Effects of arginine vasotocin (AVT) on the behavioral, cardiovascular, and corticosterone responses of starlings (Sturnus vulgaris) to crowding

Hormones and Behavior 47 (2005) 280 – 289 www.elsevier.com/locate/yhbeh

Effects of arginine vasotocin (AVT) on the behavioral, cardiovascular, and corticosterone responses of starlings (Sturnus vulgaris) to crowding Benjamin C. Nephew*, Robert S. Aaron, L. Michael Romero Department of Biology, Tufts University, Medford, MA 02155, USA Received 1 March 2004; revised 12 August 2004; accepted 16 November 2004 Available online 16 January 2005

Abstract Previous studies in European starlings have concluded that conspecific crowding can be a significant stressor that is capable of simultaneously altering behavior, heart rate, and corticosterone (CORT) concentrations. It was hypothesized that the peptide hormone arginine vasotocin (AVT) has a role in the regulation of these three types of responses to crowding. Four male and four female resident starlings were submitted to nine combinations of 3 crowding treatments (0, 1, or 5 intruder starlings) and 3 subcutaneous injections (1, 4 Ag AVT, and saline control). Resident starlings were given a treatment injection, their heart rate and behavior were monitored for 30 min, 0, 1, or 5 intruder Starlings were allowed to enter the residents cage, and HR and behavior were monitored for another 30 min. Blood samples were taken before and after all treatments to assess CORT concentrations. Exogenous AVT decreased the frequency of maintenance behaviors (feeding, drinking, preening, and beak wiping), as well as activity in resident starlings. Although aggressive behaviors upright posture, head feather expansion, and pecking) increased during crowding, these increases were significantly attenuated by AVT. Heart rate was significantly lower during these behavioral effects, and the CORT data indicate that the cardiovascular and behavioral effects are not dependent on significant increases in CORT. These data support the hypothesis that AVT’s attenuation of general behavior and crowding induced aggression are modulated by a cardiovascular mechanism. D 2004 Elsevier Inc. All rights reserved. Keywords: Arginine vasotocin; Sturnus vulgaris; Corticosterone; Behavior; Heart rate; Crowding

Introduction While the stress response is often equated with the elevation of plasma corticosterone (CORT) concentrations, independent changes in behavior and heart rate (HR) may also play significant roles in surviving exposure to noxious stimuli. Consequently, although numerous studies have investigated the behavioral, cardiovascular, or CORT responses to stressors independently, few have explored all three responses simultaneously. When examining only one facet of the stress response, however, conclusions may be unknowingly confounded by variations in the other unmeas-

* Corresponding author. Fax: +1 508 233 5298. E-mail address: [email protected] (B.C. Nephew). 0018-506X/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.yhbeh.2004.11.007

ured responses. In many species, it is unknown how cardiovascular, endocrine, and behavioral responses interact to forge an overall response to a stressor, but recent studies of starlings suggest that they are capable of modulating these three axes independently (Nephew et al., 2003). One peptide hormone which could potentially regulate all three of these responses is arginine vasotocin (AVT) and its mammalian homologue, arginine vasopressin (AVP). In this study, we tested the hypothesis that AVT regulates an integrated stress response by simultaneously modulating the behavioral, cardiovascular, and CORT responses during acute crowding in European starlings (Sturnus vulgaris). AVT/AVP have been found to have diverse cardiovascular, endocrine, and behavioral effects in many species of vertebrates (Ames et al., 1971; Baerwolff and Bie, 1988; Goecke and Goldstein, 1997; Gray et al., 1990; Robinzon

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et al., 1993; Stallone and Braun, 1985). In birds, physiologically, they are vital to maintaining fluid homeostasis and regulating blood pressure (Robinzon et al., 1993; Wilson and West, 1986). However, identifying the regulatory mechanisms of AVT on cardiovascular effects has been somewhat inconclusive. Exogenous AVT has been shown to increase (John and George, 1992), decrease (Robinzon et al., 1993), or have no effect (Hassinen et al., 1999) on HR in birds. Although there is a clear need for further study on AVT’s specific role, most studies indicate that AVT can regulate cardiovascular function. AVT and AVP also have major roles in the endocrine stress response, where they have been shown to both directly stimulate adrenocorticotropin (ACTH) release and have a synergistic effect with corticotropin releasing factor (CRF) on ACTH release at the anterior pituitary in both mammals (Aguilera et al., 1996; Antoni, 1993; Gillies et al., 1982; Wotjak et al., 1996) and birds (Castro et al., 1986). ACTH then stimulates the release of CORT, an important stress hormone in birds. During chronic stress, AVP’s resistance to CORT negative feedback provides a possible mechanism for the long-term maintenance of elevated CORT concentrations in mammals (Bartanusz et al., 1993; de Goeij et al., 1992; Scaccianoce et al., 1991), but this has yet to be shown in birds. The behavioral effects of AVT/AVP in many different species include the modulation of vocalization (Chu et al., 1998; de Kloet et al., 1993; Goodson, 1998; Maney et al., 1997; Propper and Dixon, 1997), sexual behavior (Castagna et al., 1998), learning (Davis and Pico, 1984; Engelmann and Landgraf, 1994; Landgraf et al., 1995), aggression (Delville et al., 1996a; Ferris and Delville, 1994; Goodson, 1998), and general activity level (Boyd, 1991; Buwalda et al., 1993). In birds, patterns of AVT-modulated aggression have been hypothesized to be dependent on the social organization of the species being studied (Goodson, 1998), and AVT injections decrease sexual activity in male Japanese quail (Castagna et al., 1998). While there is a wealth of data on AVT/AVP’s behavioral roles, the connections between their physiological and behavioral functions during stressful events are poorly understood, especially in avian species. Although much of the previous work on AVT’s role in social stress and aggression has been on small mammals (Delville et al., 1996b; Stribley and Carter, 1999; Winslow et al., 1993), increasing population density, or crowding, is a common event in wild starling populations (Feare, 1984). Aviary studies of starling roosting behavior demonstrated that dominance hierarchies are established by aggressive competition (Feare et al., 1995), and radio tracking studies have proposed that dominant adult males leave roosts first and feed at the highest quality feeding sites (Summers and Feare, 1995). In the field, increases in density correlate with increased aggression in multiple species of shorebirds (Burger et al., 1979), white crown sparrows (Zonotrichia leucophrys) (Slotow, 1996), and the shelduck (Tadorna

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tadorna) (Makepeace and Patterson, 1980). In European starlings, intraspecific aggression can result in increased mortality (Flux and Flux, 1992). Based on the results of these and similar studies, we suggest that crowding, and the aggression often associated with it, may be a significant stressor in the life of a starling. Starlings are an ideal species to examine responses to crowding. While starlings maintain and defend individual territories during the breeding season, they are a social species for much of the year and congregate in flocks of thousands of birds (Feare, 1984). However, it is unknown how starlings and other colonial species physiologically cope with high intraspecific densities; although previous studies indicate that crowding elicits significant changes in Starling behavior, cardiovascular physiology, and plasma CORT (Nephew and Romero, 2003). Other studies suggest that AVT can modulate Starling behavior through a CORTindependent cardiovascular mechanism (unpublished results). We hypothesized that crowding would be a significant stressor in non-breeding starlings, causing HR, activity level, and aggression to increase. Furthermore, we expected that subcutaneous AVT administration would attenuate these responses.

Methods Birds Wild, photorefractory European starlings were captured using mist nets in February of 2001 in eastern Massachusetts. Birds were initially housed communally in indoor flight aviaries for 1–3 months (43 birds evenly distributed in two 1 m  2 m  2 m enclosures) on a short day light cycle (11 L/13 D), which mimics the fall/winter photoperiod, before being transferred to the experimental room. Although we did not assess gonadal status surgically, the presence of dark beaks indicated low levels of testosterone during the experiments (conducted from March to May; Ball and Wingfield, 1987). The experimental room was equipped with a 2-way mirror, and a digital video camera outside allowed for behavioral observation without human disturbance. The crowding cage consisted of two 34  38  45 cm wire cages on top of one another with a remotely operated trap door between the cages (see Fig. 1) The lower section contained a 34-cm perch, and the upper section had a 15-cm perch, water and food dishes, and the receiver for the HR transmitter. Since starlings are social in non-breeding condition, experiments were performed simultaneously in two adjacent crowding cages. All rooms were kept at 258C and food and water were provided ad libitum (except for intruder birds, see Crowding protocol). Experiments were performed according to AALAC guidelines and approved by Tufts University Institutional Animal Care and Use Committee. Four male and four female starlings were used in this study as resident birds in the crowding experiment,

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Fig. 1. Diagram of crowding cage.

and intruder birds were randomly selected from a flock of 20–30 birds in the flight aviary. Transmitter implantation Resident starlings were anesthetized using Metofane (Pitman-Moore, Mundelein, IL) administered through a beak cone. A 15-mm incision was made in the abdominal wall perpendicular to the long axis of the body, and a 5-mm incision was made both on the side of the neck and lateral to the pygostyle for placement of the electrocardiogram (ECG) leads. The 4.0 g Data Sciences International (DSI, St. Paul, MN) transmitter, measuring 20  10  10 mm, was equipped with two 6-cm stainless steel leads wrapped in flexible polyurethane with 0.5-cm exposed ends. The leads were placed by sliding a trochar and sleeve between the skin and muscle tissue from the abdominal incision to one of the 5-mm incisions, removing the trochar, and threading the lead through the sleeve. The lead ends were first secured to muscle tissue or skin at the incision site using coated Vicryl braided suture (Ethicon, Sommerville, NJ) before the incision was closed with additional sutures. Data from pilot studies indicated that this lead placement produced the most consistent ECG signal (unpublished data). Forceps were used to create a pocket on the right side of the abdominal cavity for the body of the transmitter prior to insertion. The incision was closed, antibiotic ointment was applied to all incision sites, and the starlings were allowed to recover for 2 days in the upper sections of the crowding cages within the experimental room prior to the start of the treatments. Starlings had complete range of motion while implanted, and were fully capable of flight. Crowding protocol Experiments were conducted on weekdays (with the exception of Thursdays) between the hours of 10:00 9m

(2 h after lights on) and 12:00 pm, which corresponds to a typical feeding period at the capture site (unpublished observations). This schedule was chosen to avoid slight variations in basal HR between weekdays and weekends (observed in previous pilot studies and postulated to be due to differences in activity in the animal facility), and to allow for routine animal care on Thursdays. At least 2 h prior to the initiation of an experiment, basal blood samples (60 Al) were taken within 3 min of entering the experimental room, and the transmitter was activated by triggering a magnetic switch. Blood samples were obtained by perforating the brachial vein with a hypodermic needle and collecting the upwelling blood in microcapillary tubes. After the resident birds were bled and the transmitter activated, 1 or 5 intruder birds (random sex) were placed in the lower sections of the crowding cages, with the residents confined to the upper sections. Residents and intruders were in visual and acoustic contact throughout the experiment. Two hours after taking blood samples, both residents were given one of three subcutaneous treatments [the vehicle, lactated Ringers solution (LRS), 1 Ag AVT, or 4 Ag AVT, and behavioral and HR data acquisition commenced immediately following the injections. These doses of AVT were chosen based on previous study in the Japanese quail (Castagna et al., 1998), a similarly sized bird, and pilot studies in starlings. Pilot data indicated that these doses were behaviorally active. Thirty minutes later, the trap door between the cages was opened without entering the room, and the intruder birds flew into the upper cage (within 30 s–2 min). The trap door was immediately closed when all birds were in the upper cage. HR and behavioral data were collected for 30 min post-intrusion, and the resident bird was bled within 3 min of entering the room at the end of the post-intrusion recording. Intruder birds were then returned to the flight aviary. On subsequent experimental days, the same resident birds were exposed to the remaining combinations of intruder and AVT treatments in random order until all birds had been exposed to all nine treatments (0, 1, or 5 intruders receiving 0, 1, or 4 Ag AVT) over a period of 3 weeks, with a maximum of four treatments per week. Heart rate and activity data collection DSI model TA10EA-F20 transmitters sent HR data to a receiver connected to a Dataquest Advanced Research Technology (A.R.T.) Gold (version 1.1, St. Paul, MN) software package which recorded a continuous ECG and calculated the mean R wave frequency over a 10-s interval. The activity of each resident bird was recorded as a unitless measure by the HR transmitter software based on changes in signal strength. For the HR and activity figures, rolling means were calculated every minute for the 60-min treatments.

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Behavioral analysis The frequency of 8 behaviors from the resident starling were monitored for 30 min before and after the intrusion: feeding, drinking, preening, pecking, head feather expansion, upright posture, body contact, and general activity. Feeding and drinking events consisted of a trip to either dish where food or water was consumed. A preening event consisted of a continuous bout of preening with at least 2 s separating bouts. A peck was denoted as the use of the beak in a single jabbing motion directed towards another starling. Head feather expansion consisted of the erection of the head feathers in an attempt to give the illusion of increased size (Feare, 1984). An upright posture event has been described as vertical elongation of the torso and neck by a perching starling while facing another starling (Ellis, 1966). Body contact was the use of the body by one bird in an attempt to dislodge another bird from its perch. The frequency of body contact was too low to be analyzed statistically. Upright posture, pecking, head feather expansion, and body contact are considered aggressive behaviors (Feare, 1984). Recorded resident aggression was directed at the intruders. Due to the lack of consistent patterns in the brief aggressive interactions between residents and intruders, it was not possible to establish a dominance hierarchy. A typical aggressive interaction consisted of a resident starling approaching an intruder on a perch, assuming an upright posture and expanded head feathers, followed by pecking or body contact by either or both birds. In addition to these manually recorded variables, the activity of each resident bird was recorded with the transmitter as described previously. CORT analysis Blood samples in microhematocrit tubes were sealed on one end with clay and centrifuged at approximately 400  g for 6 min. Plasma was then removed and frozen. CORT assays were performed by RIA after extraction in dichloromethane as previously described (Wingfield et al., 1992). Briefly, tritiated CORT was added to the plasma samples to determine recovery and allowed to equilibrate with plasma lipids and binding proteins overnight. Samples were extracted with dichloromethane and dried under a stream of nitrogen. The dried extracts were redissolved in phosphate buffered saline. Samples were then split into two duplicate assay tubes, with a portion transferred to a scintillation vial to estimate recovery. Tritiated CORT and CORT antibody (Endocrine Sciences, B3-163) were added to the samples and a standard curve. Charcoal was added to adsorb all unbound steroid, the bound and unbound fractions separated by centrifugation, and the bound fraction was decanted and counted. Corticosterone concentrations were determined by comparing the bound/unbound ratio to the standard curve, and adjusted by the percent recovery. All

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samples were included in one assay, and the intraassay coefficient of variation was 5.2%. Statistics All data were analyzed using 2-way repeated measures ANOVA (comparing treatment and time); followed by a Fisher’s PLSD if a significant overall treatment effect was found. No sex differences were found in any of the variables, so data from males and females were combined for all analyses. Behavioral data were checked for homogeneity of variances. We performed repeated measures ANOVA for all behaviors with homogeneous variances. For behaviors that did not have homogeneous variances (preening, beak wiping, pecking), none of the transformations along Tukey’s Ladder of Powers resulted in homogeneous variances. Consequently, we used a rank transformation (Conover and Iman, 1981) and performed a repeated measures ANOVA.

Results Behavior Subcutaneous AVT treatment resulted in significant overall decreases in feeding (F 8,112 = 5.74, P b 0.01), drinking (F 8,112 = 2.49, P b 0.02), preening ( F 8,112 = 2.0, P b 0.05), and beak wiping (F 8,112 = 4.13, P b 0.01) during the 60-min treatments (only feeding data shown, Fig. 2A). All aggressive behaviors, including upright posture (F 1,112 = 15.0, P b 0.01) head feather expansion (F 1,112 = 8.9, P V 0.01) and pecking (F 1,112 = 15.5, P b 0.01), increased post-intrusion, but these increases were significantly attenuated by AVT (P b 0.01 for the effect of AVT and interaction between AVT and crowding for all three aggressive behaviors, Figs. 2B–D). Overall repeated measures ANOVA of the activity data indicated significant effects of treatment (F 2,3953 = 7.9, P b 0.03) and time (F 59,3953 = 12.3, P b 0.01), as well as an interaction between treatment and time (F 118,3953 = 1.5, P b 0.01). Data are summarized as means of the pre- and postintrusion periods in Fig. 3A. However, post hoc analysis of the entire 60-min treatment revealed a significant increase in activity due to crowding only in the LRS treatments (P b 0.01, Fig. 3A). There were no differences in crowdinginduced activity among the 1- or 4-Ag AVT doses (P N 0.05), except for a slight increase among the 4-Ag AVT treatments when 5 intruders was compared to 0 intruders (P b 0.03). Consequently, since crowding by itself evoked only small changes in activity, all three crowding conditions were combined for each AVT dose in Fig. 2B in order to clearly display AVT’s effects on activity. The 1-Ag VT dose had no effect (P N 0.3) and 4 Ag AVT decreased (P b 0.01) activity compared to controls (Fig. 3A). Separate analysis of the pre- and post-intrusion data indicate no significant differences among treatments

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Fig. 2. (A–D) Mean F SEM counts of resident starling feeding, upright posture, head feather expansion, and pecking behavior both pre- and post-intrusion of 0, 1, or 5 intruder starlings following subcutaneous LRS vehicle control, 1, or 4 Ag AVT. * Indicates significant overall effect of AVT; # indicates significant effect of crowding; % indicates significant interaction between AVT and crowding (n = 8, P b 0.05, repeated measures ANOVA, Fisher’s PLSD).

during the 30-min pre-intrusion period (F 8,61 = 1.34, P N 0.2), no effect of time (F 29,1769 = 1.22, P N 0.1), and no interaction between time and treatment (F 232,1769 = 1.04, P N 0.3, Fig. 3A). Although there is a trend of decreased activity during the pre-intrusion period, it does not become significant until after the intrusion (Fig. 4A). Analysis of the 31- to 35-min time frame indicates significant effects

of treatment (F 8,61 = 2.76, P b 0.02), and time (F 4,244 = 9.68, P b 0.01), as well as an interaction between the treatment and time (F 32,244 = 1.7, P b 0.02, Fig. 4A). The 4-Ag dose clearly attenuated the increase in activity immediately following the intrusion. Analysis of the entire 30-min post-intrusion period revealed significant effects of treatment (F 8,61 = 3.0, P b 0.01), time (F 29,1769 = 20.0, P b

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Fig. 3. (A and B) Mean F SEM activity (A) and heart rate [B, in beats per minute (BPM)] of resident starlings pre- and post-intrusion of 0, 1, or 5 intruder starlings following subcutaneous LRS vehicle control, 1, or 4 Ag AVT. * Indicates significant overall effect of AVT; # indicates significant effect of crowding; % indicates significant interaction between AVT and crowding (n = 8, P b 0.05, repeated measures ANOVA, Fisher’s PLSD).

0.01), and an interaction between treatment and time (F 232,1769 = 1.7, P b 0.01). However, AVT only decreased activity at the 4-Ag level (P b 0.01). To summarize AVT’s effects on behavior, AVT decreased maintenance behaviors, crowding-induced aggression, and activity in starlings. Heart rate Overall repeated measures ANOVA of the full 60 min of HR data revealed significant effects of time (F 59,4071 = 43.12, P b 0.01), treatment (F 2,4071 = 5.64, P b 0.01), and an interaction between treatment and time (F 118,4071 = 7.47, P b 0.01). Data are summarized as means of the pre and postintrusion periods in Fig. 3B. Within the three LRS treatments, crowding significantly elevated mean HR during the post-intrusion period (P b 0.01). However, there were no effects of crowding between the 1-Ag AVT doses (P N 0.3), and only the 5 intruder treatment HR was significantly elevated for the 4-Ag treatments (P b 0.04). Consequently, similar to the activity data, the three crowding treatments were combined for each AVT dose to clearly display AVT’s effects on HR (Fig. 4B). Analysis restricted to the pre-intrusion HR revealed no overall treatment effect (F 8,63 = 1.6, P N 0.1), but a

significant effect of time (F 29,1828 = 53.1, P b 0.01), and an interaction between treatment and time (F 232,1828 = 3.6, P b 0.01, Fig. 3B). This resulted from transient (initial 10 min) pre-intrusion increases in HR in the 4-Ag AVT treatments, where HR was significantly elevated compared to the LRS and 1 Ag treatments (P b 0.01 over the first 10 min, Fig. 4B). Analysis of the 31- to 35-min period indicated no effect of treatment on HR (F 8,63 = 1.8, P N 0.1), although there were significant effects of time (F 4,252 = 13.9, P b 0.01) and an interaction between time and AVT (F 32,252 = 2.8, P b 0.01, Fig. 4B). During the 30-min post-intrusion period, there were significant differences in HR due to treatment (F 8,63 = 3.7, P b 0.01), and over time (F 29,1827 = 38.7, P b 0.01), with an interaction between treatment and time (F 232,1827 = 2.4, P b 0.01 Figs. 3B and 4B). AVT significantly decreased HR during the post-intrusion period. In summary, crowding dramatically increased HR, but only in the few minutes following the intrusion. AVT had no effect on this immediate response to intrusion. However, AVT created an overall attenuation of HR over the course of the experiment. The lack of an overall AVT effect in the pre-intrusion period resulted from a transient increase in HR after the 4-Ag dose immediately post-injection.

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Fig. 4. (A and B) Mean F SEM activity (A) and heart rate [B, in beats per minute (BPM)] of resident starlings during the crowding protocol following subcutaneous LRS vehicle control, 1, or 4 Ag AVT. All crowding treatments were combined to clearly display the effects of AVT on activity and heart rate. * Indicates significant effect of 4 Ag AVT on post-intrusion activity (n = 8, P b 0.01, repeated measures ANOVA followed by Fisher’s PLSD). ** Indicates significant effect of 1 and 4 Ag subcutaneous AVT on post-intrusion heart rate (P b 0.01, repeated measures ANOVA followed by Fisher’s PLSD). Arrow indicates intrusion (initiation of crowding).

CORT

Discussion

The overall repeated measures ANOVA indicated significant treatment effects on CORT concentrations (F 9,63 = 6.74, P b 0.01 Fig. 5). 4 Ag AVT significantly increased resident CORT post-intrusion compared to both LRS controls (P b 0.01), and basal values (basal data not shown, P b 0.01, Fig. 5). In addition, CORT concentrations elicited by the 4-Ag doses were significantly higher than values after the 1-Ag doses for all three intruder treatments (P b 0.05, Fig. 5). Crowding, however, had no effect on CORT (P N 0.4 for all comparisons, Fig. 5).

Behavior AVT decreased the frequency of maintenance behaviors (feeding, drinking, preening, beak wiping) as well as activity in resident starlings. Although the aggressive behaviors (upright posture, head feather expansion, and pecking) increased following the intrusion, these increases were significantly attenuated by AVT. These data indicate that AVT not only inhibits typical maintenance behaviors, it also alters behavioral responses to an acute social stressor.

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may be an important component in the process of habituation to chronic stressors. Future studies which examine how disruptions of the normal AVT response affect behavior will greatly enhance our understanding of AVT’s role in the behavioral stress response. Heart rate

Fig. 5. Mean F SEM resident plasma CORT concentrations post-intrusion of 0, 1, or 5 intruder starlings following treatment with LRS vehicle control, 1, or 4 Ag AVT. * Indicates significant effect of 4 Ag AVT (n = 8, P b 0.05, repeated measures ANOVA followed by Fisher’s PLSD).

One potential explanation is that the behavioral decreases resulted from a general lethargy caused by the AVT-induced decrease in HR. However, although the overall activity levels following the 4-Ag doses were significantly lower than controls, there was no effect of 1 Ag AVT on activity. In contrast, 1 Ag AVT significantly altered behavior, especially aggressive behaviors. Neither dose caused a noticeable increase in diuresis, which has been observed in Japanese quail subjected to large AVT doses (Castagna et al., 1998). In addition, an equivalent maximal HR immediately following the intrusion for all treatments suggests that the cardiovascular effects of AVT do not have a sustained effect on the acute catecholamine response. Taken together, these data suggest that the behavioral effects are not directly correlated with activity, and that AVT is not affecting the typical fight-or-flight response, which might be expected if the AVT treatments were causing a general lethargy. A related hypothesis is that AVT reduced behavioral responses following crowding because of its role in the habituation to chronic stressors. Numerous mammalian studies have reported increases in AVP concentrations during the later stages of chronic stress (Bartanusz et al., 1993; de Goeij et al., 1992; Scaccianoce et al., 1991), and several avian studies have correlated HR with metabolic rate (Bevan et al., 1994, 1995; Ely et al., 1999; Gessaman, 1980; Green et al., 2001). 1 Ag AVT clearly reduced HR (Fig. 3), and if the behavioral responses were attenuated due to a cardiovascular mechanism (as these results suggest), then reduced behavior may have been a consequence of a decrease in metabolic rate. In further support of this mechanism, note the lack of strong correlation between overall activity and the other behaviors. This trend suggests that calculating energy budgets based on visual observations is not metabolically accurate, which has been proposed in earlier HR and behavior studies (Bevan et al., 1995; Ely et al., 1999). Limiting the metabolic cost of chronic stress by attenuating behavioral responses (mediated by cardiovascular output) may promote enhanced fitness in wild birds by preserving vital energy stores. Our data, along with mammalian studies reporting increases in AVP during the later stages of chronic stress (noted previously), suggest that the AVT/AVP system

Whereas AVT decreased HR over the entire 60 min, Fig. 3B reveals a transient increase in HR in the 10 min immediately following the 4-Ag AVT injections. One plausible explanation proposed by Robinzon et al. (1994) is that subcutaneous AVT elicits a short-term catecholamine response from the avian adrenal tissue. Peripheral cannulation experiments in chickens demonstrated an initial short-term tachycardia followed by bradycardia (Robinzon et al., 1994), which the authors interpreted as resulting from AVT’s pressor effects. In addition, infusion of high doses of AVT into the central nucleus of the amygdala in rats resulted in a transient increase in HR, while a lower dose induced long-lasting bradycardia, which parallels the present data (Roozendaal et al., 1993). Although it was originally thought that AVT had a vasodepressor action in birds, it is now becoming apparent that physiological doses elicit pressor effects following the initial short-term vasodepressor action. In response to this increase in blood pressure, a simultaneous decrease in heart rate has been recorded following peripheral AVT injection (Robinzon et al., 1993). Modulation of HR could clearly have an impact on the initial fight-or-flight response to stress. Although we did not see an effect of AVT on the initial HR response to the intrusions (i.e., 30 min after AVT injections), AVT did increase HR immediately following the injection. Consequently, higher AVT doses may elicit a biphasic response, with an initial enhancement of the cardiovascular response to stress followed by a general damping of cardiovascular activity (although without affecting subsequent acute HR responses). Studies have also addressed whether HR modulations are related to the behavioral effects of AVT. Studies by Castagna et al. (1998) in Japanese quail and Sodersten (Sodersten et al., 1983) in rats have been able to separate physiological vasopressor and vasodepressor effects of AVT from central behavioral effects, but this topic has yet to be addressed in many species. While Castagna et al.’s investigation demonstrated significant behavioral effects of central AVT independent of peripheral actions on diuresis, neither HR nor blood pressure was measured. It is possible that the behavioral effects of central AVT were modulated by a cardiovascular mechanism. The present study, which used lower peripheral AVT doses, showed significant effects of AVT on HR independent of noticeable diuresis. Our experimental design was unable to determine the exact mechanism of AVT’s effects on HR. However, the present data suggest that AVT does not act via CORT to alter HR. Both 1 Ag and 4 Ag VT were equally effective at depressing HR, even though 1 Ag AVT failed to significantly elevate CORT. These

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data support earlier experiments on the effects of AVT on HR (Nephew, unpublished data). Several previous studies have implicated AVT/AVP’s ability to modulate HR through vascular mechanisms (noted previously), but more recent work suggests two other sites where AVT/AVP might regulate HR: cardiac tissue (Sham et al., 1989) and specific brain nuclei (Feuerstein et al., 1984; Martin et al., 1985; Noszczyk et al., 1993). Studies in rabbits by Martin et al. (1985) revealed that administration of the central V1a, V1b antagonist d(DH2)5 Tyr(Me) vasopressin eliminated the increase in blood pressure seen following the central injection of AVT or AVP. Further cardiovascular investigation in multiple species is needed to establish if AVT is directly affecting the heart, the vasculature, or specific nuclei in the brain. CORT Based on the post-intrusion CORT concentrations, crowding did not seem to be stressful to the residents. Neither the 1 nor the 5 intruder treatments significantly altered the CORT response to AVT. These data indicate that crowding is not a significant acute stressor to resident Starlings with respect to the HPA axis, as it does not increase CORT. These results support our initial findings on the CORT response to crowding, where only the intruder birds significantly increased CORT following crowding (Nephew and Romero, 2003). In addition, it is interesting to note that the handling and injection procedure did not significantly increase CORT concentrations 60 min later. As previous experiments in our lab have shown, AVT is a potent stimulator of plasma CORT in Starlings (unpublished results). However, while the 4-Ag AVT significantly increased CORT, there was no effect of 1 Ag AVT on CORT. These data indicate that the effects of AVT on feeding, drinking, preening, beak wiping, and aggression, as well as HR, are independent of elevated CORT. While it is possible that CORT may modulate the behavioral responses at high AVT concentrations, elevated CORT does not appear to be required for the behavioral actions of AVT.

Conclusions The advantage of analyzing behavior, HR, and CORT simultaneously allowed us to evaluate the relationships between these three types of responses. While several studies have demonstrated the potency of AVT/AVP at the neural level, few have addressed the cardiovascular effects of these hormones as confounding factors. It is possible that AVT/ AVP’s behavioral effects may be due to a central behavioral mechanism , a cardiovascular mechanism (central or peripheral), or a combination of the two. However, AVT’s effects on behavior and HR do not appear to be dependent on its role in eliciting CORT release. Instead, our data support the hypothesis that AVT’s attenuation of general behavior and crowding-induced aggression are modulated by a cardiovas-

cular mechanism. Future work involving the monitoring of cardiovascular variables during central injections of AVT/ AVP and their antagonists, as well as continued mapping of the AVT/AVP brain nuclei, will greatly enhance our understanding of how this system modulates behavior, cardiovascular physiology, and the endocrine stress response.

Acknowledgments We would like to thank Wayne Rosky for caring for our captive starlings. Jim Goodson provided valuable input on AVT’s role in social behavior, and Mark Pokras of the Tufts University School of veterinary medicine advised on the blood sampling protocol. Michael Reed assisted with the statistical analyses. This project was funded by National Science Foundation (U.S.A) grants (IBN-9975502 and IBN0235044) to L.M.R.

References Aguilera, G., Rabadan-Diehl, C., Luo, X., Kiss, A., 1996. Regulation of pituitary ACTH secretion during stress: Role of corticotrophin releasing hormone and vasopressin. Gordon and Breach, New York. Ames, E., Steven, K., Skadhauge, E., 1971. Effects of arginine vasotocin on renal excretion of Na+, K+, Cl , and urea in the hydrated chicken. Am. J. Physiol. 221, 1223 – 1228. Antoni, F.A., 1993. Vasopressinergic control of pituitary adrenocorticotropin secretion comes of age. Front. Neuroendocrinol. 14, 76 – 122. Baerwolff, M., Bie, P., 1988. Effects of subpicomolar changes in vasopressin on urinary concentration. Am. J. Physiol. 255, R940 – R945. Ball, G.F., Wingfield, J.C., 1987. Changes in plasma levels of luteinizing hormone and sex steroid hormones in relation multiple-broodedness and nest-site density in male starlings. Physiol. Zool. 60, 191 – 199. Bartanusz, V., Jezova, D., Bertini, L.T., Tilders, F.J.H., Aubry, J., Kiss, J., 1993. Stress-induced increase in vasopressin and corticotropin-releasing factor expression in hypophysiotropic paraventricular neurons. Endocrinology 132, 895 – 902. Bevan, R.M., Woakes, A.J., Butler, P.J., Boyd, I.L., 1994. The use of heart rate to estimate oxygen consumption of free-ranging black-browed albatrosses (Diomedea melanophrys). J. Exp. Biol. 193, 119 – 137. Bevan, R.M., Woakes, A.J., Butler, P.J., Croxall, J.P., 1995. Heart rate and oxygen consumption of exercising Gentoo penguins. Physiol. Zool. 68, 855 – 877. Boyd, S.K., 1991. Effect of vasotocin on locomotor activity in bullfrogs varies with developmental stage and sex. Horm. Behav. 25, 57 – 69. Burger, J., Hahn, D.C., Chase, J., 1979. Aggressive interactions in mixedspecies flocks of migrating shorebirds. Anim. Behav. 27, 459 – 469. Buwalda, B., Nyakas, C., Koolhaas, J.M., Bohus, B., 1993. Neuroendocrine and behavioral effects of vasopressin in resting and mild stress conditions. Physiol. Behav. 54, 947 – 953. Castagna, C., Absil, P., Foidart, A., Balthazart, J., 1998. Systemic and intracerebroventricular injections of vasotocin inhibit appetitive and consummatory components of male sexual behavior in Japanese quail. Behav. Neurosci. 112, 233 – 250. Castro, M.G., Estivariz, F.E., Iturriza, F.C., 1986. The regulation of the corticomelanotropic cell activity in Aves—II. Effect of various peptides on the release of ACTH from dispersed, perfused duck pituitary cells. Comp. Biochem. Physiol. 83A, 71 – 75. Chu, J., Marler, C.A., Wilczynski, W., 1998. The effects of arginine vasotocin on the calling behavior of male cricket frogs in changing social contexts. Horm. Behav. 34, 248 – 261.

B.C. Nephew et al. / Hormones and Behavior 47 (2005) 280–289 Conover, W.J., Iman, R.L., 1981. Rank transformations as a bridge between parametric and nonparametric statistics. Am. Stat. 35, 124 – 129. Davis, J.L., Pico, R.M., 1984. Arginine vasotocin delays extinction of a conditioned avoidance behavior in neonatal chicks. Peptides 5, 1221 –1223. de Goeij, D.C.E., Dijkstra, H., Tilders, F.J.H., 1992. Chronic psychosocial stress enhances vasopressin, but not corticotropin-releasing factor, in the external zone of the median eminence of male rats: relationship to subordinate status. Endocrinology 131, 847 – 853. de Kloet, E.R., Elands, J., Voorhuis, D.A.M., 1993. Implication of central neurohypophyseal hormone receptor-mediated action in the timing of reproductive events: evidence from novel observations on the effect of a vasotocin analogue on singing behavior of the canary. Regul. Pept. 45, 85 – 89. Delville, Y., Mansour, K.M., Ferris, C.F., 1996a. Serotonin blocks vasopressin-facilitated offensive aggression: interactions within the ventrolateral hypothalamus of golden hamsters. Physiol. Behav. 59, 813 – 816. Delville, Y., Mansour, K.M., Ferris, C.F., 1996b. Testosterone facilitates aggression by modulating vasopressin receptors in the hypothalamus. Physiol. Behav. 60, 25 – 29. Ellis, C.R., 1966. Agonistic behavior in the male starling. Wilson Bull. 78, 208 – 224. Ely, C.R., Ward, D.H., Bollinger, K.S., 1999. Behavioral correlates of heart rates of free-living greater white-fronted geese. Condor 101, 390 – 395. Engelmann, M., Landgraf, R., 1994. Microdialysis administration of vasopressin into the septum improves social recognition in Brattleboro rats. Physiol. Behav. 55, 145 – 149. Feare, C., 1984. The Starling. Oxford Univ. Press, Oxford. Feare, C.J., Gill, E.L., Mckay, H.V., Bishop, J.D., 1995. Is the distribution of starlings (Sturnus vulgaris) within roosts determined by competition? Ibis 137, 379 – 382. Ferris, C.F., Delville, Y., 1994. Vasopressin and serotonin interactions in the control of agonistic behavior. Psychoneuroendocrinology 19, 593 – 601. Feuerstein, G., Zerbe, R.L., Faden, A.I., 1984. Central cardiovascular effects of vasotocin, oxytocin, and vasopressin in conscious rats. J. Pharmacol. Exp. Ther. 228, 348 – 353. Flux, J.E.C., Flux, M.M., 1992. Nature red in claw: how and why starlings kill each other. Notornis 39, 293 – 300. Gessaman, J.A., 1980. An evaluation of heart rate as an indirect measure of daily energy metabolism of the American kestrel (Falco sparverius). Comp. Biol. Phys. A 65, 273 – 290. Gillies, G.E., Linton, E.A., Lowry, P.J., 1982. Corticotropin releasing activity of the new CRF is potentiated several times by vasopressin. Nature 299, 355 – 357. Goecke, C.S., Goldstein, D.L., 1997. Renal glomerular and tubular effects of antidiuretic hormone and tow antidiuretic hormone analogues in house sparrows (Passer domesticus). Physiol. Zool. 70, 283 – 291. Goodson, J.L., 1998. Territorial aggression and dawn song are modulated by septal vasotocin and vasoactive intestinal polypeptide in male field sparrows. Horm. Behav. 34, 67 – 77. Gray, J.M., Yarian, D., Ramenofsky, M., 1990. Corticosterone, foraging behavior, and metabolism in dark-eyed Juncos, Junco hyemalis. Gen. Comp. Endocrinol. 79, 375 – 384. Green, J.A., Butler, P.J., Woakes, A.J., Boyd, I.L., Holder, R.L., 2001. Heart rate and rate of oxygen consumption of exercising macaroni penguins. J. Exp. Biol. 204, 673 – 684. Hassinen, E., Pyornila, A., Raimo, H., 1999. Cardiovascular and thermoregulatory responses to vasotocin and angiotensin II in the pigeon. Comp. Biochem. Physiol. 123A, 279 – 285. John, T.M., George, J.C., 1992. Effects of arginine vasotocin on cardiorespiratory and thermoregulatory responses in the pigeon. Comp. Biochem. Physiol., C 102, 353 – 359. Landgraf, R., Gerstberger, R., Montkowski, A., Probst, J.C., Wotjak, C.T., Holsboer, F., Engelmann, M., 1995. V1 vasopressin receptor antisense oligodeoxynucleotide into septum reduces vasopressin binding, social discrimination abilities, and anxiety-related behaviors in rats. J. Neurosci. 15, 4250 – 4258.

289

Makepeace, M., Patterson, I.J., 1980. Duckling mortality in the shelduck (Todorna tadorna) in relation to density, aggressive interaction, and weather. Wildfowl 31, 57 – 72. Maney, D.L., Goode, C.T., Wingfield, J.C., 1997. Intraventricular infusion of arginine vasotocin induces singing in a female songbird. J. Neuroendocrinol. 9, 487 – 491. Martin, S.M., Malkinson, T.J., Veale, W.L., Pittman, Q.J., 1985. The action of centrally administered arginine vasopressin on blood pressure in the conscious rabbit. Brain Res. 348, 137 – 145. Nephew, B.C., Romero, L.M., 2003. Behavioral, physiological, and endocrine responses of starlings to acute increases in density. Horm. Behav. 44, 222 – 232. Nephew, B.C., Kahn, S.A., Romero, L.M., 2003. Heart rate and behavior are regulated independently of corticosterone following diverse acute stressors. Gen. Comp. Endocrinol. 133, 173 – 180. Noszczyk, B., Lon, S., Szczepanskasadowska, E., 1993. Central cardiovascular effects of AVP and AVP analogs with V1, V2, and V3 agonistic or antagonistic properties in conscious dog. Brain Res. 610, 115 – 126. Propper, C.R., Dixon, T.B., 1997. Differential effects of arginine vasotocin and gonadotropin-releasing hormone on sexual behaviors in an anuran amphibian. Horm. Behav. 32, 99 – 104. Robinzon, B., Koike, T.I., Marks, P.A., 1993. At low dose, arginine vasotocin has vasopressor rather than vasodepressor effect in chickens. Gen. Comp. Endocrinol. 91, 105 – 112. Robinzon, B., Koike, T.I., Marks, P.A., 1994. Oxytocin antagonist blocks the vasodepressor but not the vasopressor effect of neurohypophysial peptides in chickens. Peptides 15, 1407 – 1413. Roozendaal, B., Schoorlemmer, G.H.M., Koolhaas, J.M., Bohus, B., 1993. Cardiac, neuroendocrine, and behavioral effects of central amygdaloid vasopressinergic and oxytocinergic mechanisms under stress-free conditions in rats. Brain Res. Bull. 32, 573 – 579. Scaccianoce, S., Muscolo, L.A.A., Cigliana, G., Navarra, D., Nicolai, R., Angelucci, L., 1991. Evidence for a specific role of vasopressin in sustaining pituitary–adrenocortical stress response in the rat. Endocrinology 128, 3138 – 3143. Sham, J.S.K., Sawyer, W.H., Pang, P.K.T., 1989. Direct cardiac stimulation by arginine vasotocin in bullfrogs (Rana catesbeiana). Am. J. Physiol. 256, R187 – R192. Slotow, R., 1996. Aggression in white-crowned sparrows: effects of distance from cover and group size. Condor 98, 245 – 252. Sodersten, P., Henning, M., Melin, P., Ludin, S., 1983. Vasopressin alters female sexual behaviour by acting n the brain independently of alterations in blood pressure. Nature 301, 608 – 610. Stallone, J.N., Braun, E.J., 1985. Contributions of glomerular and tubular mechanisms to antidiuresis in conscious domestic fowl. Am. J. Physiol. 249, F842 – F850. Stribley, J.M., Carter, C.S., 1999. Developmental exposure to vasopressin increases aggression in adult prairie voles. Proc. Natl. Acad. Sci. 96, 12601 – 12604. Summers, R.W., Feare, C.J., 1995. Roost departure by European starlings (Sturnus vulgaris): effects of competition and choice of feeding site. J. Avian Biol. 26, 289 – 295. Wilson, J.X., West, N.H., 1986. Cardiovascular responses to neurohormones in conscious chickens and ducks. Gen. Comp. Endocrinol. 62, 268 – 280. Wingfield, J.C., Vleck, C.M., Moore, M.C., 1992. Seasonal changes of the adrenocortical response to stress in birds of the Sonoran desert. J. Exp. Zool. 264, 419 – 428. Winslow, J.T., Hastings, N., Carter, C.S., Harbaugh, C.R., Insel, T.R., 1993. A role for central vasopressin in pair bonding in monogamous prairie voles. Nature 365, 545 – 548. Wotjak, C.T., Kubota, K., Masajaru, S., Liebsch, G., Montkowski, A., Holsboer, F., Neumann, I., Landgraf, R., 1996. Release of vasopressin within the rat paraventricular nucleus in response to emotional stress: a novel mechanism of regulating adrenocorticotropic hormone secretion? Neuroscience 16, 7725 – 7732.