Hormones and Behavior 65 (2014) 380–385
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Hormones and Behavior journal homepage: www.elsevier.com/locate/yhbeh
Regular article
Peripheral administration of oxytocin increases social affiliation in the naked mole-rat (Heterocephalus glaber) Skyler J. Mooney a,⁎, Natasha R. Douglas a, Melissa M. Holmes a,b a b
University of Toronto Mississauga, Department of Psychology, 3359 Mississauga Road, Mississauga, Ontario L5L 1C6, Canada University of Toronto, Department of Cell & Systems Biology, 25 Harbord Street, Toronto, Ontario M5S 3G5, Canada
a r t i c l e
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Article history: Received 11 September 2013 Revised 4 February 2014 Accepted 5 February 2014 Available online 13 February 2014 Keywords: Eusociality Heterocephalus glaber Huddling L-368,899® Naked mole-rats Oxytocin Oxytocin antagonist Social investigation Social proximity
a b s t r a c t The neuropeptide oxytocin regulates a wide variety of social behaviors across diverse species. However, the types of behaviors that are influenced by this hormone are constrained by the species in question and the social organization that a particular species exhibits. Therefore, the present experiments investigated behaviors regulated by oxytocin in a eusocial mammalian species by using the naked mole-rat (Heterocephalus glaber). In Experiment 1, adult non-breeding mole-rats were given intraperitoneal injections of either oxytocin (1 mg/kg or 10 mg/kg) or saline on alternate days. Animals were then returned to their colony and behavior was recorded for minutes 15–30 post-injection. Both doses of oxytocin increased huddling behavior during this time period. In Experiment 2, animals received intraperitoneal injections of either oxytocin (1 mg/kg), an oxytocin-receptor antagonist (0.1 mg/kg), a cocktail of oxytocin and the antagonist, or saline across 4 testing days in a counterbalanced design. Animals were placed in either a 2-chamber arena with a familiar conspecific or in a small chamber with 1 week old pups from their home colony and behaviors were recorded for minutes 15–30 post-injection. Oxytocin increased investigation of, and time spent in close proximity to, a familiar conspecific; these effects were blocked by the oxytocin antagonist. No effects were seen on pup-directed behavior. These data suggest that oxytocin is capable of modulating affiliative-like behavior in this eusocial species. © 2014 Elsevier Inc. All rights reserved.
Introduction As a member of an ancient lineage of nonapeptide genes, oxytocin (OT) shows remarkable conservation in function across vertebrate taxa (Venkatesh et al., 1997). In recent years, it has received attention for its ability to regulate social behaviors across a wide variety of species. Indeed, OT is involved in social behaviors as diverse as social recognition and parental care in rodents (Ferguson et al., 2001; Pedersen and Prange, 1979), flocking behavior in birds (Goodson et al., 2009), and response to social dominance in fish (Reddon et al., 2012). Alongside the between-species variety, OT may also play key roles in multiple types of social behavior within a species. For example, OT antagonists disrupt both partner preference (Insel and Hulihan, 1995) and alloparental behavior in the female prairie vole (Olazabal and Young, 2006). Furthermore, peripheral administration of OT increases a number of prosocial behaviors as different as food-sharing with pups and colony guarding in the meerkat (Madden and Clutton-Brock, 2011). While it is enticing to conclude that OT may serve as a conserved general sociality mechanism, the effects of OT on behavior are highly constrained by the social structure and organization of a particular species. For example, monogamous prairie voles show an increase in inter-male aggression after the ⁎ Corresponding author. E-mail address: [email protected] (S.J. Mooney).
http://dx.doi.org/10.1016/j.yhbeh.2014.02.003 0018-506X/© 2014 Elsevier Inc. All rights reserved.
first time they mate while non-monogamous montane voles do not (Winslow et al., 1993). Even low doses of OT decrease aggression following copulation in the prairie vole but do not have the same effect on postcopulatory aggression in the montane vole (Winslow et al., 1993). This has led some to suggest that nonapeptide systems such as OT may exert pleiotropic effects on behaviors that contribute to speciesspecific social organization (Goodson, 2013). OT receptor (OTR) distributions vary widely between closely related species that inhabit very different social environments, including finches of differing gregariousness (Goodson et al., 2009), colonial and solitary tuco-tucos (Beery et al., 2008), as well as eusocial and solitary mole-rats (Kalamatianos et al., 2010). These distinct distributions appear to translate into different social behaviors. For example, monogamous prairie voles show very different receptor distributions than their close relative, polygamous meadow voles. Specifically, prairie voles show much higher levels of OTR in the nucleus accumbens (NAcc) than meadow voles (Insel and Shapiro, 1992). Increasing OTR in this region via an adenovirus accelerates partner preference in the prairie vole (Ross et al., 2009) and OT antagonists infused into the NAcc block partner preference in the female prairie vole (Young et al., 2001). However, OTRs in the NAcc are not sufficient for partner preference as increasing OTR in the NAcc does not promote partner preference in the meadow vole (Ross et al., 2009). Therefore, complex networks involving OT contribute to differences in the wide variety of OT-mediated social behaviors exhibited across species.
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This species-specificity underscores the need to survey multiple species with diverse social structures. In order to develop a truly translational field of social neuroscience, we need to understand how species-specific social organization constrains the oxytocinergic regulation of social behavior. A dramatic form of sociality, eusociality, is found in some species of the Bathyergidae family. Naked mole-rat colonies, which can grow to approximately 300 animals, contain only one breeding female, called the queen, and her 1–3 male consorts (Brett, 1991; Jarvis, 1981; Lacey and Sherman, 1991). The remainder of the colony consists of reproductively suppressed subordinates. While these subordinate animals do not directly participate in the reproductive efforts of the colony, they contribute significantly to colony success through behaviors such as food finding, alloparental pup care, colony defense, and burrow/nest construction and maintenance (Jarvis, 1981; Lacey and Sherman, 1991). In the current experiments, we tested whether peripheral administration of OT would affect social behaviors in naked mole-rats. We have previously shown that subordinate naked mole-rats contain more OT-producing neurons than breeders in the paraventricular nucleus of the hypothalamus (PVN), a main area of OT production (Mooney and Holmes, 2013) and the forebrain receptor distribution of this caste has already been mapped (Kalamatianos et al., 2010). Therefore, we specifically investigated the effects of OT on the behaviors of subordinate non-breeding mole-rats. Methods Animals and housing Naked mole-rat colonies of ~20–40 animals were housed in 2 large polycarbonate cages (45.75 cm L × 24 cm W × 15.25 cm H) and 1 small cage (30 cm L× 18 cm W× 13 cm H) connected by plastic tubes (25 cm L × 5 cm D). Rooms were kept on a 12:12 light/dark cycle at 28–30 °C. Animals had ad libitum access to a diet of sweet potato and wet 19% protein mash (Harlan Laboratories, Inc.). All experimental animals ranged from 1 to 2 years of age and weighed between 27 and 57 g. Naked mole-rats typically reach adult body size within 1 year, can live for over 30 years in captivity, and do not show signs of aging well into their third decade (Buffenstein, 2005; O'Riain and Jarvis, 1998). Therefore, the experimental animals were all young adults. All procedures adhered to federal and institutional guidelines and were approved by the University Animal Care Committee.
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temperature, we removed an additional 18 animals from their colony and injected them with either 1 mg/kg OT, 10 mg/kg OT or vehicle control. We measured body temperature of these animals with a digital temperature reader (Mastercraft®) at baseline and every 10 min postinjection for 30 min. Experiment 2 In order to address potential ceiling effects of OT on social behavior in subordinates and to gain better resolution for more subtle interactions between conspecifics, we asked whether oxytocin manipulation (both agonism and antagonism) altered social behaviors in more controlled social environments. Adult subordinate naked mole-rats (N = 96) from 4 different colonies were used. Synthetic OT (Sigma) or a selective OT receptor antagonist (L-368,899®; Tocris) was dissolved in sterile saline (0.9% NaCl; Vètoquinol) to give a concentration of 0.1 mg/ml or 0.01 mg/ml, respectively. L-368,899® is highly specific to the OT receptor and is capable of crossing the blood–brain barrier (Boccia et al., 2007). Because no significant effect of OT dose was detected in Experiment 1 (see below), we used 1 mg/kg of OT for Experiment 2. Thus, each animal was given a single i.p. injection of either 1 mg/kg of OT, 0.1 mg/kg of the OT antagonist, a cocktail of OT (1 mg/kg) and the OT antagonist (0.1 mg/kg), or saline on each day of testing. Each animal received every treatment once with a rest day in between each injection. The order of administration of each drug was randomized between animals. Part A: paired interaction tests Sixty-four animals (8 males and 8 females from each colony) were used for a conspecific interaction test. Following injection, each animal was introduced to an arena containing a single familiar conspecific for 30 min. Half of the experimental animals (4 males and 4 females) from each colony were paired with a stimulus female from their home colony. The remaining animals were paired with a stimulus male. The arena was comprised of 2 polycarbonate cages (30 cm L × 18 cm W× 13 cm H) connected by a plastic tube (25 cm L× 5 cm D). Part B: pup interaction tests Beginning one week after the birth of a new litter in the colony, 32 adult subordinates (4 males and 4 females from each colony) were used for a pup-interaction test. After injection, each animal was introduced to an arena containing 4 pups from their home colony for 30 min. The arena was a single polycarbonate cage (30 cm L× 18 cm W× 13 cm H). Data collection and analyses
Experimental procedures Experiment 1 Adult subordinate naked mole-rats (N = 32) from 4 different colonies were used. Synthetic OT (Sigma) was dissolved in sterile saline (0.9% NaCl; Vètoquinol) to give a concentration of 0.1 mg/ml or 1 mg/ml. On each of 4 testing days, each animal was given a single intraperitoneal (i.p.) injection of either OT or vehicle control. Half of the animals (N = 16; 8 males and 8 females) received a low dose (1 mg/kg) of OT while the remaining animals (N = 16) received a high dose of OT (10 mg/kg). We used two doses of OT because nothing is currently known about OT action in this species and both doses have been shown to produce behavioral effects when given peripherally in other rodents (Klenerova et al., 2009; Ring et al., 2006). Every animal received each treatment (OT and saline) twice with a rest day in between each injection. The order of administration of OT and saline was counterbalanced in an ABBA fashion for half of the animals in each dosage group and a BAAB fashion for the remaining 16 animals (8 males and 8 females). In addition, equal numbers of animals within a colony were injected with each agent on each testing day. Immediately following injection, all animals within a colony were marked for visual identification with Sharpie® markers and were then returned to their home colony. The colony was then videotaped for 30 min using Sony Handycams®. To assess potential effects for OT on body
Experiment 1 Animals/treatments were coded by one experimenter and behaviors were then scored by a separate experimenter who was blind to experimental condition. Behaviors were scored for minutes 15–30 postinjection using Observer XT software (Noldus). This time period was chosen based on the results from Neumann et al. (2013) showing that i.p. injections of synthetic OT cause increases in brain levels of OT which peak in the first 30 min in mice. Behaviors of interest and their descriptions can be found in Table 1. Behaviors were averaged across the 2 days of saline administration and the 2 days of OT administration. As this experiment was exploratory in nature, we employed a liberal statistical approach; mixed-design analyses of variance (ANOVAs) were conducted for each behavior of interest with drug (OT vs. saline) as a within-subject factor and sex of the experimental animal as a between-subject factor. As there was no effect of dose, OT (1 mg/kg) and OT (10 mg/kg) were collapsed into one treatment group, therefore final N = 32 with a within-subject design for all behavioral analyses. Experiment 2 All paired- and pup-interaction tests were videotaped using Sony Handycams® and scored using the Observer XT (Noldus) software by an individual blind to experimental condition. As for Experiment 1,
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Table 1 Behaviors of interest (Experiment 1). Behavior
Description
Measurement
Digging
Any active displacement of cage bedding Any active displacement of nesting material Grasping pup in incisors Fencing with incisors, biting conspecific, open-mouth gaping, shoving Adjacent lying with conspecifics Scratching flank with hind-leg Any interaction with food
Duration
Colony maintenance Pup carrying Agonistic behavior
Huddling Grooming Feeding a
SD ± 0.75) and rose closer to baseline by 30 min post-injection (M = 29.13 ºC, SD ± 0.58). This is not surprising as naked mole-rats are poikilothermic. Importantly, no effect of treatment (F(2,15) = 0.059, p = 0.943, η2 = 0.01) and no interaction between treatment and time (F(6,45) = 0.74, p = 0.622, partial η2 = 0.090) were detected.
Duration Duration/frequency Frequencya
Duration Durationa Durationa
Very low incidence (b3).
behaviors were analyzed from minutes 15–30 post-injection. The behaviors of interest are described in Table 2. Mixed-design analyses of variance (ANOVAs) were conducted for each behavior of interest with drug (saline, OT, OT antagonist, or OT + OT antagonist) as a withinsubject factor and the sex of the experimental animal (male vs. female) as a between-subject factor. Analyses for behaviors in the paired interaction test also included the sex of the stimulus animal (opposite- vs. same-sex as experimental) as an additional between-subject factor. Eta squared (η2) was used as an estimate of effect size for main effects and interactions. When treatment effects were found, paired t-tests were conducted post hoc. Cohen's d was used as a measure of effect size for pairwise comparisons and was determined using an online calculator (http://www.cognitiveflexibility.org/effectsize).
Results Experiment 1 A significant effect of drug was found for huddling behavior (F(1,30) = 5.95, p = 0.021, η2 = 0.16) such that animals spent more time huddling with colony members after OT administration than after saline (Fig. 1A). We found no other effect of drug on behavior. An effect of sex on pup carrying duration (F(1,30) = 3.86, p = 0.059, η2 = 0.11) and frequency (F(1,30) = 2.95, p = 0.096, η2 = 0.09) approached statistical significance. Females tended to carry pups more than males (Fig. 1B). No other behavior of interest was affected by the sex of the experimental animal. Finally, there was no drug by sex interaction for any behavior measured. While we did not find any other incolony behaviors that were affected by OT administration, we cannot rule out potential ceiling effects. This possibility is supported by the fact that no effect of dose was seen for any behavior. For animals that had body temperature measured, there was an effect of time (F(3,45) = 8.62, p b 0.001, η2 = 0.34) such that temperatures dropped from baseline (M = 29.85 ºC, SD ± 1.21) in the first 10 min after being removed from the colony (M = 28.84 ºC,
Experiment 2 A significant effect of drug on proximity was detected (F(3,84) = 4.41, p = 0.006, η2 = 0.13) in the paired-interaction test. Paired t-tests revealed that experimental animals spent more time in close proximity to stimulus animals following OT treatment than any other drug treatment (Fig. 2A; all ts(31) N 2.87, all ps b 0.01, all Cohen's ds N 0.530). A similar effect of drug was seen on stimulus investigation frequency (F(3,84) = 3.67, p = 0.015, η2 = 0.11) such that experimental animals investigated the stimulus animal more on days when they received OT in comparison to all other treatment days (Fig. 2B; all ts(31) N 2.98, all ps b 0.01, all Cohen's ds N 0.510). However, the effect of drug treatment on the duration of stimulus investigation did not reach statistical significance (F(3,84) = 2.25, p = 0.089, η2 = 0.07). We found no other significant effects of drug on behavior. Importantly, co-administration of the antagonist blocked the effects of OT on these behaviors. There was an effect of sex of the experimental animal on proximity (F(1,28) = 5.03, p = 0.033, η2 = 0.15) such that female experimental animals spent more time than males in the same chamber as the stimulus animal (Fig. 2A). We found an effect of sex of the stimulus animal on mutual investigation duration (F(1,28) = 6.32, p = 0.018, η2 = 0.16) and frequency (F(1,28) = 13.06, p = 0.001, η2 = 0.29) where animals investigated each other more when animals were of the opposite sex (Figs. 3A + B). There were no other effects of the sex of the experimental animal or stimulus animal on any behavior of interest. A significant interaction between drug and the sex of the experimental animal was demonstrated for digging behavior (F(3,84) = 3.35, p = 0.023, η2 = 0.09) with females digging more than males only on days when they received the OT antagonist (Fig. 2C). We found no other interactions between drug and the sex of the experimental animal and no significant interactions between drug treatment and the sex of the stimulus animal or any 3-way interactions were detected. During the pup-interaction test, a modest effect of sex on pup carrying frequency (F(1,14) = 3.96, p = 0.067, η2 = 0.22) was detected, such that females tended to pick up pups more often than males (Fig. 2D). Duration of pup carrying was not affected by sex (F(1,14) = 1,18, p = 0.295, η2 = 0.08). No other significant main effects of sex, drug, or sex by drug interactions were seen for any other scored behavior in the pupinteraction paradigm. Discussion These data suggest that OT influences pro-social behaviors in the eusocial naked mole-rats. We found that peripheral administration of OT
Table 2 Behaviors of interest (Experiment 2). Behavior
Description
Measurement
Proximity Huddling Investigation-mutual Investigation-stimulus Investigation-experimental Agonistic behavior Digging Pup carrying Pup hovering Pup investigation
Time spent in same arena compartment as stimulus animal Adjacent lying with conspecifics Simultaneous investigation between experimental and stimulus animals Experimental animal investigates the stimulus animal Stimulus animal investigates the experimental animal Fencing with incisors, biting conspecific, open-mouth gaping, shoving Any active displacement of cage bedding Grasping pup in incisors Time spent lying on top of at least one pup Experimental animal is investigating a pup
Duration Durationa Duration/frequency Duration/frequency Duration/frequency Frequencya Duration Duration/frequency Duration Duration/frequency
a
Very low incidence (b3).
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Fig. 1. The duration of in-colony (A) huddling or (B) pup carrying (+/−SEM) for Experiment 1. Closed bars represent males and open bars represent females. * represents a treatment difference (p b 0.05). # represents a statistical trend for a sex difference (p = 0.059).
increased in-colony huddling behavior in subordinate animals, regardless of dose. Furthermore, OT increased the time spent in close proximity to, and number of investigations of, a familiar conspecific in a pairedinteraction paradigm and these effects were blocked by coadministration of an OT antagonist. While we did not see any effects of OT on pup-care behaviors either in-colony or during a pup-care paradigm, we saw subtle effects of sex where females tend to perform more pup carrying than males. Our finding that peripheral administration of OT increased huddling behavior is consistent with reports from other rodents. Central OT antagonist action decreases the total amount of huddling in rat pups, and the cohesiveness of the huddle structure and, interestingly, interferes with the ability of rat pups to use maternally-paired odor cues to guide their huddling behavior (Alberts, 2007; Kojima and Alberts, 2011). Furthermore, central OT administration increases the total amount of same-sex huddling in female prairie voles and also increases
the preference for huddling with an already familiar animal over a novel one (Beery and Zucker, 2010), while OTKO mice spend less time huddling in colonies in the visible burrow system (Pobbe et al., 2012). This effect also appears to extend to some primate species. Circulating OT is highly correlated with affiliative behavior (including huddling) in tamarins (Snowdon et al., 2010) and intranasal OT increases, while intranasal OT antagonists decrease, huddling in marmosets (Smith et al., 2010). In Experiment 2, we showed that peripheral OT administration also increased proximity of naked mole-rats. The ability of OT to regulate distance between familiar conspecifics is not specific to naked molerats. Peripheral OT antagonist administration decreases the time spent in close proximity both to a familiar cagemate and to a larger group of conspecifics in zebra finches while central mesotocin administration has the opposite effect (Goodson et al., 2009). Furthermore, OTKO mice spend more time alone and away from conspecifics in the colonial
Fig. 2. The duration of time spent in (A) close proximity or (B) digging (+/−SEM) and the number of (C) investigations of the stimulus animal or (D) pup carries (+/−SEM) for Experiment 2. Closed bars represent males and open bars represent females. * represents a difference from all other treatments (p b 0.01). ρ represents a sex difference (p b 0.05). # represents a statistical trend for a sex difference (p = 0.067).
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Fig. 3. The (A) number or (B) duration of mutual investigations (+/−SEM) in Experiment 2. Closed bars represent opposite-sex pairings and open bars represent same-sex pairings. ρ represents a sex difference (p b 0.05).
visible burrow system (Pobbe et al., 2012). Intramuscular injections of OT in the colonial meerkat did not result in spending more time close to adult conspecifics but this was likely due to an increase in the time spent close to pups that resulted from these peripheral OT injections (Madden and Clutton-Brock, 2011). We found no effect of OT on huddling in Experiment 2 (operationally defined as adjacent lying). However, while naked mole-rats form large group huddles in their colony, they rarely exhibit this behavior when placed in a novel environment for a short period (as done in the social pairing paradigm of Experiment 2) because they are highly active, exploring the new environment. During the dyadic interactions of the experimental and stimulus animals in the paired-interaction paradigm, we assessed which animal initiated each social investigation. OT administration increased the experimental-initiated investigations. This could potentially be due to the animals being in closer proximity on days in which OT was administered. However, the stimulus animal would have also been in closer proximity to the experimental animals on these days and yet they failed to show the same increase in investigation frequency. OTKO mice appear to have trouble habituating to familiar mice and so continue to investigate them at a high rate (Choleris et al., 2003). However, because OT did not alter the duration of investigation, it seems unlikely that social recognition was impaired following OT treatment. This leads us to suggest that OT administration increased interest in the social partner. Importantly, the effects of OT on proximity and investigation were blocked by co-administration of the OT antagonist, suggesting that these effects were linked to OT receptor activity. As the OT antagonist did not reduce behaviors by itself, OT action may not be necessary for these behaviors but may be capable of modulating them. Alternatively, the dosage of this particular OT antagonist may not have been high enough to independently change behavior. Other studies using the L368,899® antagonist in primates and rodents have found behavioral effects with much higher doses (Boccia et al., 2007; Klenerova et al., 2009). We employed peripheral administration of OT and the antagonist. As such, we cannot conclude which neural regions, if any, are associated with our behavioral effects. However, subordinate naked mole-rats have more OT neurons in the PVN than breeders (Mooney and Holmes, 2013) and the medial amygdala and nucleus accumbens show a high density of OT receptor binding in subordinates (Kalamatianos et al., 2010). OT activity in the medial amygdala is critical for social recognition in mice (Ferguson et al., 2001). Furthermore, OT may additionally mediate the salience of social stimuli via interactions with the dopaminergic system (Burkett and Young, 2012; Liu and Wang, 2003; Shahrokh et al., 2010; Young et al., 2001). Given that the behaviors affected by peripheral OT administration in naked mole-rats are consistent with an increase in social salience and, potentially, social recognition (e.g., the increased frequency of investigation of stimulus animals), we propose that OT coming from the PVN acts on the medial amygdala and nucleus accumbens to promote pro-social behavior in subordinate naked mole-rats.
Of course, it must be considered that the apparent increased affiliation we report is due to indirect effects of OT. For example, OT may promote social cohesion during environmental stressors (Yamasue et al., 2012). Although we did not measure glucocorticoid activity, removal from the colony is a potential stressor for this gregarious species and so it is interesting that OT would increase proximity during this situation. However, in light of the ability of OT to promote huddling behavior in-colony, this explanation seems unlikely. Alternatively, we classify huddling and proximity as pro-social behaviors but they are also a means of thermoregulation and energy conservation in this poikilothermic species (Yahav and Buffenstein, 1991). While we did not measure energy conservation, the huddling results do not appear to be due to OT effects on body temperature. Animals treated with 1 or 10 mg/kg OT did not decrease in temperature any more than the saline treated animals over a 30 minute period. We did not detect any effect of drug on pup care. While subordinate naked mole-rats perform aspects of pup care in their natal colony, breeding animals perform a higher per capita amount of these behaviors (Jarvis, 1991; Lacey and Sherman, 1991). Thus, investigating OT effects on pup-directed behavior in breeding mole-rats will be important. Somewhat surprisingly, we report a marginal effect of sex on pup carrying behavior where adult female subordinates tend to carry pups more than males do. While there was a difference between Experiments 1 and 2 in what parameter of pup carrying (frequency vs. duration) was closer to statistical significance, this was possibly due to the testing chamber dimensions. Colony chambers are larger and so pups may be carried longer distances at a time. This was not the case for the much smaller arenas in which the pup-interactions tests took place. While this putative sex effect was not related to OT manipulation, it has some implications for the naked mole-rat literature. Previous reports have either failed to identify a sex difference or did not account for sex in pup care in this species when examined in a colony setting (Jacobs and Jarvis, 1996; Jarvis, 1981, 1991; Lacey and Sherman, 1991). On a potentially related note, we also demonstrated that subordinate females may be slightly more affiliative in that they remain in closer proximity to stimulus animals more than males do. While sex differences among non-reproductive subordinates are often absent in the neural regions measured to date (Holmes et al., 2007, 2011; Seney et al., 2006), the subtle differences in behavior reported here may guide investigation into sex differences in brain physiology that have yet to be discovered in this species. Finally, in experiment 2, we found that administering the antagonist induced a sex difference in digging behavior resulting from a decrease in males and an increase in females (Fig. 2B). While it is exciting to postulate that OT acts to eliminate sex differences in digging behavior in naked mole-rats, future investigation into the role of direct OT action in the brain will help understand this effect. In summary, we show here that OT modulates a number of behaviors important for mammalian eusociality. Interest in conspecifics, the tendency to stay close to familiar conspecifics and huddling behavior
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Hormones and Behavior journal homepage: www.elsevier.com/locate/yhbeh
Regular article
Peripheral administration of oxytocin increases social affiliation in the naked mole-rat (Heterocephalus glaber) Skyler J. Mooney a,⁎, Natasha R. Douglas a, Melissa M. Holmes a,b a b
University of Toronto Mississauga, Department of Psychology, 3359 Mississauga Road, Mississauga, Ontario L5L 1C6, Canada University of Toronto, Department of Cell & Systems Biology, 25 Harbord Street, Toronto, Ontario M5S 3G5, Canada
a r t i c l e
i n f o
Article history: Received 11 September 2013 Revised 4 February 2014 Accepted 5 February 2014 Available online 13 February 2014 Keywords: Eusociality Heterocephalus glaber Huddling L-368,899® Naked mole-rats Oxytocin Oxytocin antagonist Social investigation Social proximity
a b s t r a c t The neuropeptide oxytocin regulates a wide variety of social behaviors across diverse species. However, the types of behaviors that are influenced by this hormone are constrained by the species in question and the social organization that a particular species exhibits. Therefore, the present experiments investigated behaviors regulated by oxytocin in a eusocial mammalian species by using the naked mole-rat (Heterocephalus glaber). In Experiment 1, adult non-breeding mole-rats were given intraperitoneal injections of either oxytocin (1 mg/kg or 10 mg/kg) or saline on alternate days. Animals were then returned to their colony and behavior was recorded for minutes 15–30 post-injection. Both doses of oxytocin increased huddling behavior during this time period. In Experiment 2, animals received intraperitoneal injections of either oxytocin (1 mg/kg), an oxytocin-receptor antagonist (0.1 mg/kg), a cocktail of oxytocin and the antagonist, or saline across 4 testing days in a counterbalanced design. Animals were placed in either a 2-chamber arena with a familiar conspecific or in a small chamber with 1 week old pups from their home colony and behaviors were recorded for minutes 15–30 post-injection. Oxytocin increased investigation of, and time spent in close proximity to, a familiar conspecific; these effects were blocked by the oxytocin antagonist. No effects were seen on pup-directed behavior. These data suggest that oxytocin is capable of modulating affiliative-like behavior in this eusocial species. © 2014 Elsevier Inc. All rights reserved.
Introduction As a member of an ancient lineage of nonapeptide genes, oxytocin (OT) shows remarkable conservation in function across vertebrate taxa (Venkatesh et al., 1997). In recent years, it has received attention for its ability to regulate social behaviors across a wide variety of species. Indeed, OT is involved in social behaviors as diverse as social recognition and parental care in rodents (Ferguson et al., 2001; Pedersen and Prange, 1979), flocking behavior in birds (Goodson et al., 2009), and response to social dominance in fish (Reddon et al., 2012). Alongside the between-species variety, OT may also play key roles in multiple types of social behavior within a species. For example, OT antagonists disrupt both partner preference (Insel and Hulihan, 1995) and alloparental behavior in the female prairie vole (Olazabal and Young, 2006). Furthermore, peripheral administration of OT increases a number of prosocial behaviors as different as food-sharing with pups and colony guarding in the meerkat (Madden and Clutton-Brock, 2011). While it is enticing to conclude that OT may serve as a conserved general sociality mechanism, the effects of OT on behavior are highly constrained by the social structure and organization of a particular species. For example, monogamous prairie voles show an increase in inter-male aggression after the ⁎ Corresponding author. E-mail address: [email protected] (S.J. Mooney).
http://dx.doi.org/10.1016/j.yhbeh.2014.02.003 0018-506X/© 2014 Elsevier Inc. All rights reserved.
first time they mate while non-monogamous montane voles do not (Winslow et al., 1993). Even low doses of OT decrease aggression following copulation in the prairie vole but do not have the same effect on postcopulatory aggression in the montane vole (Winslow et al., 1993). This has led some to suggest that nonapeptide systems such as OT may exert pleiotropic effects on behaviors that contribute to speciesspecific social organization (Goodson, 2013). OT receptor (OTR) distributions vary widely between closely related species that inhabit very different social environments, including finches of differing gregariousness (Goodson et al., 2009), colonial and solitary tuco-tucos (Beery et al., 2008), as well as eusocial and solitary mole-rats (Kalamatianos et al., 2010). These distinct distributions appear to translate into different social behaviors. For example, monogamous prairie voles show very different receptor distributions than their close relative, polygamous meadow voles. Specifically, prairie voles show much higher levels of OTR in the nucleus accumbens (NAcc) than meadow voles (Insel and Shapiro, 1992). Increasing OTR in this region via an adenovirus accelerates partner preference in the prairie vole (Ross et al., 2009) and OT antagonists infused into the NAcc block partner preference in the female prairie vole (Young et al., 2001). However, OTRs in the NAcc are not sufficient for partner preference as increasing OTR in the NAcc does not promote partner preference in the meadow vole (Ross et al., 2009). Therefore, complex networks involving OT contribute to differences in the wide variety of OT-mediated social behaviors exhibited across species.
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This species-specificity underscores the need to survey multiple species with diverse social structures. In order to develop a truly translational field of social neuroscience, we need to understand how species-specific social organization constrains the oxytocinergic regulation of social behavior. A dramatic form of sociality, eusociality, is found in some species of the Bathyergidae family. Naked mole-rat colonies, which can grow to approximately 300 animals, contain only one breeding female, called the queen, and her 1–3 male consorts (Brett, 1991; Jarvis, 1981; Lacey and Sherman, 1991). The remainder of the colony consists of reproductively suppressed subordinates. While these subordinate animals do not directly participate in the reproductive efforts of the colony, they contribute significantly to colony success through behaviors such as food finding, alloparental pup care, colony defense, and burrow/nest construction and maintenance (Jarvis, 1981; Lacey and Sherman, 1991). In the current experiments, we tested whether peripheral administration of OT would affect social behaviors in naked mole-rats. We have previously shown that subordinate naked mole-rats contain more OT-producing neurons than breeders in the paraventricular nucleus of the hypothalamus (PVN), a main area of OT production (Mooney and Holmes, 2013) and the forebrain receptor distribution of this caste has already been mapped (Kalamatianos et al., 2010). Therefore, we specifically investigated the effects of OT on the behaviors of subordinate non-breeding mole-rats. Methods Animals and housing Naked mole-rat colonies of ~20–40 animals were housed in 2 large polycarbonate cages (45.75 cm L × 24 cm W × 15.25 cm H) and 1 small cage (30 cm L× 18 cm W× 13 cm H) connected by plastic tubes (25 cm L × 5 cm D). Rooms were kept on a 12:12 light/dark cycle at 28–30 °C. Animals had ad libitum access to a diet of sweet potato and wet 19% protein mash (Harlan Laboratories, Inc.). All experimental animals ranged from 1 to 2 years of age and weighed between 27 and 57 g. Naked mole-rats typically reach adult body size within 1 year, can live for over 30 years in captivity, and do not show signs of aging well into their third decade (Buffenstein, 2005; O'Riain and Jarvis, 1998). Therefore, the experimental animals were all young adults. All procedures adhered to federal and institutional guidelines and were approved by the University Animal Care Committee.
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temperature, we removed an additional 18 animals from their colony and injected them with either 1 mg/kg OT, 10 mg/kg OT or vehicle control. We measured body temperature of these animals with a digital temperature reader (Mastercraft®) at baseline and every 10 min postinjection for 30 min. Experiment 2 In order to address potential ceiling effects of OT on social behavior in subordinates and to gain better resolution for more subtle interactions between conspecifics, we asked whether oxytocin manipulation (both agonism and antagonism) altered social behaviors in more controlled social environments. Adult subordinate naked mole-rats (N = 96) from 4 different colonies were used. Synthetic OT (Sigma) or a selective OT receptor antagonist (L-368,899®; Tocris) was dissolved in sterile saline (0.9% NaCl; Vètoquinol) to give a concentration of 0.1 mg/ml or 0.01 mg/ml, respectively. L-368,899® is highly specific to the OT receptor and is capable of crossing the blood–brain barrier (Boccia et al., 2007). Because no significant effect of OT dose was detected in Experiment 1 (see below), we used 1 mg/kg of OT for Experiment 2. Thus, each animal was given a single i.p. injection of either 1 mg/kg of OT, 0.1 mg/kg of the OT antagonist, a cocktail of OT (1 mg/kg) and the OT antagonist (0.1 mg/kg), or saline on each day of testing. Each animal received every treatment once with a rest day in between each injection. The order of administration of each drug was randomized between animals. Part A: paired interaction tests Sixty-four animals (8 males and 8 females from each colony) were used for a conspecific interaction test. Following injection, each animal was introduced to an arena containing a single familiar conspecific for 30 min. Half of the experimental animals (4 males and 4 females) from each colony were paired with a stimulus female from their home colony. The remaining animals were paired with a stimulus male. The arena was comprised of 2 polycarbonate cages (30 cm L × 18 cm W× 13 cm H) connected by a plastic tube (25 cm L× 5 cm D). Part B: pup interaction tests Beginning one week after the birth of a new litter in the colony, 32 adult subordinates (4 males and 4 females from each colony) were used for a pup-interaction test. After injection, each animal was introduced to an arena containing 4 pups from their home colony for 30 min. The arena was a single polycarbonate cage (30 cm L× 18 cm W× 13 cm H). Data collection and analyses
Experimental procedures Experiment 1 Adult subordinate naked mole-rats (N = 32) from 4 different colonies were used. Synthetic OT (Sigma) was dissolved in sterile saline (0.9% NaCl; Vètoquinol) to give a concentration of 0.1 mg/ml or 1 mg/ml. On each of 4 testing days, each animal was given a single intraperitoneal (i.p.) injection of either OT or vehicle control. Half of the animals (N = 16; 8 males and 8 females) received a low dose (1 mg/kg) of OT while the remaining animals (N = 16) received a high dose of OT (10 mg/kg). We used two doses of OT because nothing is currently known about OT action in this species and both doses have been shown to produce behavioral effects when given peripherally in other rodents (Klenerova et al., 2009; Ring et al., 2006). Every animal received each treatment (OT and saline) twice with a rest day in between each injection. The order of administration of OT and saline was counterbalanced in an ABBA fashion for half of the animals in each dosage group and a BAAB fashion for the remaining 16 animals (8 males and 8 females). In addition, equal numbers of animals within a colony were injected with each agent on each testing day. Immediately following injection, all animals within a colony were marked for visual identification with Sharpie® markers and were then returned to their home colony. The colony was then videotaped for 30 min using Sony Handycams®. To assess potential effects for OT on body
Experiment 1 Animals/treatments were coded by one experimenter and behaviors were then scored by a separate experimenter who was blind to experimental condition. Behaviors were scored for minutes 15–30 postinjection using Observer XT software (Noldus). This time period was chosen based on the results from Neumann et al. (2013) showing that i.p. injections of synthetic OT cause increases in brain levels of OT which peak in the first 30 min in mice. Behaviors of interest and their descriptions can be found in Table 1. Behaviors were averaged across the 2 days of saline administration and the 2 days of OT administration. As this experiment was exploratory in nature, we employed a liberal statistical approach; mixed-design analyses of variance (ANOVAs) were conducted for each behavior of interest with drug (OT vs. saline) as a within-subject factor and sex of the experimental animal as a between-subject factor. As there was no effect of dose, OT (1 mg/kg) and OT (10 mg/kg) were collapsed into one treatment group, therefore final N = 32 with a within-subject design for all behavioral analyses. Experiment 2 All paired- and pup-interaction tests were videotaped using Sony Handycams® and scored using the Observer XT (Noldus) software by an individual blind to experimental condition. As for Experiment 1,
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Table 1 Behaviors of interest (Experiment 1). Behavior
Description
Measurement
Digging
Any active displacement of cage bedding Any active displacement of nesting material Grasping pup in incisors Fencing with incisors, biting conspecific, open-mouth gaping, shoving Adjacent lying with conspecifics Scratching flank with hind-leg Any interaction with food
Duration
Colony maintenance Pup carrying Agonistic behavior
Huddling Grooming Feeding a
SD ± 0.75) and rose closer to baseline by 30 min post-injection (M = 29.13 ºC, SD ± 0.58). This is not surprising as naked mole-rats are poikilothermic. Importantly, no effect of treatment (F(2,15) = 0.059, p = 0.943, η2 = 0.01) and no interaction between treatment and time (F(6,45) = 0.74, p = 0.622, partial η2 = 0.090) were detected.
Duration Duration/frequency Frequencya
Duration Durationa Durationa
Very low incidence (b3).
behaviors were analyzed from minutes 15–30 post-injection. The behaviors of interest are described in Table 2. Mixed-design analyses of variance (ANOVAs) were conducted for each behavior of interest with drug (saline, OT, OT antagonist, or OT + OT antagonist) as a withinsubject factor and the sex of the experimental animal (male vs. female) as a between-subject factor. Analyses for behaviors in the paired interaction test also included the sex of the stimulus animal (opposite- vs. same-sex as experimental) as an additional between-subject factor. Eta squared (η2) was used as an estimate of effect size for main effects and interactions. When treatment effects were found, paired t-tests were conducted post hoc. Cohen's d was used as a measure of effect size for pairwise comparisons and was determined using an online calculator (http://www.cognitiveflexibility.org/effectsize).
Results Experiment 1 A significant effect of drug was found for huddling behavior (F(1,30) = 5.95, p = 0.021, η2 = 0.16) such that animals spent more time huddling with colony members after OT administration than after saline (Fig. 1A). We found no other effect of drug on behavior. An effect of sex on pup carrying duration (F(1,30) = 3.86, p = 0.059, η2 = 0.11) and frequency (F(1,30) = 2.95, p = 0.096, η2 = 0.09) approached statistical significance. Females tended to carry pups more than males (Fig. 1B). No other behavior of interest was affected by the sex of the experimental animal. Finally, there was no drug by sex interaction for any behavior measured. While we did not find any other incolony behaviors that were affected by OT administration, we cannot rule out potential ceiling effects. This possibility is supported by the fact that no effect of dose was seen for any behavior. For animals that had body temperature measured, there was an effect of time (F(3,45) = 8.62, p b 0.001, η2 = 0.34) such that temperatures dropped from baseline (M = 29.85 ºC, SD ± 1.21) in the first 10 min after being removed from the colony (M = 28.84 ºC,
Experiment 2 A significant effect of drug on proximity was detected (F(3,84) = 4.41, p = 0.006, η2 = 0.13) in the paired-interaction test. Paired t-tests revealed that experimental animals spent more time in close proximity to stimulus animals following OT treatment than any other drug treatment (Fig. 2A; all ts(31) N 2.87, all ps b 0.01, all Cohen's ds N 0.530). A similar effect of drug was seen on stimulus investigation frequency (F(3,84) = 3.67, p = 0.015, η2 = 0.11) such that experimental animals investigated the stimulus animal more on days when they received OT in comparison to all other treatment days (Fig. 2B; all ts(31) N 2.98, all ps b 0.01, all Cohen's ds N 0.510). However, the effect of drug treatment on the duration of stimulus investigation did not reach statistical significance (F(3,84) = 2.25, p = 0.089, η2 = 0.07). We found no other significant effects of drug on behavior. Importantly, co-administration of the antagonist blocked the effects of OT on these behaviors. There was an effect of sex of the experimental animal on proximity (F(1,28) = 5.03, p = 0.033, η2 = 0.15) such that female experimental animals spent more time than males in the same chamber as the stimulus animal (Fig. 2A). We found an effect of sex of the stimulus animal on mutual investigation duration (F(1,28) = 6.32, p = 0.018, η2 = 0.16) and frequency (F(1,28) = 13.06, p = 0.001, η2 = 0.29) where animals investigated each other more when animals were of the opposite sex (Figs. 3A + B). There were no other effects of the sex of the experimental animal or stimulus animal on any behavior of interest. A significant interaction between drug and the sex of the experimental animal was demonstrated for digging behavior (F(3,84) = 3.35, p = 0.023, η2 = 0.09) with females digging more than males only on days when they received the OT antagonist (Fig. 2C). We found no other interactions between drug and the sex of the experimental animal and no significant interactions between drug treatment and the sex of the stimulus animal or any 3-way interactions were detected. During the pup-interaction test, a modest effect of sex on pup carrying frequency (F(1,14) = 3.96, p = 0.067, η2 = 0.22) was detected, such that females tended to pick up pups more often than males (Fig. 2D). Duration of pup carrying was not affected by sex (F(1,14) = 1,18, p = 0.295, η2 = 0.08). No other significant main effects of sex, drug, or sex by drug interactions were seen for any other scored behavior in the pupinteraction paradigm. Discussion These data suggest that OT influences pro-social behaviors in the eusocial naked mole-rats. We found that peripheral administration of OT
Table 2 Behaviors of interest (Experiment 2). Behavior
Description
Measurement
Proximity Huddling Investigation-mutual Investigation-stimulus Investigation-experimental Agonistic behavior Digging Pup carrying Pup hovering Pup investigation
Time spent in same arena compartment as stimulus animal Adjacent lying with conspecifics Simultaneous investigation between experimental and stimulus animals Experimental animal investigates the stimulus animal Stimulus animal investigates the experimental animal Fencing with incisors, biting conspecific, open-mouth gaping, shoving Any active displacement of cage bedding Grasping pup in incisors Time spent lying on top of at least one pup Experimental animal is investigating a pup
Duration Durationa Duration/frequency Duration/frequency Duration/frequency Frequencya Duration Duration/frequency Duration Duration/frequency
a
Very low incidence (b3).
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Fig. 1. The duration of in-colony (A) huddling or (B) pup carrying (+/−SEM) for Experiment 1. Closed bars represent males and open bars represent females. * represents a treatment difference (p b 0.05). # represents a statistical trend for a sex difference (p = 0.059).
increased in-colony huddling behavior in subordinate animals, regardless of dose. Furthermore, OT increased the time spent in close proximity to, and number of investigations of, a familiar conspecific in a pairedinteraction paradigm and these effects were blocked by coadministration of an OT antagonist. While we did not see any effects of OT on pup-care behaviors either in-colony or during a pup-care paradigm, we saw subtle effects of sex where females tend to perform more pup carrying than males. Our finding that peripheral administration of OT increased huddling behavior is consistent with reports from other rodents. Central OT antagonist action decreases the total amount of huddling in rat pups, and the cohesiveness of the huddle structure and, interestingly, interferes with the ability of rat pups to use maternally-paired odor cues to guide their huddling behavior (Alberts, 2007; Kojima and Alberts, 2011). Furthermore, central OT administration increases the total amount of same-sex huddling in female prairie voles and also increases
the preference for huddling with an already familiar animal over a novel one (Beery and Zucker, 2010), while OTKO mice spend less time huddling in colonies in the visible burrow system (Pobbe et al., 2012). This effect also appears to extend to some primate species. Circulating OT is highly correlated with affiliative behavior (including huddling) in tamarins (Snowdon et al., 2010) and intranasal OT increases, while intranasal OT antagonists decrease, huddling in marmosets (Smith et al., 2010). In Experiment 2, we showed that peripheral OT administration also increased proximity of naked mole-rats. The ability of OT to regulate distance between familiar conspecifics is not specific to naked molerats. Peripheral OT antagonist administration decreases the time spent in close proximity both to a familiar cagemate and to a larger group of conspecifics in zebra finches while central mesotocin administration has the opposite effect (Goodson et al., 2009). Furthermore, OTKO mice spend more time alone and away from conspecifics in the colonial
Fig. 2. The duration of time spent in (A) close proximity or (B) digging (+/−SEM) and the number of (C) investigations of the stimulus animal or (D) pup carries (+/−SEM) for Experiment 2. Closed bars represent males and open bars represent females. * represents a difference from all other treatments (p b 0.01). ρ represents a sex difference (p b 0.05). # represents a statistical trend for a sex difference (p = 0.067).
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Fig. 3. The (A) number or (B) duration of mutual investigations (+/−SEM) in Experiment 2. Closed bars represent opposite-sex pairings and open bars represent same-sex pairings. ρ represents a sex difference (p b 0.05).
visible burrow system (Pobbe et al., 2012). Intramuscular injections of OT in the colonial meerkat did not result in spending more time close to adult conspecifics but this was likely due to an increase in the time spent close to pups that resulted from these peripheral OT injections (Madden and Clutton-Brock, 2011). We found no effect of OT on huddling in Experiment 2 (operationally defined as adjacent lying). However, while naked mole-rats form large group huddles in their colony, they rarely exhibit this behavior when placed in a novel environment for a short period (as done in the social pairing paradigm of Experiment 2) because they are highly active, exploring the new environment. During the dyadic interactions of the experimental and stimulus animals in the paired-interaction paradigm, we assessed which animal initiated each social investigation. OT administration increased the experimental-initiated investigations. This could potentially be due to the animals being in closer proximity on days in which OT was administered. However, the stimulus animal would have also been in closer proximity to the experimental animals on these days and yet they failed to show the same increase in investigation frequency. OTKO mice appear to have trouble habituating to familiar mice and so continue to investigate them at a high rate (Choleris et al., 2003). However, because OT did not alter the duration of investigation, it seems unlikely that social recognition was impaired following OT treatment. This leads us to suggest that OT administration increased interest in the social partner. Importantly, the effects of OT on proximity and investigation were blocked by co-administration of the OT antagonist, suggesting that these effects were linked to OT receptor activity. As the OT antagonist did not reduce behaviors by itself, OT action may not be necessary for these behaviors but may be capable of modulating them. Alternatively, the dosage of this particular OT antagonist may not have been high enough to independently change behavior. Other studies using the L368,899® antagonist in primates and rodents have found behavioral effects with much higher doses (Boccia et al., 2007; Klenerova et al., 2009). We employed peripheral administration of OT and the antagonist. As such, we cannot conclude which neural regions, if any, are associated with our behavioral effects. However, subordinate naked mole-rats have more OT neurons in the PVN than breeders (Mooney and Holmes, 2013) and the medial amygdala and nucleus accumbens show a high density of OT receptor binding in subordinates (Kalamatianos et al., 2010). OT activity in the medial amygdala is critical for social recognition in mice (Ferguson et al., 2001). Furthermore, OT may additionally mediate the salience of social stimuli via interactions with the dopaminergic system (Burkett and Young, 2012; Liu and Wang, 2003; Shahrokh et al., 2010; Young et al., 2001). Given that the behaviors affected by peripheral OT administration in naked mole-rats are consistent with an increase in social salience and, potentially, social recognition (e.g., the increased frequency of investigation of stimulus animals), we propose that OT coming from the PVN acts on the medial amygdala and nucleus accumbens to promote pro-social behavior in subordinate naked mole-rats.
Of course, it must be considered that the apparent increased affiliation we report is due to indirect effects of OT. For example, OT may promote social cohesion during environmental stressors (Yamasue et al., 2012). Although we did not measure glucocorticoid activity, removal from the colony is a potential stressor for this gregarious species and so it is interesting that OT would increase proximity during this situation. However, in light of the ability of OT to promote huddling behavior in-colony, this explanation seems unlikely. Alternatively, we classify huddling and proximity as pro-social behaviors but they are also a means of thermoregulation and energy conservation in this poikilothermic species (Yahav and Buffenstein, 1991). While we did not measure energy conservation, the huddling results do not appear to be due to OT effects on body temperature. Animals treated with 1 or 10 mg/kg OT did not decrease in temperature any more than the saline treated animals over a 30 minute period. We did not detect any effect of drug on pup care. While subordinate naked mole-rats perform aspects of pup care in their natal colony, breeding animals perform a higher per capita amount of these behaviors (Jarvis, 1991; Lacey and Sherman, 1991). Thus, investigating OT effects on pup-directed behavior in breeding mole-rats will be important. Somewhat surprisingly, we report a marginal effect of sex on pup carrying behavior where adult female subordinates tend to carry pups more than males do. While there was a difference between Experiments 1 and 2 in what parameter of pup carrying (frequency vs. duration) was closer to statistical significance, this was possibly due to the testing chamber dimensions. Colony chambers are larger and so pups may be carried longer distances at a time. This was not the case for the much smaller arenas in which the pup-interactions tests took place. While this putative sex effect was not related to OT manipulation, it has some implications for the naked mole-rat literature. Previous reports have either failed to identify a sex difference or did not account for sex in pup care in this species when examined in a colony setting (Jacobs and Jarvis, 1996; Jarvis, 1981, 1991; Lacey and Sherman, 1991). On a potentially related note, we also demonstrated that subordinate females may be slightly more affiliative in that they remain in closer proximity to stimulus animals more than males do. While sex differences among non-reproductive subordinates are often absent in the neural regions measured to date (Holmes et al., 2007, 2011; Seney et al., 2006), the subtle differences in behavior reported here may guide investigation into sex differences in brain physiology that have yet to be discovered in this species. Finally, in experiment 2, we found that administering the antagonist induced a sex difference in digging behavior resulting from a decrease in males and an increase in females (Fig. 2B). While it is exciting to postulate that OT acts to eliminate sex differences in digging behavior in naked mole-rats, future investigation into the role of direct OT action in the brain will help understand this effect. In summary, we show here that OT modulates a number of behaviors important for mammalian eusociality. Interest in conspecifics, the tendency to stay close to familiar conspecifics and huddling behavior
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