Beyond the binary: Characterizing the relationships between sex and neuropeptide receptor binding density measures in the rat brain.

Sex differences exist in numerous parameters of the brain. Yet, sex-related factors are part of a large set of variables that interact to affect many aspects of brain structure and function. This raises questions regarding how to interpret findings of sex differences at the level of single brain measures and the brain as a whole. In the present study, we reanalyzed two datasets consisting of measures of oxytocin, vasopressin V1a, and mu opioid receptor binding densities in multiple brain regions in rats. At the level of single brain measures, we found that sex differences were rarely dimorphic and were largely persistent across estrous stage and parental status but not across age or context. At the level of aggregates of brain measures showing sex differences, we tested whether individual brains are 'mosaics' of female-typical and male-typical measures or are internally consistent, having either only female-typical or only male-typical measures. We found mosaicism for measures showing overlap between females and males. Mosaicism was higher a) with a larger number of measures, b) with smaller effect sizes of the sex difference in these measures, and c) in rats with more diverse life experiences. Together, these results highlight the limitations of the binary framework for interpreting sex effects on the brain and suggest two complementary pathways to studying the contribution of sex to brain function: (1) focusing on measures showing dimorphic and persistent sex differences and (2) exploring the relations between specific brain mosaics and specific endpoints.

Anxiety, aggression, reward sensitivity, and forebrain dopamine receptor expression in a laboratory rat model of early-life disadvantage.

Despite early-life disadvantage (ELD) in humans being a highly heterogenous construct, it consistently predicts negative neurobehavioral outcomes. The numerous environmental contributors and neural mechanisms underlying ELD remain unclear, though. We used a laboratory rat model to evaluate the effects of limited resources and/or heavy metal exposure on mothers and their adult male and female offspring. Dams and litters were chronically exposed to restricted (1-cm deep) or ample (4-cm deep) home cage bedding postpartum, with or without lead acetate (0.1%) in their drinking water from insemination through 1-week postweaning. Restricted-bedding mothers showed more pup-directed behaviors and behavioral fragmentation, while lead-exposed mothers showed more nestbuilding. Restricted bedding-raised male offspring showed higher anxiety and aggression. Either restricted bedding or lead exposure impaired goal-directed performance in a reinforcer devaluation task in females, whereas restricted bedding alone disrupted it in males. Lead exposure, but not limited bedding, also reduced sucrose reward sensitivity in a progressive ratio task in females. D1 and D2 receptor mRNA in the medial prefrontal cortex and nucleus accumbens (NAc) were each affected by the early-life treatments and differently between the sexes. Most notably, adult males (but not females) exposed to both early-life treatments had greatly increased D1 receptor mRNA in the NAc core. These results illuminate neural mechanisms through which ELD threatens neurobehavioral development and highlight forebrain dopamine as a factor.

Vasopressin regulates social play behavior in sex-specific ways through glutamate modulation in the lateral septum.

Social play is a highly rewarding behavior that is essential for the development of social skills. Social play is impaired in children diagnosed with autism, a disorder with a strong sex bias in prevalence. We recently showed that the arginine vasopressin (AVP) system in the lateral septum (LS) regulates social play behavior sex-specifically in juvenile rats: Administration of a AVP 1a receptor (V1aR) antagonist increased social play behavior in males and decreased it in females. Here, we demonstrate that glutamate, but not GABA, is involved in the sex-specific regulation of social play by the LS-AVP system. First, males show higher extracellular glutamate concentrations in the LS than females while they show similar extracellular GABA concentrations. This resulted in a baseline sex difference in excitatory/inhibitory balance, which was eliminated by V1aR antagonist administration into the LS: V1aR antagonist increased extracellular glutamate release in females but not in males. Second, administration of the glutamate receptor agonist L-glutamic acid into the LS prevented the V1aR antagonist-induced increase in social play behavior in males while mimicking the V1aR antagonist-induced decrease in social play behavior in females. Third, administration of the glutamate receptor antagonists AP-5 and CNQX into the LS prevented the V1aR antagonist-induced decrease in social play behavior in females. Last, both sexes showed increases in extracellular LS-GABA release upon V1aR antagonist administration into the LS and decreases in social play behavior upon administration of the GABA-A receptor agonist muscimol into the LS, suggesting that GABA is not involved in the sex-specific regulation of social play by the LS-AVP system. Finally, to start identifying the cellular mechanism mediating the sex-specific effects of the LS-AVP system on LS-glutamate, we determined the presence of potential sex differences in the type of LS cells expressing V1aR. However, no sex differences were found in the percentage of Avpr1a+ LS cells expressing markers for either GABAergic neurons, somatostatin-expressing neurons, calbindin 1-expressing neurons, or astrocytes. In conclusion, these findings demonstrate that the LS-AVP system regulates social play sex-specifically via differential local glutamatergic neurotransmission in male and female juvenile rats. Further research is required to uncover the underlying cellular mechanism.

Development of social recognition ability in female rats: Effect of pubertal ovarian hormones.

The ability to recognize previously encountered conspecifics is crucial for social interaction. This social recognition ability is well characterized in adult rodents of both sexes but remains largely unexplored in juveniles. Using the social discrimination test of social recognition with short intervals (30 min and 1 h), we first found that juvenile female rats do not display a difference in investigation directed toward a novel vs. familiar stimulus rat. Using the social discrimination test with a 30-minute interval, we then showed that social recognition is established by the time of adolescence in female rats. Based on these findings, we hypothesized that social recognition is dependent on the initiation of ovarian hormone release during puberty. To test this, we ovariectomized females prior to puberty and found that prepubertal ovariectomy prevented the development of social recognition ability in adulthood. Administration of estradiol benzoate, 48 h prior to testing, to juvenile females or prepubertally ovariectomized adult females did not restore social recognition, suggesting that ovarian hormones organize the neural circuitry regulating this behavior during adolescence. These findings provide the first evidence of an effect of pubertal development on social recognition ability in female rats and highlight the importance of considering sex and age when interpreting results from behavioral paradigms initially designed for use in adult males.

The social versus food preference test: A behavioral paradigm for studying competing motivated behaviors in rodents.

Behavior is influenced by a combination of factors, with the expression of the appropriate behavior dependent on an individual's current motivational state and the presence of stimuli in their surrounding environment. Thus far, most laboratory studies have focused on uncovering the peripheral and central systems that regulate the expression of a single behavior or the expression of a suite of behaviors associated with a single motivational state. In natural settings, however, an individual can be simultaneously experiencing multiple motivational states with multiple choices of how to act. Yet, the direct assessment of the roles of peripheral and central systems in coordinating motivated behavioral choice is largely understudied. This may be due to a lack of behavioral tests that are suitable for such investigations. Here, we describe a recently developed behavioral paradigm, hereafter called the Social versus Food Preference Test. This behavioral paradigm was validated in both rats and mice and is highly flexible, which will allow addressing of a wide range of research questions concerning the peripheral and central systems that coordinate the choice to seek social interaction versus the choice to seek food.•This paradigm was validated in rats and mice, the two most commonly used nonhuman species in behavioral research, but could be adapted for use in other rodent models.•The specific social and food stimuli used can be selected based on the research question.•Three-chamber apparatuses can be custom-constructed.

Involvement of orexin/hypocretin in the expression of social play behaviour in juvenile rats.

Social play is a highly rewarding and motivated behaviour displayed by juveniles of many mammalian species. We hypothesized that the orexin/hypocretin (ORX) system is involved in the expression of juvenile social play behaviour because this system is interconnected with brain regions that comprise the social behaviour and mesocorticolimbic reward networks. We found that exposure to social play increased recruitment of ORX-A neurons in juvenile rats. Furthermore, central administration of ORX-A decreased social play duration, while central blockade of ORX-1 receptors differentially altered social play duration in juvenile rats with low versus high baseline levels of social play (increasing social play in low baseline social play individuals and decreasing social play in high baseline social play individuals). Together, our results provided the first evidence of a role for the ORX system in the modulation of juvenile social play behaviour.

Wistar rats and C57BL/6 mice differ in their motivation to seek social interaction versus food in the Social versus Food Preference Test.

Here we characterized the Social versus Food Preference Test, a behavioral paradigm designed to investigate the competition between the choice to seek social interaction versus the choice to seek food. We assessed how this competition was modulated by internal cues (social isolation, food deprivation), external cues (stimulus salience), sex (males, females), age (adolescents, adults), and rodent model (Wistar rats, C57BL/6 mice). We found that changes in stimulus preference in response to the internal and external cue manipulations were similar across cohorts. Specifically, social over food preference scores were reduced by food deprivation and social familiarly in Wistar rats and C57BL/6 mice of both sexes. Interestingly, the degree of food deprivation-induced changes in stimulus investigation patterns were greater in adolescents compared to adults in Wistar rats and C57BL/6 mice. Strikingly, baseline stimulus preference and investigation times varied greatly between rodent models: across manipulations, Wistar rats were generally more social-preferring and C57BL/6 mice were generally more food-preferring. Adolescent Wistar rats spent more time investigating the social and food stimuli than adult Wistar rats, while adolescent and adult C57BL/6 mice investigated the stimuli a similar amount. Social isolation did not alter behavior in the Social versus Food Preference Test. Together, our results indicate that the Social versus Food Preference Test is a flexible behavioral paradigm suitable for future interrogations of the peripheral and central systems that can coordinate the expression of stimulus preference related to multiple motivated behaviors.

Involvement of ventral pallidal vasopressin in the sex-specific regulation of sociosexual motivation in rats.

The ventral pallidum (VP) is a critical node of the mesocorticolimbic reward circuit and is known to modulate social behaviors in rodents. Arginine vasopressin (AVP) signaling via the V1A receptor (V1AR) within the VP is necessary for the expression of socially motivated affiliative behaviors in monogamous voles. However, whether the VP-AVP system regulates socially motivated behaviors in non-monogamous species remains unknown. Here, we determined the extent of AVP fiber innervation in the VP as well as the involvement of the VP-AVP system in sociosexual motivation in adult male and female rats. We found that males have nearly twice the density of AVP-immunoreactive (AVP-ir) fibers in the VP compared to females, suggesting the possibility that males experience enhanced AVP signaling in the VP. We further found that this sex difference in VP-AVP-ir fiber density likely arises from an observed sex difference (males > females) in the percentage of VP-projecting AVP-ir cell bodies located in the bed nucleus of the stria terminalis and medial amygdala. To determine the behavioral implications of this sex difference, we next blocked AVP signaling in the VP by antagonizing VP-V1ARs in male and female rats and tested their preference to investigate an unfamiliar male rat or unfamiliar estrus female rat confined to corrals located on opposite ends of a three-chamber apparatus. Under vehicle conditions, males showed a significantly greater innate preference to investigate an opposite sex over same sex conspecific than estrus females. Interestingly, VP-V1AR antagonism significantly reduced males' opposite sex preference, while enhancing estrus females' opposite sex preference. Importantly, all subjects reliably discriminated between male and female stimulus rats regardless of drug treatment, demonstrating a change in motivational state rather than a perceptual impairment induced by VP-V1AR blockade. These results provide a novel functional link between a sex difference in ventral pallidal AVP fiber density and the sex-specific regulation of a sexually motivated behavior necessary for reproductive success.

Effects of oxytocin receptor antagonism on social function and corticosterone release after adolescent social instability in male rats.

Oxytocin influences social behaviour and hypothalamic-pituitary-adrenal (HPA) function. We previously found that social instability stress (SS) from postnatal day 30 to 45 increased oxytocin receptor (OTR) densities in the lateral septum and nucleus accumbens of adolescent male rats. Here, we investigated social behaviour and HPA function in adolescent male SS rats compared with age- and sex-matched controls after intraperitoneal treatment with an OTR antagonist L-368,899 (OTR-A). Regardless of OTR antagonism, adolescent SS rats spent more time in social approach (investigation through wire mesh) but less time in social interaction (physical interaction) with unfamiliar same-sex and same-age peers than did controls. However, OTR-A-treatment caused SS rats to be more socially avoidant than OTR-A-treated controls and saline-treated rats of the same condition. Additionally, the predicted rise in plasma corticosterone in response to OTR-A treatment was blunted in SS rats. Fos immunoreactivity (IR) was used as a marker of neural activation in social brain regions and oxytocin-IR was examined in the paraventricular nucleus of the hypothalamus (PVN) in response to interacting with unfamiliar peers in SS and control rats after OTR-A treatment. OTR-A treatment had little effect on Fos-IR and oxytocin-IR in the analyzed brain regions, but SS rats had lower Fos-IR and oxytocin-IR in the PVN and greater Fos-IR in subregions of the prefrontal cortex, and hippocampus, and lateral septum than did controls. Finally, binding density of OTR was measured in the PVN and hippocampus, and greater OTR binding density was found in the PVN of SS rats. Together, these data demonstrate a greater influence of OTR antagonism on social behaviour and a reduced influence of OTR antagonism on HPA responses after adolescent SS in male rats. The results also suggest that differences in neural functioning in the prefrontal cortex, hippocampus and lateral septum of adolescent SS rats may be involved in their altered social behaviour relative to that of controls.

Oestrogen and androgen receptor activation contribute to the masculinisation of oxytocin receptors in the bed nucleus of the stria terminalis of rats.

Oxytocin (OT) often regulates social behaviours in sex-specific ways, and this may be a result of sex differences in the brain OT system. Adult male rats show higher OT receptor (OTR) binding in the posterior bed nucleus of the stria terminalis (pBNST) than adult female rats. In the present study, we investigated the mechanisms that lead to this sex difference. First, we found that male rats have higher OTR mRNA expression in the pBNST than females at postnatal day (P) 35 and P60, which demonstrates the presence of the sex difference in OTR binding density at message level. Second, the sex difference in OTR binding density in the pBNST was absent at P0 and P3, but was present by P5. Third, systemic administration of the oestrogen receptor (ER) antagonist fulvestrant at P0 and P1 dose-dependently reduced OTR binding density in the pBNST of 5-week-old male rats, but did not eliminate the sex difference in OTR binding density. Fourth, pBNST-OTR binding density was lower in androgen receptor (AR) deficient genetic male rats compared to wild-type males, but higher compared to wild-type females. Finally, systemic administration of the histone deacetylase inhibitor valproic acid at P0 and P1 did not alter pBNST-OTR binding density in 5-week-old male and female rats. Interestingly, neonatal ER antagonism, AR deficiency, and neonatal valproic acid treatment each eliminated the sex difference in pBNST size. Overall, we demonstrate a role for neonatal ER and AR activation in setting up the sex difference in OTR binding density in the pBNST, which may underlie sexual differentiation of the pBNST and social behaviour.

Comparing vasopressin and oxytocin fiber and receptor density patterns in the social behavior neural network: Implications for cross-system signaling.

Vasopressin (AVP) and oxytocin (OXT) regulate social behavior by binding to their canonical receptors, the vasopressin V1a receptor (V1aR) and oxytocin receptor (OTR), respectively. Recent studies suggest that these neuropeptides may also signal via each other's receptors. The extent to which such cross-system signaling occurs likely depends on anatomical overlap between AVP/OXT fibers and V1aR/OTR expression. By comparing AVP/OXT fiber densities with V1aR/OTR binding densities throughout the rat social behavior neural network (SBNN), we propose the potential for cross-system signaling in four regions: the medial amygdala (MeA), bed nucleus of the stria terminalis (BNSTp), medial preoptic area, and periaqueductal grey. We also discuss possible implications of corresponding sex (higher in males versus females) and age (higher in adults versus juveniles) differences in AVP fiber and OTR binding densities in the MeA and BNSTp. Overall, this review reveals the need to unravel the consequences of potential cross-system signaling between AVP and OXT systems in the SBNN for the regulation of social behavior.

Involvement of dopamine, but not norepinephrine, in the sex-specific regulation of juvenile socially rewarding behavior by vasopressin.

Social play is a highly rewarding behavior displayed mostly during the juvenile period. We recently showed that vasopressin V1a receptor (V1aR) blockade in the lateral septum (LS) enhances social play in male juvenile rats, but reduces it in females. Here, we determined whether the LS-AVP system modulates dopamine (DA) and/or norepinephrine (NE) neurotransmission in the LS to regulate social play behavior in sex-specific ways. Using microdialysis combined with retrodialysis, we demonstrated that both LS-AVP administration and social play exposure increased extracellular LS-DA release in females, but not in males. Pharmacological blockade of LS-DA receptors reduced social play in both sexes, but required a higher dose in females. This suggests that baseline LS-DA release is sufficient for social play in males, while increased LS-DA release is necessary for social play in females. Administration of a V1aR antagonist into the LS inhibited the social play-induced increase in extracellular LS-DA release in females. Furthermore, co-administration of the DA agonist apomorphine prevented the LS-V1aR blockade-induced decrease in social play in females. This suggests that LS-V1aR blockade reduces social play in females by dampening the rise in LS-DA release. Extracellular LS-NE release was enhanced in response to pharmacological manipulations of the LS-AVP system and to social play in males and/or females, but pharmacological blockade or stimulation of LS-NE receptors did not alter social play in either sex. Overall, we define a mechanism by which the LS-AVP system alters LS-DA neurotransmission differently in males than females resulting in the sex-specific regulation of juvenile social play behavior.

Sex differences in the regulation of social and anxiety-related behaviors: insights from vasopressin and oxytocin brain systems.

To understand how the brain regulates behavior, many variables must be taken into account, with sex as a prominent variable. In this review, we will discuss recent human and rodent studies showing the sex-specific involvement of the neuropeptides vasopressin and oxytocin in social and anxiety-related behaviors. We discuss that sex differences can be evident at pre-pubertal ages as seen in the sex-specific regulation of social recognition, social play, and anxiety by the vasopressin system in juvenile rats. We further discuss that the oxytocin system in humans and rodents alters brain activation, anxiety, and sociosexual motivation in sex-specific ways. Finally, we propose that knowledge of vasopressin and oxytocin mediated sex-specific brain mechanisms can provide essential insights into how these neuropeptide systems contribute to sex-specific vulnerability as well as resilience to perturbations, with subsequent relevance to social and emotional disorders.

Author Correction: Hippocampal oxytocin receptors are necessary for discrimination of social stimuli.

The original version of this Article contained an error in the spelling of the author Alexa H. Veenema, which was incorrectly given as Alexa Veenema. This has now been corrected in both the PDF and HTML versions of the Article.

Nucleus accumbens mu opioid receptors regulate context-specific social preferences in the juvenile rat.

The µ opioid receptor (MOR) in the nucleus accumbens (NAc) is involved in assigning pleasurable, or hedonic value to rewarding stimuli. Importantly, the hedonic value of a given rewarding stimulus likely depends on an individual's current motivational state. Here, we examined the involvement of MORs in the motivation to interact with a novel or a familiar (cage mate) conspecific in juvenile rats. First, we demonstrated that the selective MOR antagonist CTAP administered into the NAc reduces social novelty preference of juvenile males, by decreasing the interaction time with the novel conspecific and increasing the interaction time with the cage mate. Next, we found that a 3-h separation period from the cage mate reduces social novelty preference in both juvenile males and females, which was primarily driven by an increase in interaction time with the cage mate. Last, we showed that MOR agonism (intracerebroventricularly or in the NAc) restored social novelty preference in juvenile males that did not show social novelty preference following social isolation. Taken together, these data support a model in which endogenous MOR activation in the NAc facilitates the relative hedonic value of novel over familiar social stimuli. Our results may implicate the MOR in neuropsychiatric disorders characterized by altered social motivation, such as major depression and autism spectrum disorder.

Robust age, but limited sex, differences in mu-opioid receptors in the rat brain: relevance for reward and drug-seeking behaviors in juveniles.

In the brain, the µ-opioid receptor (MOR) is involved in reward-seeking behaviors and plays a pivotal role in the mediation of opioid use disorders. Furthermore, reward-seeking behaviors and susceptibility to opioid addiction are particularly evident during the juvenile period, with a higher incidence of opioid use in males and higher sensitivity to opioids in females. Despite these age and sex differences in MOR-mediated behaviors, little is known regarding potential age and sex differences in the expression of MORs in the brain. Here, we used receptor autoradiography to compare MOR binding densities between juvenile and adult male and female rats. Age differences were found in MOR binding density in 12 out of 33 brain regions analyzed, with 11 regions showing higher MOR binding density in juveniles than in adults. These include the lateral septum, as well as sub-regions of the bed nucleus of the stria terminalis, hippocampus, and thalamus. Sex differences in MOR binding density were observed in only two brain regions, namely, the lateral septum (higher in males) and the posterior cortical nucleus of the amygdala (higher in females). Overall, these findings provide an important foundation for the generation of hypotheses regarding differential functional roles of MOR activation in juveniles versus adults. Specifically, we discuss the possibility that higher MOR binding densities in juveniles may allow for higher MOR activation, which could facilitate behaviors that are heightened during the juvenile period, such as reward and drug-seeking behaviors.

Hippocampal oxytocin receptors are necessary for discrimination of social stimuli.

Oxytocin receptor (Oxtr) signaling in neural circuits mediating discrimination of social stimuli and affiliation or avoidance behavior is thought to guide social recognition. Remarkably, the physiological functions of Oxtrs in the hippocampus are not known. Here we demonstrate using genetic and pharmacological approaches that Oxtrs in the anterior dentate gyrus (aDG) and anterior CA2/CA3 (aCA2/CA3) of mice are necessary for discrimination of social, but not non-social, stimuli. Further, Oxtrs in aCA2/CA3 neurons recruit a population-based coding mechanism to mediate social stimuli discrimination. Optogenetic terminal-specific attenuation revealed a critical role for aCA2/CA3 outputs to posterior CA1 for discrimination of social stimuli. In contrast, aCA2/CA3 projections to aCA1 mediate discrimination of non-social stimuli. These studies identify a role for an aDG-CA2/CA3 axis of Oxtr expressing cells in discrimination of social stimuli and delineate a pathway relaying social memory computations in the anterior hippocampus to the posterior hippocampus to guide social recognition.

Social instability stress in adolescent male rats reduces social interaction and social recognition performance and increases oxytocin receptor binding.

Social experiences in adolescence are essential for displaying context-appropriate social behaviors in adulthood. We previously found that adult male rats that underwent social instability stress (SS) in adolescence had reduced social interactions with unfamiliar peers compared with non-stressed controls (CTL). Here we determined whether SS altered social recognition and social reward and brain oxytocin and vasopressin receptor density in adolescence. We confirmed that SS rats spent less time interacting with unfamiliar peers than did CTL rats (p=0.006). Furthermore, CTL rats showed a preference for novel over familiar conspecifics in a social recognition test whereas SS rats did not, which may reflect reduced recognition, impaired memory, or reduced preference for novelty in SS rats. The reward value of social interactions was not affected by SS based on conditioned place preference tests and based on the greater time SS rats spent investigating stimulus rats than did CTL rats when the stimulus rat was behind wire mesh (p=0.03). Finally, oxytocin receptor binding density was higher in the dorsal lateral septum and nucleus accumbens shell in SS rats compared with CTL rats (p=0.02, p=0.01, respectively). No effect of SS was found for vasopressin 1a receptor binding density in any of the brain regions analyzed. We discuss the extent to which the differences in social behavior exhibited after social instability in adolescence involve changes in social salience and social competency, and the possibility that changes in oxytocin signaling in the brain underlie the differences in social behavior.

Involvement of the oxytocin system in the nucleus accumbens in the regulation of juvenile social novelty-seeking behavior.

Exploration of novel environments, stimuli, and conspecifics is highly adaptive during the juvenile period, as individuals transition from immaturity to adulthood. We recently showed that juvenile rats prefer to interact with a novel individual over a familiar cage mate. However, the neural mechanisms underlying this juvenile social novelty-seeking behavior remain largely unknown. One potential candidate is the oxytocin (OXT) system, given its involvement in various motivated social behaviors. Here, we show that administration of the specific oxytocin receptor antagonist desGly-NH2,d(CH2)5-[Tyr(Me)2,Thr4]OVT reduces social novelty seeking-behavior in juvenile male rats when injected into the nucleus accumbens (10ng/0.5µl/side). The same drug dose was ineffective at altering social novelty-seeking behavior when administered into the lateral septum or basolateral amygdala. These results are the first to suggest the involvement of the OXT system in the nucleus accumbens in the regulation of juvenile social novelty-seeking behavior.

Sex differences in neural activation following different routes of oxytocin administration in awake adult rats.

The neuropeptide oxytocin (OT) regulates social behavior in sex-specific ways across species. OT has promising effects on alleviating social deficits in sex-biased neuropsychiatric disorders. However little is known about potential sexually dimorphic effects of OT on brain function. Using the rat as a model organism, we determined whether OT administered centrally or peripherally induces sex differences in brain activation. Functional magnetic resonance imaging was used to examine blood oxygen level-dependent (BOLD) signal intensity changes in the brains of awake rats during the 20min following intracerebroventricular (ICV; 1µg/5µl) or intraperitoneal (IP; 0.1mg/kg) OT administration as compared to baseline. ICV OT induced sex differences in BOLD activation in 26 out of 172 brain regions analyzed, with 20 regions showing a greater volume of activation in males (most notably the nucleus accumbens and insular cortex), and 6 regions showing a greater volume of activation in females (including the lateral and central amygdala). IP OT also elicited sex differences in BOLD activation with a greater volume of activation in males, but this activation was found in different and fewer (10) brain regions compared to ICV OT. In conclusion, exogenous OT modulates neural activation differently in male versus female rats with the pattern and magnitude, but not the direction, of sex differences depending on the route of administration. These findings highlight the need to include both sexes in basic and clinical studies to fully understand the role of OT on brain function.