Not just for bonding: Nucleus accumbens oxytocin receptors facilitate huddling with strangers and feeding in male spiny mice

This is the author accepted manuscript of Fricker BA, Boender AJ, Young LJ, Kelly AM. Not just for bonding: Nucleus accumbens oxytocin receptors facilitate huddling with strangers and feeding in male spiny mice. Psychoneuroendocrinology. 2025 Aug;178:107496. doi: 10.1016/j.psyneuen.2025.107496. Epub 2025 May 15. PMID: 40403453. Not just for bonding: Nucleus accumbens oxytocin receptors facilitate huddling with strangers and feeding in male spiny mice Brandon A. Fricker1, Arjen J. Boender2, Larry J. Young2,3, Aubrey M. Kelly1* Affiliations: 1. Department of Psychology, Emory University, Atlanta, Georgia 30322 [email protected] (B.A.F.) [email protected] (A.M.K.) 2. Center for Translational Social Neuroscience, Emory University, Atlanta GA 30329 USA 3. Department of Psychiatry and Behavioral Sciences, Emory University School of Medicine, Atlanta GA 30322 USA [email protected] (A.J.B.) *Corresponding author: Aubrey M. Kelly [email protected] Key words: oxytocin receptor, huddling, sociality, nucleus accumbens Abstract Although oxytocin receptors (OXTRs) in the nucleus accumbens (NAc) are well-known for their contributions to bonding in mating and parental contexts, little is known about how accumbal OT signaling modulates nonreproductive social behavior. Here we used the communal spiny mouse and viralmediated CRISPR/Cas9 to decrease OXTR expression in the NAc of males to determine the direct contributions of accumbal OXTRs to behavior during interactions with novel, same-sex conspecifics. To determine whether NAc OXTRs specifically regulate social behaviors, feeding behavior was also assessed in a mealworm eating test. Males with reduced NAc OXTR expression exhibited less huddling with novel, same-sex conspecifics and consumed fewer mealworms compared to control males. These findings suggest that accumbal OXTRs do not specifically modulate social behaviors and that there is strong evolutionary conservation of NAc OXTR social function, such that these receptors facilitate prosocial behavior across rodent species that vary in breeding system and group structure. 1. Introduction Oxytocin (OT) signaling is well known for modulating bonding, particularly in mating and parental contexts (Bosch and Young, 2018; Froemke and Young, 2021). Although OT receptors (OXTRs) are widely distributed throughout the brain (Freeman et al., 2020), a critical brain region for OT action that regulates prosocial behavior is the nucleus accumbens (NAc), a key node in reward circuitry (Volkow et al., 2017; Klawonn and Malenka, 2018). Indeed, OT interacts with other neuromodulators to regulate social reward. For example, OT interacts with dopamine to regulate pair bond formation in female prairie voles (Keebaugh et al., 2015) and OXTRs and serotonin 1b receptors are required for social conditioned place preference (with cagemates) in male mice (Dolen et al., 2013). Most studies examining NAc OXTR function use the socially monogamous prairie vole. For example, knockdown of accumbal OXTRs disrupts partner preference formation and reduces alloparental behavior (Keebaugh et al., 2015), whereas overexpression of OXTRs in the NAc enhances partner preference formation and alloparental care in female prairie voles (Keebaugh and Young, 2011). Notably, NAc OXTRs similarly modulate maternal care in transgenic mice (Witchey et al., 2024), demonstrating that accumbal OT-mediated prosocial behavior is not unique to voles and/or socially monogamous species. Recent studies have elucidated a more complex function of accumbal OXTRs, such that socio-sexual experience alters oxytocinergicmodulated signaling in the NAc (Borie et al., 2022) and development with or without OXTRs differentially influences NAc signaling in a sex-specific manner in prairie voles (Long et al.). To date, most studies have identified prosocial functions of NAc OXTRs in female rodents, as well as in species that are territorial in contexts outside of mating and parenting. How accumbal OT signaling modulates behavior in highly social, communal species largely remains unknown. Spiny mice (Acomys dimidiatus) are a communally breeding rodent native to Africa, the Middle East, and Southeast Asia (Haughton et al., 2016). This species naturally lives in large groups (Haughton et al., 2016), and in the lab both males and females exhibit high degrees of prosociality and very low levels of aggression with conspecifics regardless of novelty/familiarity, kinship status, or reproductive context (Fricker et al., 2021; Gonzalez Abreu et al., 2022; Fricker et al., 2023). Spiny mice also welcome unrelated newcomers to an established group (Cizkova et al., 2011). Therefore, spiny mice are an excellent organism for examining the contributions of accumbal OT signaling to prosocial behavior in nonreproductive contexts, particularly with strangers. We previously mapped OXTR distributions throughout the basal forebrain and midbrain of spiny mice and found that they exhibit robust OXTR expression in the NAc (Powell et al., 2022). While there has been no direct comparison of spiny mouse NAc OXTR expression with other mammals, species such as prairie voles, California mice, and SpragueDawley rats show OXTR expression in both the shell and core of NAc (Bernheim et al., 2017; Williams et al., 2020; Inoue et al., 2022), whereas OXTR expression is largely restricted to the NAc shell of spiny mice (Powell et al., 2022). Further, although NAc OXTR densities distinguish mating systems in voles, with monogamous prairie voles having more NAc OXTRs than polygamous montane voles (Insel and Shapiro, 1992), eusocial naked mole rats (a non-monogamous rodent) also exhibit robust NAc OXTR expression (Mooney et al., 2015; Freeman et al., 2020). The dense OXTR binding observed in species like naked mole rats and spiny mice demonstrates that NAc OXTR function is not restricted to behaviors associated with monogamy. Although, to our knowledge, no studies have examined the role of nonapeptide receptors in spiny mouse behavior, we previously examined a role for OT in social behavior for this species. OT neurons in the paraventricular nucleus of the hypothalamus (PVN) are more responsive to interactions with a novel, same-sex conspecific compared to a novel object (i.e., rubber duck); additionally, PVN OT neural responses positively correlate with nonreproductive prosocial behavior with a stranger as well as with tyrosine hydroxylase neural responses in the ventral tegmental area (Gonzalez Abreu et al., 2022). These findings suggest that PVN OT may gate social reward in nonreproductive contexts via connections with reward circuitry in spiny mice. Indeed, retrobead tracing demonstrated that PVN OT neurons send axonal projections to the VTA (Gonzalez Abreu et al., 2022) and the NAc (unpub obs) of spiny mice, as has been observed in lab mice (Xiao et al., 2017; He et al., 2021). This prior data in spiny mice suggests that the OT system may modulate social reward in this species. Yet, how accumbal OT signaling contributes toward behavior in spiny mice remains unknown. Here we used viral-mediated CRISPR/Cas9 to decrease OXTR expression in the NAc of male spiny mice (previously validated in Boender et al. (Boender et al., 2023)) to determine the direct contributions of accumbal OXTRs to behavior during interactions with novel conspecifics. We previously found that male spiny mice are more prosocial with novel kin than novel non-kin in a complex group interaction (Fricker et al., 2023) and, therefore, tested subjects in social interaction tests with novel kin as well as novel nonkin. To determine whether NAc OXTRs modulate affiliative preferences with strangers, we also conducted a social preference test where subjects had the choice of interacting with a novel kin or novel non-kin same-sex conspecific. Lastly, to examine whether NAc OXTRs specifically modulate behavior associated with social contexts, we conducted a giant mealworm eating test. Although prior studies have shown that central administration of OT reduces feeding (Sabatier et al., 2013), extremely few studies have specifically examined the influence of NAc OXTRs on feeding behavior. If NAc OXTRs facilitate prosocial behavior across species, then these receptors will modulate prosocial behaviors like huddling in the communal spiny mouse outside the contexts of mating/pair bonding and parenting. Thus, we hypothesized that spiny mice with reduced OXTR expression in the NAc would exhibit less prosocial behavior with strangers compared to control animals. Because spiny mice may find interactions with novel kin more rewarding than with novel non-kin (Fricker et al., 2023), we predicted that a reduction in NAc OXTRs would abolish a preference to affiliate with novel kin. Further, because conditional knockout of NAc OXTRs in female mice does not influence feeding in the homecage (Witchey et al., 2024), we predicted that a reduction of NAc OXTR expression in spiny mice would not influence consumption of giant mealworms. 2. Methods 2.1 Animals 24 adult male (post-natal day (PND) 250-500; ages were evenly distributed across treatment groups) spiny mice (Acomys dimidiatus) from our breeding colony were used as subjects for the study; males were randomly assigned to either an OXTR knockdown group or a control group. Sex was determined by examination of external genitalia. All animals were group-housed (2-3) in standard rat polycarbonate cages (40.64cm X 20.32cm X 20.32cm) that contained Sani-Chips bedding. Spiny mice were provided with rodent igloos and shepherd shacks and were able to obtain food and water ad libitum. Animals were kept on a 14-h light:10-h dark cycle with an ambient temperature of 24 + 2C. The circadian rhythm of Acomys species can vary depending on environmental conditions (Levy et al., 2007; Cohen et al., 2009); spiny mice in our colony exhibit diurnal activity patterns and were therefore tested during the day under the light phase. All procedures were approved by the Institutional Animal Care and Use Committee of Emory University. Due to a lack of female spiny mouse availability in our colony, we were only able to conduct this study in males. Therefore, future studies are needed to examine the contribution of NAc OXTRs to female spiny mouse behavior. 4 animals were removed from the dataset due to inaccurate surgical targeting, resulting in a final sample size of 10 males in the OXTR knockdown group (AAV- OXTR) and 10 males in the control group (AAV-CTRL). 2.2 Experimental Design Subjects were randomly assigned to either an AAV-OXTR (i.e., OXTR knockdown) group or an AAVCTRL (i.e., control) group. After 4-6 weeks of viral incubation, subjects underwent behavioral testing for two days. Subjects were tested in two tests a day, with 1-2 hours in-between tests. Behavioral tests included: (1) a social interaction with a novel, same-sex kin conspecific, (2) a social interaction with a novel, same-sex non-kin conspecific, (3) a social preference test, and (4) a mealworm eating test. The order of behavior tests was randomized across subjects. Figure 1. NAc surgical targeting and CRISPR/Cas9 AAV efficacy. (A) Schematic illustrating viral-induced enhanced green fluorescent protein (eGFP) fluorescence in a spiny mouse subject (left) and representative section from the Allen Mouse Brain Atlas (right). Note that spiny mouse brains are shaped slightly differently than lab mouse brains. (B) A representative autoradiogram from a spiny mouse from the prior validation study (Boender et al., 2023) demonstrating OXTR expression reduction on the side of the brain injected with AAV-OXTR but not on the side of the brain injected with AAV-CTRL. 2.3 Stereotaxic Injections Subjects in the OXTR knockdown group received intracranial injections targeting the NAc of a CRISPR/Cas9 AAV targeting the gRNA OXTR.2 sequence (referred to as AAV-OXTR; Figure 1A), whereas subjects in the control group received injections of a control AAV-gRNA (referred to as AAVCTRL). These viruses were previously generated and validated in the NAc of spiny mice, with the AAV- OXTR significantly reducing OXTR expression compared to the AAV-CTRL (Boender et al., 2023). From this validation study, the hemisphere that expressed the AAV-OXTR exhibited 95% less OXTR binding compared to the control hemisphere (Boender et al., 2023)(Figure 1B). Spiny mice received a 1.5mg/kg dose of meloxicam orally 30 minutes prior to anesthetization with 4% isoflurane and were maintained with 2% isoflurane during the surgery. Subjects were fitted onto a stereotax (Sotocinal et al.) and all AAVs were delivered via a pulled glass pipette at a rate of 1nl/sec using a nanosyringe (Drummond Nanoject III). 250nL of virus was injected at each site. Pipettes were held at the injection site for 10 min following AAV release. Coordinates (referenced to Bregma) for bilaterally targeting the NAc were: 2.55mm anterior, +0.80mm lateral, 4.70mm depth and 2.95mm anterior, +0.65mm lateral, 4.60 depth. Viral vectors were allowed to express for 4-6 weeks prior to behavioral testing. During this time, all subjects were group-housed with 1-2 other untreated, same-sex littermates. Confirmation of surgical targeting was confirmed by the presence of fluorescent virus; only subjects with accurate targeting of the NAc and no viral spread were retained for analyses. It should be noted that the eGFP and Cas9 viruses are premixed and are therefore co-injected through the same needle, and thus the spread of eGFP and Cas9 should be similar. However, it is possible that some cells may contain eGFP but no Cas9 and vice versa; such cells likely represent a small minority as this tool achieves >90% knockdown over the whole area of eGFP positive tissue (Boender et al., 2023). Although not perfect, based on our observations, the fluorescent signal gives an adequate indication as to whether the right area of the brain was targeted, as was previously demonstrated by Boender and colleagues (Boender et al., 2023). Accurate surgical targeting for all subjects was determined by examination of viral eGFP labeling post mortem; note that the AAVs contained an eGFP tag. Surgical targeting was considered an accurate “hit” if eGFP was detected only around the NAc where OXTRs are present as indicated in Figure 1. 4 animals exhibited mistargeting and/or viral leakage from when the glass pipette was removed from the brain after injection (i.e., creating a trail of virus from the NAc to the top of the cortex); these 4 animals were removed from the dataset. 2.4 Social Interactions Tests To determine the contribution of NAc OXTRs to behavior during freely behaving interactions with novel conspecifics, subjects underwent two social interaction tests – one with a novel, same-sex kin conspecific and one with a novel, same-sex non-kin conspecific. The subject and an ageand weight-matched stimulus animal were simultaneously placed into a clean rat cage via plastic beakers and were allowed to freely interact for 10 min. The cages contained no enrichment or food/water. Tests were video recorded using Sony Handycam cameras. Stimulus animals were previously marked with a rodent marker to distinguish subjects from stimuli in video recordings. Huddling, investigation, and autogrooming were scored using BORIS (Friard and Gamba, 2016). Huddling was defined as the subject and stimulus animal resting together with bodies touching and/or overlapping. Investigation was defined as the subject’s nose making contact with (i.e., sniffing) the head, flank, or rear of the stimulus animal. No aggression (i.e., chasing, biting, lunging) was observed for any animal. 2.5 Social Preference Test To examine whether male spiny mice exhibit a preference for spending time near or investigating a novel, same-sex kin conspecific or a novel, same-sex non-kin conspecific, subjects were tested in a social preference test. The testing chamber (previously used in (Fricker et al., 2024)) consisted of an initial acrylic chamber (15.24cm X 15.24cm X 45.72cm) that releases subjects into a large, opaque acrylic chamber (55.88cm X 71.12cm X 45.72cm) divided into two identical sections. Each section can hold up to 8 stimulus animals or objects, separated from the subject by clear acrylic with 0.12 cm diameter holes. When the subject is in one section, their view of the other section is entirely obstructed. One section contained a novel, same-sex kin conspecific whereas the other section contained a novel, same-sex nonkin conspecific. For a schematic of the chamber, see Figure S1. Stimulus sides were randomized across subjects to account for any side biases. For the preference test, the subject was transferred via a beaker into the initial chamber. Once released into the large chamber, the subject was allowed to freely explore for 10 min. Tests were video recorded, and the amount of time spent near each stimulus animal and the time spent investigating each stimulus animal (i.e., subject’s nose made contact with the walls containing a stimulus animal) was scored in BORIS. Ethovision XT 17 (Noldus, Virginia, USA) was used for automated tracking to obtain the distanced traveled and velocity during the 10 min test. 2.6 Mealworm Eating Test To determine whether NAc OXTRs modulate nonsocial behavior in male spiny mice, subjects underwent a mealworm eating test. Spiny mice eagerly consume mealworms,; mealworms are provided as enrichment to spiny mice in our colony once a month. For the test, subjects were transferred via a beaker into one side of a medium acrylic chamber (81.28cm X 30.48cm X 40.64cm) that contained a weigh-boat of 10 same-length, giant mealworms at the opposite end of the chamber. The number of mealworms consumed during a 10 min period was recorded. 2.7 Histology After behavioral testing, subjects were euthanized via isoflurane overdose and were transcardially perfused with 0.1M phosphate buffered saline (PBS) followed by 4% paraformaldehyde dissolved in 0.1M borate buffer (pH 9.5). Brains were post-fixed in 4% paraformaldehyde overnight before cryoprotection in 30% sucrose dissolved in PBS for 48 h. Brains were sectioned at 40um using a Leica cryostat. The AAVs contain an eGFP tag, and thus surgical targeting was confirmed post mortem on a Zeiss AxioImager II microscope. 2.8 Statistics Social interaction tests were analyzed using linear mixed models (LMMs) with Treatment (AAV-CTRL or AAV-OXTR) and Stimulus Type (kin or non-kin) as fixed factors and Subject as a random factor to account for repeated testing. Social interaction data analyzed for control subjects only was analyzed with Wilcoxon Signed Ranks tests. Data for the social preference test and feeding test were normally distributed and therefore analyzed using independent t-tests or paired sample t-tests. All posthoc pairwise comparison were corrected using the Sidak correction. All data were analyzed using SPSS 29 (IBM Analytics, USA) and graphs were made using Prism 10 (GraphPad, USA). 3. Results 3.1 OXTRs in the NAc facilitate huddling with novel, same-sex strangers To determine the contributions of NAc OXTRs to behavior during social interactions with novel, samesex conspecifics, male spiny mice were tested in two social interaction tests – one with a novel kin and another with a novel non-kin conspecific. A linear mixed model (LMM) with Treatment (AAV-CTRL or AAV-OXTR) and Stimulus Type (kin or non-kin) as fixed factors and Subject as a random factor yielded a main effect of Treatment for huddling (F(36) = 6.61, p = 0.01; Figure 2A), with AAV-OXTR subjects that had the NAc OXTR population knocked down exhibiting less huddling behavior with novel conspecifics, regardless of whether they were kin or non-kin. We observed no effects of or interactions with Stimulus Type (all p > 0.36) on huddling. Additionally, we found no effects of or interactions between Treatment and Stimulus Type for investigative behavior (all p > 0.32; see Figure 2B as an example for the lack of effect of Treatment). However, we did find that NAc OXTRs influenced autogrooming during social interactions. A main effect of Treatment (F(18) = 7.94, p = 0.01; Figure 2C) revealed that AAV-OXTR subjects exhibited more autogrooming than control subjects, whereas Stimulus Type did not influence autogrooming (all p > 0.64). Although we did not observe an influence of Stimulus Type for any of the behaviors examined, behavior broken down by Stimulus Type can be found in Figure S2. Figure 2. Social Interaction Test. (A) Compared to male spiny mice in the AAV-CTRL group (cream), AAV- OXTR males (sage green) spent less time huddling with a novel, same-sex conspecific. (B) Investigation of stimulus animals did not differ between AAV-CTRL and AAV-OXTR males. (C) AAV-OXTR subjects spent more time autogrooming than AAV-CTRL animals. Note that data from the two social interaction tests (one with kin (black dots), one with non-kin (maroon dots)) are combined here to reflect the LMM main effect of Treatment. Individual dots represent subjects; each subject has two dots, one black and one maroon, per bar. Data are represented as mean + SEM. An asterisk indicates statistical significance of p = 0.01. We previously found that male spiny mice were more prosocial with novel kin than novel, non-kin (Fricker et al., 2023). Because we did not observe any effects of or interactions with Stimulus Type in the LMMs that included all subjects, we specifically examined whether spiny mice exhibited differences in huddling or investigation with novel kin and non-kin only in control subjects. However, a Wilcoxon Signed Ranks test showed no differences between kin and non-kin for huddling (z = -0.56, p = 0.58), investigation (z = -0.26, p = 0.80), or a combined prosocial metric (i.e., huddling and investigation, as was previously used in Fricker et al. [15]) (z = -0.45, p = 0.88). This lack of replication of our previous finding is discussed below in the Discussion. 3.2 Male spiny mice do not exhibit a preference for novel kin or novel non-kin in a social preference test Because no studies have specifically examined whether spiny mice exhibit a preference for novel kin or novel non-kin in a preference test, male subjects were run in a social preference test in which they had the choice to investigate and/or spend time near a novel, same-sex kin peer or a novel, same-sex non-kin peer. In control subjects only, we observed no difference in the time spent investigating (t(9) = -0.14, p = 0.45) or near (t(9) = 0.05, p = 0.48) either stimulus animal, suggesting that males either do not have a preference for novel kin or novel non-kin or that we did not detect any such preference in our test. To further examine whether NAc OXTR knockdown influences behavior in the social preference test, we generated normalized preference scores (e.g., (time investigating kin minus time investigating non-kin) divided by (time investigating kin plus time investigating non-kin); same equation for time near stimuli). Independent t-tests yielded no differences between control and AAV-OXTR subjects for a near preference score (t(18) = -0.15, p = 0.44; Figure 3A) or an investigation preference score (t(18) = 0.58, p = 0.29; Figure 3B). Together these findings suggest that male spiny mice do not exhibit a preference for novel kin or novel non-kin and that knockdown of NAc OXTRs does not induce such a preference. Figure 3. Social Preference Test. 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