucidating the rewarding components of vocal communication. Finally, other neurochemicals such as GABA, endocannabinoids and substance P that affect analgesia [66,67,68] responses should be investigated in a behavioral context.

As in the previous chapter, the correlational studies (protein expression; IHC) presented in this dissertation should up followed up with more direct, quantitative measurements. For example, utilizing real-time PCR (mRNA expression) to investigate the relative number of receptors in a given brain region. Additionally, future studies are needed to explore further the possibility that 150 opioids differentially modify singing behavior in low and high singers by using an agonist and also investigating central manipulations in males singing undirected song.

Functional Significance

The neural systems that regulate motivation and rewarding behaviors are highly conserved across vertebrate taxa. Together, the aims of my research will provide insight into where and how opioids regulate context-appropriate social vocal communication. In the future, these data may lead to novel treatments targeting human communication disorders. Social withdrawal, social disconnection, depression, anxiety and other communication deficits are symptoms of several mental disorders, including ASDs. ASDs are a complex class of developmental brain disorders that are often characterized by deficits in communication, socially inappropriate behavior, and social withdrawal which are often times context specific. Dysregulation of opioid systems has been proposed to underlie at least some of the deficits observed in individuals with ASDs

[69,70,71]. For example, the opioid-excess theory of autism suggests that opioids are involved in the patho-physiological mechanisms of the disease (reviewed in [72,73]). Within a given population, some individuals show little to no motivation to communicate, others communicate at high rates yet the communication occurs in response to inappropriate stimuli or social context.

Overall, these findings suggest that mu-opioid receptors may be sites of action on which pharmacological agents may provide effective treatments for individuals diagnosed with these communication disorders. 151

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