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Social selectivity and social motivation in voles

  1. Annaliese K Beery  Is a corresponding author
  2. Sarah A Lopez
  3. Katrina L Blandino
  4. Nicole S Lee
  5. Natalie S Bourdon
  1. Department of Integrative Biology, University of California Berkeley, United States
  2. Program in Neuroscience, Departments of Psychology and Biology, Smith College, United States
  3. Neuroscience and Behavior Graduate Program, University of Massachusetts, United States
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Cite this article as: eLife 2021;10:e72684 doi: 10.7554/eLife.72684

Abstract

Selective relationships are fundamental to humans and many other animals, but relationships between mates, family members, or peers may be mediated differently. We examined connections between social reward and social selectivity, aggression, and oxytocin receptor signaling pathways in rodents that naturally form enduring, selective relationships with mates and peers (monogamous prairie voles) or peers (group-living meadow voles). Female prairie and meadow voles worked harder to access familiar versus unfamiliar individuals, regardless of sex, and huddled extensively with familiar subjects. Male prairie voles displayed strongly selective huddling preferences for familiar animals, but only worked harder to repeatedly access females versus males, with no difference in effort by familiarity. This reveals a striking sex difference in pathways underlying social monogamy and demonstrates a fundamental disconnect between motivation and social selectivity in males—a distinction not detected by the partner preference test. Meadow voles exhibited social preferences but low social motivation, consistent with tolerance rather than reward supporting social groups in this species. Natural variation in oxytocin receptor binding predicted individual variation in prosocial and aggressive behaviors. These results provide a basis for understanding species, sex, and individual differences in the mechanisms underlying the role of social reward in social preference.

eLife digest

What factors drive the formation of social relationships can vary greatly in animals. While some individuals may be motivated to find social partners, others may just tolerate being around others. A desire to avoid strangers may also lead an individual to seek out acquaintances or friends. Sometimes a mix of these factors shape social behavior.

Studying motivation for social relationships in the laboratory is tricky. Traditional laboratory animals like mice and rats do not bond with specific peers or mates. But small burrowing rodents called voles are a more relationship-oriented alternative to mice and rats. Prairie voles form selective and enduring preferences for both their mates and familiar same-sex peers. Meadow voles on the other hand, live alone much of the year but move in with other animals over the winter.

Beery et al. show that social motivation in voles varies by relationship type, species and sex. In the experiments, voles were first trained to press a lever to get a food reward. Then, the food reward was swapped with access to familiar or unfamiliar voles. Female prairie voles strived to be with animals they knew rather than to be with strangers, while male prairie voles tried hard to access any female. In contrast, meadow voles did not overly exert themselves to access other animals.

Beery et al. then measured oxytocin receptor levels in the brains of prairie voles. Prairie voles that had more receptors for oxytocin in part of their brain known as the nucleus accumbens worked harder to access their familiar partner. But individuals with more oxytocin receptors in the bed nucleus of the stria terminalis were more likely to attack an unfamiliar animal.

The meadow voles’ behavior suggests that they are more motivated by tolerance of familiar animals, while the female prairie voles may find it rewarding to be with animals they have bonded with. These differences may help explain why these two species of vole have evolved different social behaviors. The experiments also suggest that oxytocin – which is linked with maternal behavior – plays an important role in social motivation. Learning more about the biological mechanisms that underlie vole social behaviors may help scientists identify fundamental aspects of social behavior that may apply to other species including humans.

Introduction

The brain regions and neurochemicals involved in social behaviors show remarkable conservation across species (O’Connell and Hofmann, 2011). At the same time, social behavior is not a unified construct, with different species exhibiting distinct social structures and behavioral repertoires. The formation of selective social relationships is a particular hallmark of both human and prairie vole societies. Such relationships are difficult to study in traditional lab rodents because mice, rats, and other rodents typically do not form preferences for known peers or mates (Triana-Del Rio et al., 2015; Schweinfurth et al., 2017; Beery et al., 2018; Cymerblit-Sabba et al., 2020; Insel et al., 2020; Beery and Shambaugh, 2021). In species that form specific relationships, selectivity may be based on reward and prosocial motivation toward specific individuals, or on avoidance (fear, aggression) of unfamiliar individuals. The role of social motivation and tolerance may also differ by familiarity, sex, and type of relationship (e.g. same-sex peer versus opposite-sex mate). Voles provide an opportunity to probe the role of selectivity and social reward across relationship types and social organization.

The reinforcing properties of social interaction have been demonstrated in a variety of rodent species and contexts, often through conditioned place preference for a socially associated environmental cue (e.g. Panksepp and Lahvis, 2007; Dölen et al., 2013; Goodwin et al., 2019). Operant conditioning for access to a social stimulus has been used to more directly measure motivation for specific types of social interaction, particularly access to pups, social play, and sexual opportunities (reviewed in Trezza et al., 2011). Social motivation has also been assessed with access to novel same-sex peers (Martin and Iceberg, 2015; Achterberg et al., 2016; Borland et al., 2017). Often social interactions are affiliative, but in some contexts animals will work for access to aggressive interactions (Azrin et al., 1965; Falkner et al., 2016; Golden et al., 2017). To date, only one study has examined the role of familiarity in social motivation, in novelty-preferring female rats (Hackenberg et al., 2021), and none have done so with mate relationships.

Prairie voles, Microtus ochrogaster, and meadow voles, Microtus pennsylvanicus, both form selective social relationships but exhibit different social organization and mating systems. Prairie voles are socially monogamous, forming long-term selective relationships between males and females that have been studied for decades (Carter et al., 1995; Walum and Young, 2018). Prairie voles also form selective relationships with familiar same-sex cage-mate ‘peers’ (DeVries et al., 1997; Beery et al., 2018; Lee et al., 2019). Meadow voles are promiscuous breeders that transition to living in social groups and sharing nests during winter (Getz, 1972; Madison and Mcshea, 1987). Under conditions of short daylength in the laboratory, female (but not male) meadow voles exhibit greater social huddling and less aggression than their long daylength counterparts (Beery et al., 2008b; Lee et al., 2019). These vole species thus allow comparison of the properties of peer relationships across species (prairie vole peers versus meadow vole peers) and relationship type within species (prairie vole mates versus prairie vole peers).

Prairie voles exhibit socially conditioned place preferences (sCPP) for familiar opposite-sex mates (Ulloa et al., 2018; Goodwin et al., 2019), and in some circumstances for same-sex peers (Lee and Beery, 2021). In contrast, meadow voles do not form sCPP and may even condition away from social cues (Goodwin et al., 2019). Neurochemical pathways underlying social reward also vary between species and relationship type; dopamine is necessary for the formation of opposite-sex pair bonds in prairie voles (Aragona and Wang, 2009), but is not necessary for the formation of same-sex peer preferences in meadow or prairie voles (Beery and Zucker, 2010; Lee and Beery, 2021). These initial findings suggest that social selectivity may result from differential social motivation and tolerance in these species.

Voles demonstrate striking preferences for familiar versus novel peers and mates, assessed using the partner preference test (Williams et al., 1992b; Beery, 2021). This test quantifies preference, but as no effort is required to access a conspecific, it cannot distinguish between prosocial motivation and avoidance of unfamiliar conspecifics. To examine the role of motivation in relationships, we assessed effort expended by voles of different sexes (male, female), relationship types (same-sex, opposite-sex), and species (prairie vole, meadow vole) to reach social targets in an operant conditioning paradigm. Because the seasonal transition from solitary to social is most pronounced in female meadow voles in the field and laboratory (Madison and Mcshea, 1987; Beery et al., 2009), only females of this species were used. Subjects underwent >60 active training and testing days (Figure 1). Responses (lever presses) in lightly food-restricted voles were shaped and reinforced using a food reward, followed by 8 days of pressing for a food reward on a progressive ratio 1 (PR-1) schedule. Social testing consisted of 8 consecutive test days in which each reward consisted of 1 min of access to the familiar (same- or opposite-sex) partner, and 8 test days for which rewards consisted of access to different sex-matched strangers (order balanced within groups). We assessed effort expended to access familiar and novel social stimuli in four groups of prairie voles (Figure 1): females lever pressing for a female conspecific (F➤F), females pressing for a male conspecific (F➤M), males pressing for a male conspecific (M➤M), and males pressing for a female conspecific (M➤F). Meadow vole females (F➤F) were also trained and tested for 8 days of familiar and 8 days of novel vole exposure, counterbalanced. A subset of voles was used to explore the reward value of an empty chamber, extinguishing timelines, and relationships between oxytocin receptor (OTR) density and behavior.

Overview of apparatuses, timeline, and testing groups.

(A) Lever pressing in voles was shaped and trained using food reinforcement. (B,C) In social operant testing a lever operated a motorized door, providing 1 min access to a conspecific tethered in a connected compartment. (D) Five groups were tested, abbreviated here as focal sex-partner sex-species abbreviation (e.g. FF Prairie indicates a female prairie vole trained as a lever presser and housed with a female partner). Prairie = prairie vole (Microtus ochrogaster); Meadow = meadow vole (Microtus pennsylvanicus). Black lines connect testing phases completed by all study subjects; gray lines connect additional phases completed by a subset of subjects.

Oxytocin is involved in social recognition as well as in preference for familiar individuals (reviewed in Anacker and Beery, 2013), and in many instances, oxytocin signaling alters the rewarding properties of social stimuli (Dölen et al., 2013; Borland et al., 2018). We conducted receptor autoradiography to assess variation in neural OTR density in female prairie voles. (OTR was not analyzed in male brains; following early results, later males were used to pilot a two-choice social operant paradigm.)

Together these studies allowed us to examine how the reward value of social contact differs between male and female prairie voles, between opposite-sex and same-sex pairings, and between meadow and prairie vole FF pairings. We found both similarities in and striking differences between social motivation across species, sexes, and pairing types. Detailed examination of social behaviors during social access further underscored the distinction between social motivation and familiarity preference, especially in males. In addition to these group differences in social motivation, individual differences in OTR density were related to aggressive and prosocial behaviors.

Results

Sex-specific patterns of social effort in prairie voles

In order to assess motivation for different kinds of social stimuli across groups, lever pressing responses were quantified on a progressive ratio schedule (PR-1). Males and females showed qualitatively different response patterns in the social chambers, as well as significant interaction between sex and variables of interest in a model screening for sex differences (sex*stimulus type (p = 0.01), sex*stimulus familiarity (p = 0.09)), so responses were further analyzed separately by sex (Beery, 2018; Beltz et al., 2019). For each sex, two-way repeated measures ANOVA (RM-ANOVA) was performed with familiarity of the tethered stimulus (partner/stranger) as the within-subjects/repeated measure, and sex of the tethered stimulus (opposite-sex/same-sex) as a between-subjects measure. Female prairie voles pressed more for familiar partners than unfamiliar strangers, with no effect of opposite-sex versus same-sex pairings (Figure 2A, effect of stimulus familiarity: F(1, 14) = 15.17, p = 0.0016, ηp2p20.52; no effect of stimulus sex: F(1, 14) = 0.44, p = 0.51, ηp2p20.03; subject matching: F(14, 14) = 4.2, p = 0.0057, ηp2p20.81, no significant interaction). Paired t-tests were used for within-group comparisons of responses for the partner or stranger: familiarity preferences were significant in females paired with males (t(7) = –2.7, p = 0.03, d = 0.96) as well as in females paired with females (t(7) = –4.1, p = 0.0048, d = 1.43). The mapping from response count to the corresponding PR-1 breakpoint (i.e. the maximum number of responses exhibited to achieve a reward) is shown in Figure 2A and applies to all response count figures.

Figure 2 with 1 supplement see all
Sex-specific patterns of effort expended to access different social stimuli on a progressive ratio 1 (PR-1) schedule.

(A) Female prairie voles responded more for familiar than unfamiliar voles of either sex. (B) Male prairie voles pressing for females responded more than did males pressing for males, regardless of familiarity. Dots represent mean number of responses across eight 30 min PR-1 sessions for each vole. Bars represent group means. PR-1 breakpoint thresholds are listed in italics next to the corresponding number of responses on the interior y-axis of panel A and apply to all lever pressing data (e.g. a vole that presses 55 times should receive 10 rewards, the last of which takes 10 responses to gain). Asterisks indicate significant familiarity preferences within groups (paired t-tests). *p < 0.05, **p < 0.01.

Male prairie voles pressed at a higher rate for opposite-sex social stimuli regardless of familiarity (effect of stimulus sex: F(1, 14) = 17.4, p = 0.0009, ηp2p20.71; no effect of familiarity, F(1, 14) = 0.013, p = 0.91, ηp2p20.00, no significant effects of subject matching or interaction).

Because each vole was tested in eight consecutive sessions of each type, familiarity preference could also be assessed within individuals across days. Significant within-vole familiarity preferences were present in more female pressers (6/8 F➤M and 3/8 F➤F) than males (1/8 M➤F and 0/8 M➤M pairs) (Figure 2—figure supplement 1; p = 0.0059 Fisher’s exact test). One male in a M➤F pair exhibited a significant preference for stranger females (Figure 2—figure supplement 1), and mounted/copulated with strangers in multiple test sessions.

Social motivation and behavior were parallel in female but not male prairie voles

In female prairie voles, the familiarity preference for both mates and peers in lever pressing was mirrored in cohabitation time and huddling. Even when these behaviors were scaled relative to lever presses (and thus access time), females spent a significantly higher fraction of the available time in the social chamber (time in social chamber/access time) when it was occupied by a familiar vole rather than a novel one (effect of familiarity F(1,14)=95.06, p < 0.0001, ηp2p20.87; subject matching F(14,14) = 2.789, p = 0.03, ηp2p20.73; others NS; two-way RM-ANOVA, Figure 3A). Females also spent more of the available time huddling (time spent in immobile side-by-side contact/access time) with familiar rather than unfamiliar conspecifics of either sex (effect of familiarity: F(1,14) = 25.82, p = 0.0002, ηp2p20.65; others NS; two-way RM-ANOVA, Figure 3C). Within-group matched comparisons of time spent with a partner or stranger also revealed that females exhibited significant familiarity preferences in time spent in the social chamber or huddling with the stimulus animal relative to time with access (time in social chamber/access time: FF: p < 0.0001, d = 3.51; FM: p = 0.0006, d = 2.12; time huddling/access time: FF: p = 0.0090, d = 1.26; FM: p = 0.0083, d = 1.29; paired t-tests).

Affiliative and aggressive interactions with stimulus voles.

Data represent the 8-day testing mean for each vole (n = 8/group, ± SEM). (A,B) Percent of time focal voles spent in the social chamber relative to time when the door was open, allowing chamber access. Females shown in A, males in B. (C,D) Percent time spent huddling out of access time (i.e. when the door was raised). Significant effects of two-way repeated measures ANOVA (RM-ANOVA) are reported above each graph. Asterisks represent the results of within-groups paired t-tests. (E,F) Prairie voles exhibited significantly more bouts of aggression toward strangers (p < 0.0001), and there were no significant effects of sex of the presser or of the social target. (G) No relationship was present between daily lever pressing for access to strangers and aggression scaled by access time in male or female prairie voles. NS = not significant, *p < 0.05, **p < 0.01, ***p < 0.001.

In contrast, while males exhibited no familiarity preferences in lever pressing responses, they still exhibited strong familiarity preferences in social interaction. Males spent more of the available time in the social chamber when the tethered stimulus was familiar (effect of familiarity F(1,14) = 6.33, p = 0.02, ηp2p20.31; subject matching F(14,14) = 4.459, p = 0.0042, ηp2p20.24; others NS; two-way RM-ANOVA, Figure 3B), and huddling behavior was even more specific, with a strong effect of stimulus familiarity (partner versus stranger) and no effect of stimulus sex (opposite- versus same-sex) on the percent of [time huddling]/[time with access to the social chamber] (effect of familiarity: F(1,14) = 25.27, p = 0.0002, ηp2p20.64; all else NS; Figure 3D). Within-group matched comparisons also revealed significant familiarity preferences in huddling time relative to access (huddling/access time: MM: p = 0.0177, d = 1.09, MF p = 0.0022, 1.66), with lesser or no familiarity preference in chamber time (time in social chamber/access time: MM: p = 0.0390, d = 0.90, MF: p = 0.56, d = 0.21; paired t-tests). There was no apparent sex difference in huddling behavior between male and female prairie voles, confirmed by pooling males and females in a three-way ANOVA (effect of focal sex NS, p = 0.91; significant effect of stimulus familiarity (F(1,56) = 48.03, p < 0.0001, ηp2p20.46); effect of stimulus sex; NS, no significant interactions).

Other social/sexual behaviors in prairie voles

Aggressive behavior was exhibited by prairie voles in all groups during social operant sessions and was analyzed by RM-ANOVA on all voles tested with partners and strangers (between-subjects factors: sex of presser (M/F)*pairing type [same/opposite sex]; within-subjects factor: target familiarity). Both males and females engaged in far more bouts of aggression with strangers than familiar partners (F(1,29) = 30.22, p < 0.0001, ηp2p20.51, Figure 3E and F). There was no significant effect of sex of the presser (F(1, 29) = 3.36, p = 0.077, ηp2p20.10), pairing type (same-sex or opposite-sex), or interactions between these variables.

Because aggression was primarily targeted at strangers, we asked whether stranger aggression might be motivating: that is, whether aggression was associated with greater lever pressing for strangers. Correlation of daily stranger lever pressing with bouts of aggression was not significant across females (R = 0.14, p = 0.10), but was significant across males (R = 0.25, p = 0.004). Because more time with access to a stranger provides more opportunity for aggression to occur, aggressive bouts were also scaled relative to access time, as was done for the other social measures. Across groups there were no relationships between stranger-directed daily lever pressing and aggression/access time in either male or females (males: R = 0.04, p = 0.64; females: R = 0.12, p = 0.16, Figure 3G).

Mounting behavior was present in five prairie voles, all of which were male prairie voles tested with novel (unfamiliar) female voles. This distribution was significantly non-random across the eight testing combinations used in prairie voles (e.g. male with female partner, male with female stranger, etc.) (χ2(7) = 37.97, p < 0.0001). These five voles exhibited an average of 6 bouts of mounting per testing session.

Neural OTR density related to behavior and housing

OTR density was associated with both motivated and aggressive social behaviors in different brain regions in female prairie voles (males not assayed). There was a strong positive correlation between OTR density and lever presses for same-sex partners in the nucleus accumbens (NAcc) core (R = 0.959, p = 0.0098) and shell (R = 0.948, p = 0.0141, Figure 4A). There was also a strong positive correlation between mean bouts of stranger-directed aggression and OTR density in the bed nucleus of the stria terminalis (BNST) in female prairie voles (R = 0.719, p = 0.0126), again connecting receptor binding to behavior. Binding density in the BNST was not associated with stranger approach or avoidance, operationalized as time spent in the stranger’s social chamber relative to access time (R = 0.350, p = 0.29), or lever presses for the stranger’s chamber (R = 0.264, p = 0.43).

Oxytocin receptor (OTR) density, housing, and social behavior.

(A) Representative images of 125I-OVTA binding patterns in the brains of female prairie voles. The top row shows binding at the level of the nucleus accumbens (NAcc), and an adjacent acetylcholinesterase (AChE) stained section for anatomical verifications. The bottom row shows binding at the level of the bed nucleus of the stria terminalis (BNST) and late lateral septum, as well as an adjacent section from the same brain processed for non-specific binding using the highly selective OTR antagonist [Thr4Gly7]-oxytocin. (B) OTR binding in the NAcc core and shell were strongly correlated with individual variation in lever pressing for a partner. (C) OTR binding in the BNST was positively correlated with stranger-directed aggression in females across pair types. (D) OTR density also varied in response to housing/pairing condition (opposite-sex versus same-sex). *p < 0.05, +p < 0.1.

OTR density varied with housing condition. Females housed with same-sex cage-mates showed no difference in OTR density in the NAcc or lateral septum (LS), higher OTR density in the BNST (t(8.99) = 2.93, p = 0.0167, d = 1.78), and a non-significant trend in the central amygdala (t(8.71) = 1.92, p = 0.0883, d = 1.17) compared to females housed with opposite-sex cage-mates (Figure 4C).

Interspecific comparisons: responses were reward-specific and comparable across species and sexes

Lever pressing responses in prairie voles were compared to those of a related non-monogamous vole species (the meadow vole) that exhibits group living during winter months. Female meadow voles are territorial and aggressive in summer or long daylengths in the lab, but socially tolerant in winter or short days. Because male meadow voles do not undergo this transition (Madison and Mcshea, 1987; Beery et al., 2009), we focused on comparison of social motivation in female meadow voles relative to female prairie voles. Prior to making this comparison, we assessed whether species and sexes differed in their lever pressing effort in response to a common reward (food). There were no sex or species differences in the number of lever pressing responses for a food reward (PR-1 schedule; 8 days averaged per subject) between female prairie voles, male prairie voles, and female meadow voles (F(2,40) = 1.18, p = 0.32, η2 = 0.56; one-way ANOVA; Figure 5A). Food responses and social responses were converted to response rates for comparison across trials with different active lever pressing periods: individual response rates for a food reward did not predict response rates during social testing for either the partner (p = 0.78) or the stranger (p = 0.98), indicating that responses were not subject-specific across reward types (Figure 5B). These findings validate the specificity of comparisons across species, sexes, and reward types.

Figure 5 with 1 supplement see all
Quantifying responses across species, sexes, and reward types.

(A) Responses for a food reward did not significantly differ between prairie voles of different sexes or between meadow and prairie vole females. Each data point represents the 8-day mean of responses from a vole tested using a progressive ratio 1 (PR-1) schedule in 30-min sessions. (B) Food response rate did not predict social response rate for familiar or unfamiliar stimuli. Data points show prairie vole response rates for food pellets on a PR-1 schedule (8-day mean for each vole) versus social reward (black: partner; gray: stranger) on a PR-1 schedule (8-day mean for each vole). (C) Meadow voles, like prairie voles, pressed more for a partner than a stranger, but pressed significantly less overall. (D) Social pressing for a partner in meadow voles was no higher than pressing for an empty chamber, and stranger pressing was similar to the minimum achieved by extinction. (E) Extinction profile over 10 days for each species and sex tested. Lever presses diminished rapidly over the first 4–5 days of testing with an inactive lever.

Meadow voles exhibited familiarity preferences but low social response rates

Female meadow voles pressed significantly more for familiar females than novel females (t(6)=3.637, p = 0.0109, d = 1.37, paired t-test, Figure 5C; males not tested). This preference was individually significant within four of the seven meadow voles (Figure 5—figure supplement 1). Comparisons of time spent with a partner or stranger when the door was up also revealed significant familiarity preferences (P versus S for social chamber/access time: p = 0.0351, d = 1.02; P versus S for huddling/access time: p = 0.0357, d = 1.02; paired t-tests).

Despite familiarity preference, meadow vole response rate for both partners and strangers was low. Direct comparison with female prairie voles tested under the same conditions reveals that while both groups pressed more for familiar partners than for strangers, there was significantly less lever pressing in female meadow voles (two-way ANOVA, effect of target familiarity: F(1,13) = 29.51, p < 0.001, ηp 2 = 0.69, effect of species: F(1,13) = 9.71, p < 0.01, ηp2p20.43, Figure 5C). Comparison of lever presses between social conditions and non-social ‘empty control’ conditions indicates that, for female meadow voles, the partner was not more rewarding than the empty chamber control, stranger pressing was significantly lower than empty control, and it was similar to the post-extinction level of pressing (Figure 5D).

Other social/sexual behaviors in meadow voles

Aggression was rare in meadow vole trials (mean 0.3 bouts/trial), and as in our prior studies (Lee et al., 2019) it was significantly less frequent than aggression between female prairie voles (mean 2.3 bouts/trial, species difference: t(3.83), p = 0.001). No mounting behavior was observed in meadow vole tests, all of which were conducted in female voles.

Empty chamber control and extinction

At the conclusion of social testing, all voles from cohorts 4 to 7 were tested for effort expended to explore an empty chamber without a tethered partner or stranger for 8 days each (n = 6 meadow females, 10 prairie females, and 14 prairie males). Voles were distributed across all housing types. There was no species difference in pressing for the empty chamber (meadow vole female versus prairie vole female). In both male and female prairie voles, the extent of lever pressing for the control chamber was correlated with pressing for the stranger (females: R = 0.75, p = 0.013; males: R = 0.71, p < 0.005) but not with lever pressing for the partner.

The same cohorts were then tested for extinction of lever pressing over 10 days of trials in which the door was closed and the lever did not activate the motor. All groups extinguished lever pressing behavior within ~5 days of testing (Figure 5). Repeated measures analysis revealed a significant effect of day of testing on pressing (F(9,21) = 3.72, p = 0.0063, ηp2p20.61) but no significant effect of the testing group on extinction (F(2,29) = 0.76, p = 0.48, ηp2p20.05).

Discussion

Male and female prairie voles worked for brief access to conspecifics, but exhibited quantitatively and qualitatively different patterns of pressing, indicating striking sex differences in social motivation. In females, lever pressing effort was based on familiarity of the social target (partner versus stranger), but did not differ between same-sex (FF) and opposite-sex (FM) housed pairs. Because testing occurred with only the partner or stranger present at any given time, failure to spend time in the stranger chamber indicates lack of interest in the stranger, as opposed to relative preference for a better option. Females also exhibited extensive partner huddling and time spent in the chamber of a partner but not a stranger. These preferences persisted when scaled by rewards (i.e. time the subject was available), indicating strong selectivity in social preferences. Social motivation thus paralleled social selectivity in females.

Male prairie voles exhibited similarly strong selectivity in huddling and chamber preferences, consistent with decades of work showing partner preferences in both male and female prairie voles. In contrast, males showed no propensity to work harder to access a familiar vole than an unfamiliar social target, but instead worked significantly harder to access an opposite-sex social target than a same-sex social target. This reveals a dissociation between social motivation and markers of social bond formation such as huddling in males. This sex difference in motivated behavior is consistent with the hypothesis that outwardly similar partner preferences in males and females result from latent differences in underlying signaling pathways (De Vries, 2004). Oxytocin, dopamine, and opioid signaling all affect partner preferences in males and females (Williams et al., 1992a; Gingrich et al., 2000; Aragona et al., 2003; Burkett et al., 2011; Resendez et al., 2012; Johnson et al., 2016), but prairie voles also exhibit sex differences in these signaling pathways (e.g. Winslow et al., 1993; Martin et al., 2015; Ulloa et al., 2018). These latent differences in mechanisms underlying social bonding may support similar partner preference behavior while promoting sex differences in social motivation.

Factors that may particularly motivate males to access unfamiliar females include opportunities for mating and aggression. Male prairie voles exhibit multiple mating strategies in field settings, including both a socially monogamous ‘resident’ partner strategy, and a ‘wanderer’ strategy; however, even residents engage in extra-pair copulations (Madrid et al., 2020). Interest in non-partners can also result from motivation for aggressive behavior; for example, the opportunity for aggression is rewarding in dominant male mice (reviewed in Golden et al., 2019). That does not seem to be the case in male prairie voles, however. Aggression toward partners was rare, and response rate was not correlated with aggression toward a stranger in male prairie voles tested with females or males. Aggressive bouts in male prairie voles tested with strangers initially appeared correlated with lever pressing effort, but this effect disappeared when scaled by access time, unlike effects reported for huddling/access time. When social interest is high (e.g. males for unfamiliar females), it is still possible that males would press more for their partners if placed in direct opposition to a stranger, and this is an avenue for future investigation.

The lack of consistent mapping between effort in the operant task and partner preferences in male huddling highlights a disconnect between social reward and the selectivity of huddling preferences. This disconnect is further underscored by the presence of robust partner preferences in female meadow voles despite no evidence of social reward in the operant task or in sCPP tests (Goodwin et al., 2019). Thus, partner preference does not imply social reward, nor does social reward imply selective preference. These behavioral findings are consistent with the lack of effects of dopamine antagonists on same-sex peer partner preferences in female meadow voles as well as prairie voles (Beery and Zucker, 2010; Lee and Beery, 2021). While dopamine signaling is not necessary for peer partner preference expression, it can enhance preferences (Lee and Beery, 2021) and may play a more fundamental role in pair bonding with mates (Aragona and Wang, 2009). Because partner preference does not indicate behavioral reward, the partner preference test and other tests of social approach in the absence of work likely reflect different combinations of partner tolerance, partner reward, and stranger aversion.

OTR signaling differs by relationship type and by individual social behaviors

Strong relationships were present between OTR density, housing differences, and behavior, highlighting connections across levels of organization. Variation in OTR density by relationship type has not been previously assessed, although OTR density or mRNA levels differ in response to early-life housing manipulations in prairie voles, such as presence of a father and single versus group housing (Prounis et al., 2015) as well as chronic social isolation in adulthood (Pournajafi-Nazarloo et al., 2013).

Oxytocin signaling plays a role in diverse social behaviors in prairie voles, including pair bond formation, consolation behavior, and alloparental care (Williams et al., 1992a; Olazábal and Young, 2006; Bales et al., 2007; Burkett et al., 2016). Furthermore, oxytocin signaling has been related to social reward in non-selective mice and hamsters (Dölen et al., 2013; Song et al., 2016; Borland et al., 2018). Strong correlations between NAcc OTR and lever pressing for the partner in the present study provide additional support for the role of NAcc OTR in social reward. Neural OTR was related to aggressive behavior as well as prosocial behavior, underscoring the complexity of oxytocin signaling in different brain regions (van Anders et al., 2013; Beery, 2015).

Species differences

Social pressing differed quantitatively but not qualitatively by species in meadow and prairie voles. Females of both species pressed more for partners than for strangers, but responses were lower in meadow voles, indicative of the lack of social reward. This is consistent with prior findings from sCPP tests, in which meadow voles did not condition toward a bedding associated with social contact, and in one setting conditioned away from it (Goodwin et al., 2019). These findings are also in line with results from the sole prior study of operant responses in voles. Matthews et al., 2013, tested prairie voles and meadow voles housed in long daylengths to determine whether they would learn to lever press for stranger voles. Only prairie voles demonstrated clear learning in this scenario, consistent with low stranger interest in meadow voles housed in the long daylengths that promote territorial behavior in this species (Beery et al., 2008b). Nonetheless, even under pro-social short daylength conditions used in the present study, social pressing was low in meadow voles. Comparison of short daylength-housed female meadow vole responses for the partner chamber, stranger chamber, and an empty chamber in different trial blocks revealed equivalent levels of pressing for a partner or an empty chamber and less for the stranger. This suggests that decreased pressing for the stranger represents avoidance, but that pressing for the partner may indicate tolerance more than reward. Female (short daylength-housed) meadow voles also exhibited lower aggression than female prairie voles, consistent with social tolerance, and with prior descriptions of their behavior (Lee et al., 2019).

Comparability across vole species and sexes

Lever pressing was demonstrated to be an effective metric to compare effort exerted to reach different social stimuli in voles; voles of each species and sex tested pressed at comparable rates for food reward, indicating a lack of major differences in task learning, and thus that social lever pressing can be assessed and compared across groups. Subject response rates were not consistently high or low across reward conditions, indicating that responses are reward-specific. Extinction was effective, with all subjects decreasing lever pressing behavior by more than half their baseline response count. Differences in lever pressing effort between groups could therefore be attributed to reward-specific differences in social motivation.

Implications for the evolution of social relationships

Persistent relationships within specific pairs or groups of conspecifics are present throughout the animal kingdom, including species of invertebrates, fishes, amphibians, reptiles, birds, and mammals (Bales et al., 2021). While the nature and extent of these relationships vary considerably, they share in common the specificity of social preferences that leads to repeated association. They may differ, however, in the mechanisms that influence familiar approach and unfamiliar avoidance. In particular, familiar individuals—whether mates or peers—may or may not be socially rewarding, and unfamiliar individuals may or may not be aversive.

Even within closely related vole species, we see evidence that only some relationships involve selective social reward, for example, mate relationships in female prairie voles, while others—such as peer relationships in winter phenotype meadow voles—involve selectivity without appreciable reward. Selectivity in the absence of reward may rely instead on changing social anxiety and aggression (Beery, 2019). For example, when exposed to the short, winter photoperiods associated with the transition from solitary to group living in the wild, meadow voles undergo changes in CRF (corticotropin-releasing factor) receptor densities, glucocorticoid secretion, behavioral indicators of anxiety, and aggression (Ossenkopp et al., 2005; Beery et al., 2014; Anacker et al., 2016). More research is needed to establish causal links between these changes and the transition to group living. More broadly, it remains to be determined to what extent social monogamy and pair bonding with mates shares mechanisms across species (Goodson, 2013), and to what extent different types of relationships (e.g. with peers or mates) share foundations, or differ in their regulation. Ultimately, these studies should help us understand how selective relationships of different types evolve.

Conclusions

While other studies have assessed social reward in rodents, few have considered the role of stimulus familiarity, likely because laboratory rodents do not exhibit familiarity preferences under normal conditions (reviewed in Beery and Shambaugh, 2021). In social choice tests, mice and young rats often prefer social novelty (Moy et al., 2004; Smith et al., 2015), and relative preference for a social stimulus versus a food stimulus is greater when novel rats are presented (Reppucci et al., 2020). Indeed, in operant trials in which rats had simultaneous access to familiar and unfamiliar same-sex conspecifics, rats expended more effort to access unfamiliar conspecifics (Hackenberg et al., 2021). In the present study, female prairie voles exhibited similar partner preferences but higher social motivation and aggression compared to female meadow voles. Social motivation and selectivity were not linked in male prairie voles, and there was a striking sex difference in the reward value of mates and peers in prairie voles. OTR binding revealed connections between social environment, receptor density, and prosocial behavior, illustrating the importance of this system across levels of biological organization. Better understanding of the interface between social motivation and social selectivity will thus be key to improving our understanding of the nature of social relationships.

Materials and methods

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Chemical compound, drug(Thr⁴,Gly⁷)-OxytocinBachem4013837
Chemical compound, drug125I-OVTA; 125I-ornithine vasotocin analog; vasotocin, d(CH2)5 [Tyr(Me)2,Thr4,Orn8,(125I)Tyr9-NH2]Perkin ElmerNEX254050UC
Chemical compound, drugTestosteroneSigma-AldrichT1500
Software, algorithmMED-PC IVMed AssociatesSOF-735

Animal subjects

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Prairie voles and meadow voles from in-house colonies were bred in a long photoperiod (14 hr light:10 hr dark; lights off at 17:00 EST; described further in Lee et al., 2019). Meadow voles were weaned into the winter-like short photoperiods associated with group living in this species (10:14 light:dark; lights off at 17:00 EST). Voles were pair-housed in clear plastic cages with aspen bedding and an opaque plastic hiding tube. Food (5015 supplemented with rabbit chow; LabDiet, St Louis, MO) and water were provided ad libitum, except during food restriction (described below). All procedures adhered to federal and institutional guidelines and were approved by the Institutional Animal Care and Use Committee at Smith College.

Timeline and groups

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Training began in adulthood at 62 ± 1.3 days of age (mean ± SEM, range 41–76). Operant conditioning training and testing consisted of multiple phases described briefly here and in greater detail in subsequent sections. Responses (lever presses) were shaped and trained using a food reward on a fixed ratio 1 (FR-1) schedule. Animals that met training criteria progressed to the experimental testing sequence, beginning with 8 days of pressing for a food reward on a PR-1 schedule (Figure 1). Subjects in opposite-sex pairs were placed with either a tubally ligated, hormonally intact female mate, or a castrated and testosterone implanted male mate 5–10 days prior to the start of social habituation and testing. Subjects in same-sex pairs remained with their cage-mate. Social testing consisted of 8 days of PR-1 with rewards yielding access to the familiar (same- or opposite-sex) partner, and 8 days with access to a sex-matched stranger (order balanced within groups). Voles were trained and tested over seven cohorts; group membership was distributed across cohorts, and voles were assigned to groups within sex without knowledge of their response rates in the training phase. A subset of voles (those in cohorts 4–7) continued in empty chamber control and/or extinguishing tests as described below. Voles were sacrificed at the conclusion of testing, and brains were stored at –80°C.

We tested four groups of prairie voles (Figure 1): females lever pressing for a female conspecific (F➤F), females pressing for a male conspecific (F➤M), males pressing for a male conspecific (M➤M), and males pressing for a female conspecific (M➤F). Each group consisted of eight focal voles, tested for 8 days with their partner and for 8 days with a series of novel ‘strangers’, sex-matched to the partner. The order of testing (partner then stranger or stranger then partner) was counterbalanced within groups. Some voles did not complete both partner and stranger testing, in which case additional voles were added up to 8/group. Meadow vole females (F➤F-Mp, n = 7) were also trained and tested for 8 days of familiar and 8 days of novel vole exposure, with order counterbalanced within the group.

Operant conditioning and testing with food reward

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Subjects were weighed for 3 consecutive days to establish baseline body weights, then food-restricted to a target weight of 90% baseline to enhance motivation for the food reward. Weights were recorded daily after training or testing, prior to being returned to their home-cages. Any vole that dropped to or below 85% of the baseline weight was returned to ad libitum food to avoid long-term health consequences. Perforated cage dividers were used during food restriction to ensure each vole had access to its specific ration (0.3–1 food pellets and ~4 g [half] of a baby carrot). Food restriction ended when subjects transitioned to social testing.

Operant conditioning was conducted in mouse-sized modular test chambers (30.5 cm × 24.1 cm × 21.0 cm) outfitted with a response lever, clicker, modular pellet dispenser for mouse, and pellet receptacle (Med Associates Inc, St Albans, VT, Figure 1A). Data were acquired using the MED-PC-IV program running training protocols coded by experimenters. Sessions lasted 30 min and took place between 0900 and 1700. Vole behavior was shaped using manual reinforcement by an experimenter until a subject met the training criterion of 3 days in a row of ≥5 responses without manual reinforcement on an FR-1 schedule. One 20 mg food pellet (Dustless Precision Pellet Rodent Grain Based Diet; Bio-Serv, Flemington, NJ) was dispensed as each reward. Animals that did not learn to consistently lever press within ~20 days were used as partners or strangers for future social testing. Subjects that met the training criterion transitioned to a PR-1 schedule with each successive reward requiring an additional response. The progressive ratio has been shown to be a better indicator of motivation than FR programs (Hodos and Kalman, 1963; Weatherly et al., 2003). PR-1 testing was conducted for 8 days, at the conclusion of which all focal animals were returned to ad lib food, and cage dividers were removed.

Testing with social rewards

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Social reward testing was conducted in mouse-sized modular test chambers, custom-equipped with a motorized door (Med Associates Inc, St Albans, VT) for access to a second ‘social’ chamber (Figure 1B). This chamber was constructed of clear plastic (15 cm × 20.5 cm × 13 cm) and contained an eye-bolt for tethering a stimulus vole (Figure 1C). A clear plastic tunnel (2.54 cm diameter, 5.5 cm long) connected the operant chamber to the social chamber, and the entire apparatus was fixed to a mounting board. Lever presses were rewarded by door opening and chamber access; the door remained raised for 1 min, after which the experimenter returned the focal vole to the operant chamber. Sessions lasted 30 min and were video-recorded for quantification of additional behaviors.

Subjects transitioned to social testing following a habituation session and two FR-1 sessions. Habituation to the social apparatus took place with the door open and the lever covered: voles explored the apparatus for 15 min with an empty social chamber, and 15 min with the partner tethered in the social chamber. Two days of FR-1 pressing for a tethered vole followed habituation to ensure that subjects associated lever pressing with access to the social chamber and a stimulus vole.

Social testing took part in two phases: pressing for a partner vole on a PR-1 schedule and pressing for a stranger on a PR-1 schedule. Each phase lasted 8 days. The order of testing was counterbalanced within groups and subjects completed both phases. Social stimulus animals were tethered to the end of the social chamber. During the 8 days of stranger testing, the focal vole was tested against a novel vole each session to prevent familiarity between conspecifics.

Non-social conditions

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Empty chamber testing took place after social testing to avoid altering lever pressing for the social stimuli. The empty chamber control was run to assess the value of apparatus exploration: 30 voles (10 female prairie voles, 14 male prairie voles, 6 female meadow voles) pressed the lever for 8 successive days on a PR-1 schedule to access the adjacent chamber when no stimulus vole was present. Sessions lasted 30 min and video was recorded and scored for behavior after testing. For the extinction phase, 31 voles (13 female prairie voles, 11 male prairie voles, 7 female meadow voles) were tested in the social chamber with an unrewarded lever for 10 successive days (30 min sessions).

Behavioral scoring

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Counts of responses (lever presses) and rewards (food pellets or door raises) were automatically recorded during each test. In all social trials (16/vole) and all empty chamber control trials (8/vole), behavior in the ‘social’ chamber was also filmed with a portable digital video camera. Videos were scored using a custom perl script (OperantSocialTimer; https://github.com/BeeryLab/Operant/, Beery, 2017) to determine time in the social chamber, time in side-by-side contact with the tethered vole (huddling), and bouts of aggression. These values could also be reported relative to other intervals (e.g. time huddling/access time when the door was up, or time huddling/time in the social chamber). Non-social/empty chamber trials (8 days/vole) were also videotaped and analyzed for time in the social chamber/available time with the door raised.

Castration and tubal ligation

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At least 1 week prior to pairing, the future ‘partner’ of each opposite-sex prairie vole pair was surgically altered to prevent pregnancies during testing. Female partners of male focal voles underwent tubal ligation. Dorsal incisions were made over each ovary. Two knots were placed below each ovary at the top of the uterine horn. The wound was closed using a sterile suture. Male partners of female focal voles were castrated and implanted with testosterone capsules. Testes were accessed by midline incision, and the blood supply was cut-off through a tie at the testicular artery. Testes were removed and the muscle wall and skin were closed using sterile suture. A testosterone capsule was implanted subcutaneously between the scapulae. Capsules contained 4 mm of crystalline testosterone (Sigma-Aldrich, St Louis, MO) in silastic tubing (ID 1.98 mm, OD 3.18 mm; Dow Corning, Midland, MO) as in Costantini et al., 2007. Capsules were sealed with silicone, dried, and soaked in saline for 24 hr prior to insertion. A subset of strangers was also castrated or ligated, with no effect on focal behavior. Surgical procedures were performed under isoflurane anesthesia. Voles received 0.05 mg/kg buprenorphine and 1.0 mg/kg metacam subcutaneously prior to surgery, and again the following day. Post-operative wound checks continued for up to 10 days post-surgery.

Receptor autoradiography

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OTR binding density was assessed in the brains of 11 female prairie voles at the conclusion of the study (males were used for an additional pilot study). Frozen brains were sectioned coronally at 20 μm, thaw-mounted on Super-frost Plus slides (Fisher, Inc), and stored at –80°C until processing (as in Beery et al., 2008a; Beery and Zucker, 2010; Mooney et al., 2015). Briefly, slides were thawed until dry, then fixed for 2 min in fresh, chilled 0.1% paraformaldehyde in 0.1 M PBS. Sections were rinsed 2 × 10 min in 50 mM Tris (pH 7.4), and incubated for 60 min at room temperature in a solution (50 mM Tris, 10 mM MgCl2, 0.1% BSA, 0.05% bacitracin, 50 pM radioligand) containing the radioactively labeled 125I-ornithine vasotocin analog vasotocin, d(CH2)5 [Tyr(Me)2,Thr4,Orn8,(125I)Tyr9-NH2] (125I-OVTA, PerkinElmer, Inc). An adjacent series of slides, processed for non-specific binding, was incubated with an additional 50 nM non-radioactive ligand [Thr4Gly7]-oxytocin (Bachem). All slides were rinsed 3 × 5 min in chilled Tris–MgCl2 (50 mM Tris, 10 mM MgCl2, pH 7.4), dipped in cold distilled water, and air-dried. Sections were apposed to Kodak BioMax MR film (Kodak, Rochester, NY) for 3 days and subsequently developed. Radioligand binding density in each brain region was quantified in samples of uniform area from three adjacent sections for each brain region and averaged for each brain. Non-specific binding was subtracted from total binding to yield specific binding values.

Statistical analyses

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Social data were analyzed for all subjects completing both partner and stranger phases of testing (n = 8 prairie vole M➤M pairs, 8 prairie vole M➤F pairs, 8 prairie vole F➤F pairs, 8 prairie voles F➤M pairs, and 7 meadow vole F➤F pairs). Four additional female prairie voles completed testing with a partner or stranger only: data from these subjects was included in analysis of food responses and food versus social response rates. Group differences in single variables (e.g. food responses) were assessed by one-way ANOVA. Two-way RM-ANOVA was used to assess the effects of social factors, with stimulus familiarity [partner, stranger] as a within-subjects repeated measure, stimulus type [same-sex, opposite-sex] as a between-subjects (non-repeated) measure, a test for interaction effects [stimulus familiarity*stimulus type], and for subject matching. Paired t-tests were used within groups for comparison of behavior toward the partner versus stranger. Response count (i.e. lever presses) and breakpoint (i.e. number of rewards achieved) are highly correlated; detailed results are therefore shown for only one measure (response count). Response rate (responses/active session time) was used when comparing food responses to social responses, as the lever was continuously active during food-rewarded testing (active session time = 30 min), but was not capable of raising the door when it was already up (active session time = 30 min with the door up). Autoradiography data were collected in multiple brain regions, and comparisons were performed by two-way ANOVA (group*brain region). Statistical analyses were performed in JMP 15.0 (SAS, Inc) and Prism 9 (GraphPad Software Inc). Effect sizes were calculated in Excel. Cohen’s d for paired t-tests used the mean of partner-stranger differences/standard deviation of partner-stranger differences. Eta squared (η2) and partial eta squared (ηp2) were reported for one-way and two-way ANOVAs, respectively (Lakens, 2013). Pearson’s product-moment correlation coefficient was reported for correlations. All tests were two-tailed, and results were deemed significant at p < 0.05. Number of animals: social operant studies in rats have been successful with six subjects (Tan and Hackenberg, 2016; Hiura et al., 2018; Hackenberg et al., 2021). We used 30% more subjects as a buffer (eight females or males in each condition), as operant behavior in voles was not well characterized.

Data availability

Data have been deposited in a project folder on the Open Science Framework website, available at: https://osf.io/g2jf7/.

The following data sets were generated
    1. Beery A
    (2021) Open Science Framework
    ID g2jf7. Social selectivity and social motivation in voles.

References

Decision letter

  1. Brian Trainor
    Reviewing Editor; University of California, Davis, United States
  2. Michael A Taffe
    Senior Editor; University of California, San Diego, United States
  3. Karen Bales
    Reviewer; University of California, Davis

Our editorial process produces two outputs: (i) public reviews designed to be posted alongside the preprint for the benefit of readers; (ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.

Acceptance summary:

This paper introduces a new method to measure motivation to engage with familiar or unfamiliar individuals in prairie voles, a widely used animal model system for studying social relationships. The authors show that females will work harder to access familiar individuals (either pair-bonded males or same-sex females) while males will work to access females regardless of whether they are pair-bonded or unfamiliar. These results cast a new light on decades of work based partner-preference tests that assess pair bonds without considering motivation.

Decision letter after peer review:

Thank you for submitting your article "Social selectivity and social motivation in voles" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Michael Taffe as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Karen Bales (Reviewer #3).

Overall, the reviewers were enthusiastic about the application of operant methods to study attachments in the different species of voles.

The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.

Essential revisions:

1) The lack of data using a progressive ratio schedule was viewed as a weakness for evaluating sex differences in motivation. If these data can be collected the reviewers agreed that this would significantly strengthen the claims in the manuscript. If these data can not be collected quickly the authors should address this limitation in the discussion.

2) The absence of male oxytocin receptor autoradiography data was also viewed as a weakness. If these data are available (null or otherwise) they should be included. The genotyping data were viewed as being tangential to the main theme of the paper and reviewers agreed these data should be removed.

Reviewer #1 (Recommendations for the authors):

My main suggestion is for the authors to highlight how their results change how we view the results of partner preference tests in the abstract and discussion. I think it would be helpful for the authors to go into a little more depth about the difference between actively seeking out a partner (or female) versus more passively huddling (in the case of animals with an opposite sex partner) when the partner is available. Do the authors expect dopaminergic mechanisms to play a stronger role in lever pressing than huddling (similar to liking/wanting hypotheses)?

Reviewer #2 (Recommendations for the authors):

Many of my concerns were listed in the public review.

Including the missing male groups would improve the paper.

I am not sure the value of the genotype comparisons given the limited behavioral data that can be assessed. It might strengthen the paper to remove it (and perhaps the data could serve as the basis for a different paper).

It would be great to run progressive ratio on a subset of subjects where differences were found. For example, run progressive ratio for female prairie voles with a partner that is familiar vs. novel (e.g., female partners only) and male prairie voles for a partner that is male vs. female (e.g., familiar only). I think this could be done with relatively few animals but would allow for conclusions about motivation.

The claim "sex-specific" should be limited to when an analysis comparing sexes was actually conducted (e.g., the first title of the Results section is inaccurate).

The corresponding correlation in males for Figure 3G should be shown.

Reviewer #3 (Recommendations for the authors):

In general, I found the paper to be quite well-written and I enjoyed reading it. I have the following recommendations:

Because some sample sizes were relatively small, the chance of Type II error is higher. Reporting effect sizes would be helpful.

Caption for Figure 5a: should specify that this statement refers to a food reward.

Line 296: In Ahern and Young 2009, I do not believe that there were actually any differences in OTR binding due to early experience. I couldn't check the other two references for that statement because neither was actually in the reference list (see below).

Check reference list and text for correspondence: Pournajafi-Nazarloo 2013 missing from ref list; Prounis also missing; there may be more, those are just the two I noticed.

Line 377-378: "infertile but sexually active" is odd and imprecise wording.

Finally, I felt like the discussion could be broader in its consideration of the implications of these findings for the evolution of social bonding.

https://doi.org/10.7554/eLife.72684.sa1

Author response

Essential revisions:

1) The lack of data using a progressive ratio schedule was viewed as a weakness for evaluating sex differences in motivation. If these data can be collected the reviewers agreed that this would significantly strengthen the claims in the manuscript. If these data can not be collected quickly the authors should address this limitation in the discussion.

We agree that a progressive ratio schedule is preferable for evaluating differences in motivation, and this is the schedule already used throughout the study. We also cite references supporting the preferability of progressive ratio schedules in our description of the methods. A fixed ratio schedule was used in some parts of training (initial introduction to food rewards and habituation to the social testing paradigm), but a progressive ratio was used for all experimental phases.

We have identified two ways to make this information more accessible throughout the manuscript.

1. The testing protocol was described in greatest detail in the methods section which appears after the Results section. We have thus added text highlighting the progressive ratio protocol in the Results section, which now begins with: “In order to assess motivation for different kinds of social stimuli across groups, lever pressing responses were quantified on a progressive ratio schedule (PR-1).” We have also added this information to the caption of figure 2 and the y-axis of figures 2 and 5.

2. Progressive ratio results can be reported as breakpoints (which only represent PR designs) or lever pressing responses (which can be used for PR or FR studies), but as these metrics are highly correlated, only one is typically analyzed. We reported lever pressing responses as it captures additional information from incomplete reward series (detailed circa line 720), so to aid in interpreting/translating the data in terms of breakpoints, we have added the mapping of lever presses to breakpoints for a PR-1 schedule to the y-axis of Figure 2A. This is also now articulated in the text: “The mapping from response count to the corresponding PR-1 breakpoint (i.e. the maximum number of responses exhibited to achieve a reward) is shown in figure 2A and applies to all response count figures.” As well as in the figure 2 legend: “PR-1 breakpoint thresholds are listed in italics next to the corresponding number of responses on the interior y-axis of panel A and apply to all lever pressing data (e.g. a vole that presses 55 times should receive 10 rewards, the last of which takes 10 responses to gain).”

2) The absence of male oxytocin receptor autoradiography data was also viewed as a weakness. If these data are available (null or otherwise) they should be included. The genotyping data were viewed as being tangential to the main theme of the paper and reviewers agreed these data should be removed.

Male oxytocin receptor data is not available. When we discovered males were not working harder to access familiar females, we diverted later study males to pilot a social choice variant of the operant setup (and thus stopped collecting their brains at the conclusion of the study). That pilot led us to conduct a new study using a two-choice operant apparatus (now in review), and the tissues from that second study could enable a similar analysis in both male and female brains. I will strongly consider running that assay once I have autoradiography set up in my new lab.

We have clarified that male data were not omitted null data, but rather not assayed: “We conducted receptor autoradiography to assess variation in neural oxytocin receptor density in female prairie voles. (OTR was not analyzed in male brains; following early results, later males were used to pilot a two-choice social operant paradigm).”

We have removed all genotyping data as requested.

Reviewer #1 (Recommendations for the authors):

My main suggestion is for the authors to highlight how their results change how we view the results of partner preference tests in the abstract and discussion. I think it would be helpful for the authors to go into a little more depth about the difference between actively seeking out a partner (or female) versus more passively huddling (in the case of animals with an opposite sex partner) when the partner is available. Do the authors expect dopaminergic mechanisms to play a stronger role in lever pressing than huddling (similar to liking/wanting hypotheses)?

Thank you for this suggestion – we have added material to both the abstract and discussion.

Abstract: we were at the word limit, so we cut a few words and added a minimal statement:

“This reveals a striking sex difference in pathways underlying social monogamy, and demonstrates a fundamental disconnect between motivation and social selectivity in males—a distinction not detected by the partner preference test.”

Discussion (new paragraph):

“The lack of consistent mapping between effort in the operant task and partner preferences in male huddling highlights a disconnect between social reward and the selectivity of huddling preferences. This disconnect is further underscored by the presence of robust partner preferences in female meadow voles despite no evidence of social reward in the operant task or in socially conditioned place preference tests (Goodwin et al., 2019). Social reward thus does not imply selective preference, nor does partner preference imply social reward. These behavioral findings are consistent with the lack of effects of dopamine antagonists on same-sex peer partner preferences in female meadow voles as well as prairie voles (Beery and Zucker, 2010; Lee and Beery, 2021). While dopamine signaling is not necessary for peer partner preference expression, it can enhance preferences (Lee and Beery, 2021), and may play a more fundamental role in pair bonding with mates (Aragona and Wang, 2009). Because partner preference does not indicate behavioral reward, the partner preference test and other tests of social approach in the absence of work likely reflect different combinations of partner tolerance, partner reward, and stranger aversion.”

We have also added a related section to the discussion “Implications for the evolution of social relationships” (see response to reviewer #3).

Reviewer #2 (Recommendations for the authors):

Including the missing male groups would improve the paper.

I have added better explanations for their omission in these two instances. In the case of meadow voles, the seasonal transition to sociality only occurs in females (or predominantly in females) in both the field and laboratory, thus the following text has been added:

Introduction: “Because the seasonal transition from solitary to social is most pronounced in female meadow voles in the field and laboratory (Madison and McShea, 1987; Beery et al., 2009), only females of this species were used.”

Results: “Lever pressing responses in prairie voles were compared to those of a related non-monogamous vole species (the meadow vole) that exhibits group living during winter months. Female meadow voles are territorial and aggressive in summer or long day lengths in the lab, but socially tolerant in winter or short days. Because male meadow voles do not undergo this transition (Madison and McShea, 1987; Beery et al., 2009), we focused on comparison of social motivation in female meadow voles relative to female prairie voles.”

Regarding neural OTR data: We conducted receptor autoradiography to assess variation in neural oxytocin receptor density in female prairie voles. OTR was not analyzed in male brains as several male subjects were used to pilot a follow-up study using a two-choice social operant paradigm (in review).”

In prior studies in voles in our lab and others, sex differences in OTR density have not been found (e.g. Insel et al., 1992, Ahern et al., 2021), with signaling differences hypothesized to be largely mediated through sex differences in peptide distribution. Nonetheless it would have been informative to correlate individual binding data with behavior in the males, and we will consider this for a second study we conducted that yielded more male brains.

I am not sure the value of the genotype comparisons given the limited behavioral data that can be assessed. It might strengthen the paper to remove it (and perhaps the data could serve as the basis for a different paper).

We have removed the genotype comparisons as suggested. They are stronger with a larger sample set, so we have added them to our second study data set for which we also collected genotype data.

It would be great to run progressive ratio on a subset of subjects where differences were found. For example, run progressive ratio for female prairie voles with a partner that is familiar vs. novel (e.g., female partners only) and male prairie voles for a partner that is male vs. female (e.g., familiar only). I think this could be done with relatively few animals but would allow for conclusions about motivation.

A progressive ratio was used for all testing. This has been clarified in several locations in the manuscript and hopefully addresses this concern.

The claim "sex-specific" should be limited to when an analysis comparing sexes was actually conducted (e.g., the first title of the Results section is inaccurate).

We have now added quantitative comparison of the sexes to supplement the qualitative description of the difference in response pattern. Specifically, we screened for sex differences using a generalized linear model with stimulus type (same/opposite sex), stimulus familiarity (partner/stranger), sex of the presser (female/male), sex of the presser*stimulus type, and sex of the presser*stimulus familiarity. This yielded interactions of sex with stimulus type (p=.0112) and familiarity (p=0.09), providing quantitative evidence for sex differences not in the amount but in the stimulus specificity of pressing, supporting the decision to analyze the results separately by sex. We have added this result to the manuscript. We believe “Sex-specific patterns of social effort in prairie voles” is therefore accurate. If needed, this section could be re-titled “Social effort in males and females was influenced by different aspects of the social stimulus.”

The corresponding correlation in males for Figure 3G should be shown.

Both male and female data now appear in Figure 3G. We also took this opportunity to change the graph from total bouts of aggression to aggression/access time, as was already displayed for all other social comparisons (e.g. huddling/access time, social chamber/access time). We originally presented both versions in the text, emphasized the scaled findings in the text, but displayed the unscaled findings in the figure. We continue to present both unscaled and scaled findings in the text, but now display the scaled findings we believe are more meaningful.

In addition, we added further detail regarding which voles underwent control and extinguishing testing (all voles in cohorts 4, 5, 6, and 7, which represented >2/3 of the voles in the study and were distributed across groups).

Reviewer #3 (Recommendations for the authors):

In general, I found the paper to be quite well-written and I enjoyed reading it. I have the following recommendations:

Because some sample sizes were relatively small, the chance of Type II error is higher. Reporting effect sizes would be helpful.

We have added effect sizes throughout the manuscript. For pairwise comparisons we have added Cohen’s d; for 1-way and 2-way ANOVAs we now present eta squared and partial eta squared, and for correlations we already listed the Pearson product-moment correlation coefficient alongside statistical significance. We selected the sample sizes for this study in consultation with operant expert Dr. Tim Hackenberg, who assured us that the repeated testing involved in social operant studies allowed him to use samples sizes as small as 4-6 per group (we used 8). Although only 8 subjects were tested per group, each subject’s data represented the mean of 8 days of sampling in each of the three conditions, enhancing the reliability of data for each subject and reducing the chance of type II error.

Caption for Figure 5a: should specify that this statement refers to a food reward.

Fixed.

Line 296: In Ahern and Young 2009, I do not believe that there were actually any differences in OTR binding due to early experience. I couldn't check the other two references for that statement because neither was actually in the reference list (see below).

Thank you for catching this – it looks like the difference in Ahern and Young 2009 was in OT, not OTR, so this reference has been removed. The other two references show similar findings, and I’ve added more study-specific detail so this now reads:

“Variation in OTR density by relationship type has not been previously assessed, although oxytocin receptor density or mRNA levels differ in response to early-life housing manipulations in prairie voles, such as presence of a father and single versus group housing (Prounis et al., 2015) as well as chronic social isolation in adulthood (Pournajafi-Nazarloo et al., 2013).”

Check reference list and text for correspondence: Pournajafi-Nazarloo 2013 missing from ref list; Prounis also missing; there may be more, those are just the two I noticed

These references have also been added to the bibliography, and one other that was also previously missing. A few other references appeared to be missing from the LaTeX formatted file that are present in the Word file for reasons I cannot explain, so I have included all revisions on the Word version of the file.

Line 377-378: "infertile but sexually active" is odd and imprecise wording.

Agreed. In lieu of finding better language that could apply to both males and females, surgical manipulations of males and females are now specified separately:

“Subjects in opposite-sex pairs were placed with either a tubally ligated, hormonally intact female mate, or a castrated and testosterone implanted male mate”

Finally, I felt like the discussion could be broader in its consideration of the implications of these findings for the evolution of social bonding.

We have added a section at the end of the Discussion section:

Implications for the evolution of social relationships

Persistent relationships within specific pairs or groups of conspecifics are present throughout the animal kingdom, including select invertebrates, fishes, reptiles and amphibians, birds, and mammals (Bales et al., 2021). While the nature and extent of these relationships vary considerably, they share in common the specificity of social preferences that leads to repeated association. They may differ, however, in the mechanisms that influence familiar approach and unfamiliar avoidance. In particular, familiar individuals—whether mates or peers—may or may not be socially rewarding, and unfamiliar individuals may or may not be aversive.

Even within closely related vole species, we see evidence that only some relationships involve selective social reward, for example mate relationships in female prairie voles, while others—such as peer relationships in winter phenotype meadow voles—involve selectivity without appreciable reward. Selectivity in the absence of reward may rely instead on changing social anxiety and aggression (Beery, 2019). For example, when exposed to the short, winter photoperiods associated with the transition from solitary to group living in the wild, meadow voles undergo changes in CRF receptor densities, glucocorticoid secretion, behavioral indicators of anxiety, and aggression (Ossenkopp et al., 2005; Beery et al., 2014; Anacker et al., 2016). More research is needed to establish causal links between these changes and the transition to group living. More broadly, it remains to be determined to what extent social monogamy and pair bonding with mates shares mechanisms across species (Goodson, 2013), and to what extent different types of relationships (e.g. with peers or mates) share foundations, or differ in their regulation. Ultimately these studies should help us understand how selective relationships of different types evolve.

https://doi.org/10.7554/eLife.72684.sa2

Article and author information

Author details

  1. Annaliese K Beery

    1. Department of Integrative Biology, University of California Berkeley, Berkeley, United States
    2. Program in Neuroscience, Departments of Psychology and Biology, Smith College, Northampton, United States
    3. Neuroscience and Behavior Graduate Program, University of Massachusetts, Amherst, MA, United States
    Contribution
    Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Supervision, Validation, Visualization, Writing – original draft, Writing – review and editing
    For correspondence
    abeery@berkeley.edu
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-1249-9182
  2. Sarah A Lopez

    Program in Neuroscience, Departments of Psychology and Biology, Smith College, Northampton, United States
    Contribution
    Data curation, Formal analysis, Investigation, Methodology, Writing – original draft, Writing – review and editing
    Competing interests
    No competing interests declared
  3. Katrina L Blandino

    Program in Neuroscience, Departments of Psychology and Biology, Smith College, Northampton, United States
    Contribution
    Investigation, Methodology, Writing – review and editing
    Competing interests
    No competing interests declared
  4. Nicole S Lee

    Neuroscience and Behavior Graduate Program, University of Massachusetts, Amherst, MA, United States
    Contribution
    Data curation, Formal analysis, Investigation, Writing – review and editing
    Competing interests
    No competing interests declared
  5. Natalie S Bourdon

    Program in Neuroscience, Departments of Psychology and Biology, Smith College, Northampton, United States
    Contribution
    Investigation, Writing – review and editing
    Competing interests
    No competing interests declared

Funding

National Institutes of Health (R15MH113085)

  • Annaliese K Beery

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

We are extremely grateful to Dr Tim Hackenberg for advising us on the design, physical setup, and analysis of the studies described here. Lab members Emily Halstead, Amelia Windorski, Rose Hatem, Madeleine Lerner, and Marcela Rodrigues-Guimaraes assisted with behavioral testing and video scoring, and Karina Lieb assisted with brain tissue preparation. Dale Renfrow (Smith Center for Design and Fabrication) assisted with building social testing chambers. We thank the staff of the Smith College Animal Care Facility for animal care and colony maintenance. This research was supported by the National Institute of Mental Health of the National Institutes of Health under Award Number R15MH113085. Publication made possible in part by support from the Berkeley Research Impact Initiative (BRII) sponsored by the UC Berkeley Library.

Ethics

This study was carried out in accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. Animals were handled according to a research protocol (ASAF 272) approved by the Institutional Care and use committee of Smith College.

Senior Editor

  1. Michael A Taffe, University of California, San Diego, United States

Reviewing Editor

  1. Brian Trainor, University of California, Davis, United States

Reviewer

  1. Karen Bales, University of California, Davis

Publication history

  1. Preprint posted: July 31, 2021 (view preprint)
  2. Received: August 1, 2021
  3. Accepted: October 19, 2021
  4. Accepted Manuscript published: November 2, 2021 (version 1)
  5. Version of Record published: November 16, 2021 (version 2)

Copyright

© 2021, Beery et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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