Limosilactobacillus reuteri administration alters the gut-brain-behavior axis in a sex-dependent manner in socially monogamous prairie voles.

Research on the role of gut microbiota in behavior has grown dramatically. The probiotic L. reuteri can alter social and stress-related behaviors - yet, the underlying mechanisms remain largely unknown. Although traditional laboratory rodents provide a foundation for examining the role of L. reuteri on the gut-brain axis, they do not naturally display a wide variety of social behaviors. Using the highly-social, monogamous prairie vole (Microtus ochrogaster), we examined the effects of L. reuteri administration on behaviors, neurochemical marker expression, and gut-microbiome composition. Females, but not males, treated with live L. reuteri displayed lower levels of social affiliation compared to those treated with heat-killed L. reuteri. Overall, females displayed a lower level of anxiety-like behaviors than males. Live L. reuteri-treated females had lower expression of corticotrophin releasing factor (CRF) and CRF type-2-receptor in the nucleus accumbens, and lower vasopressin 1a-receptor in the paraventricular nucleus of the hypothalamus (PVN), but increased CRF in the PVN. There were both baseline sex differences and sex-by-treatment differences in gut microbiome composition. Live L. reuteri increased the abundance of several taxa, including Enterobacteriaceae, Lachnospiraceae NK4A136, and Treponema. Interestingly, heat-killed L. reuteri increased abundance of the beneficial taxa Bifidobacteriaceae and Blautia. There were significant correlations between changes in microbiota, brain neurochemical markers, and behaviors. Our data indicate that L. reuteri impacts gut microbiota, gut-brain axis and behaviors in a sex-specific manner in socially-monogamous prairie voles. This demonstrates the utility of the prairie vole model for further examining causal impacts of microbiome on brain and behavior.

Anti-inflammatory effects of ß-FNA are sex-dependent in a pre-clinical model of LPS-induced inflammation.

BACKGROUND: Inflammation is present in neurological and peripheral disorders. Thus, targeting inflammation has emerged as a viable option for treating these disorders. Previous work indicated pretreatment with beta-funaltrexamine (ß-FNA), a selective mu-opioid receptor (MOR) antagonist, inhibited inflammatory signaling in vitro in human astroglial cells, as well as lipopolysaccharide (LPS)-induced neuroinflammation and sickness-like-behavior in mice. This study explores the protective effects of ß-FNA when treatment occurs 10 h after LPS administration and is the first-ever investigation of the sex-dependent effects of ß-FNA on LPS-induced inflammation in the brain and peripheral tissues, including the intestines. RESULTS: Male and female C57BL/6J mice were administered LPS followed by treatment with ß-FNA-immediately or 10 h post-LPS. Sickness- and anxiety-like behavior were assessed using an open-field test and an elevated-plus-maze test, followed by the collection of whole brain, hippocampus, prefrontal cortex, cerebellum/brain stem, plasma, spleen, liver, large intestine (colon), proximal small intestine, and distal small intestine. Levels of inflammatory chemokines/cytokines (interferon γ-induced-protein, IP-10 (CXCL10); monocyte-chemotactic-protein 1, MCP-1 (CCL2); interleukin-6, IL-6; interleukin-1ß, IL-1ß; and tumor necrosis factor-alpha, TNF-α) in tissues were measured using an enzyme-linked immunosorbent assay. Western blot analysis was used to assess nuclear factor-kappa B (NF-κB) expression. There were sex-dependent differences in LPS-induced inflammation across brain regions and peripheral tissues. Overall, LPS-induced CXCL10, CCL2, TNF-α, and NF-κB were most effectively downregulated by ß-FNA; and ß-FNA effects differed across brain regions, peripheral tissues, timing of the dose, and in some instances, in a sex-dependent manner. ß-FNA reduced LPS-induced anxiety-like behavior most effectively in female mice. CONCLUSION: These findings provide novel insights into the sex-dependent anti-inflammatory effects of ß-FNA and advance this agent as a potential therapeutic option for reducing both neuroinflammation an intestinal inflammation.

Anti-inflammatory actions of ß-funaltrexamine in a mouse model of lipopolysaccharide-induced inflammation.

Neuroinflammation is involved in a wide range of brain disorders, thus there is great interest in identifying novel anti-inflammatory agents to include in therapeutic strategies. Our previous in vitro studies revealed that beta-funaltrexamine (ß-FNA), a well-characterized selective mu-opioid receptor (MOR) antagonist, inhibits inflammatory signaling in human astroglial cells, albeit through an apparent MOR-independent mechanism. We also previously determined that lipopolysaccharide (LPS)-induced sickness behavior and neuroinflammation in mice are prevented by pretreatment with ß-FNA. Herein we investigated the temporal importance of ß-FNA treatment in this pre-clinical model of LPS-induced neuroinflammation. Adult, male C57BL/6J mice were administered an i.p. injection of LPS followed by treatment (i.p. injection) with ß-FNA immediately or 4 h post-LPS. Sickness behavior was assessed using an open-field test, followed by assessment of inflammatory signaling in the brain, spleen, and plasma. Levels of inflammatory chemokines/cytokines (interferon γ-induced protein, CXCL10; monocyte chemotactic protein 1, CCL2; and interleukin-6, IL-6) in tissues were measured using an enzyme-linked immunosorbent assay and nuclear factor-kappa B (NFκB), p38 mitogen activated kinase (p38 MAPK), and glial fibrillary acidic protein (GFAP) expression were measured by western blot. LPS-induced sickness behavior and chemokine expression were inhibited more effectively when ß-FNA treatment occurred immediately after LPS administration, as opposed to 4 h post-LPS; and ß-FNA-mediated effects were time-dependent as evidenced by inhibition at 24 h, but not at 8 h. The inhibitory effects of ß-FNA on chemokine expression were more evident in the brain versus the spleen or plasma. LPS-induced NFκB-p65 and p38 MAPK expression in the brain and spleen were inhibited at 8 and 24 h post-LPS. These findings extend our understanding of the anti-inflammatory effects of ß-FNA and warrant further investigation into its therapeutic potential.

Open-source method of image cytometry in dorsal root ganglia tissue with immunofluorescence.

Immunohistochemistry (IHC) is a valuable tool in clinical and biological research for evaluating proteins and other antigens in spatially bound tissue. In neuroinflammatory pain research, primary afferent neurons of the dorsal root ganglion (DRG) are studied to understand molecular signaling mechanisms involved in nociception (pain) and inflammation. Measuring IHC (immunofluorescence) in DRG neurons requires manual hand tracing of nuclear and somatic boundaries, which is laborious, error-prone, and may require several weeks to collect the appropriate sample size with a mouse or pen-input display monitor. To overcome these limitations and increase standardization of sampling and measurement, we employed a reliable neuronal cytoplasmic reporter, exclusive to DRG neuronal soma, in a semi-automated algorithm-based approach of Image Cytometry in rat DRG (IC-DRG). The resulting output images are binary nuclear and somatic masks of DRG neurons, defining boundaries of measurement for CellProfiler and manually scored at 94% accurate. Herein, we successfully show a novel approach of automated image analysis for DRG neurons using a robust ImageJ/FIJI script, overcoming morphological variability and imaging artifacts native to imaging frozen tissue sections processed with immunofluorescence.

Selected omissions and capstone presentations: a new approach to student-centered integrative physiology education.

Here, we describe a pedagogical approach that combines didactics with active learning to facilitate integration across physiological systems in a team-taught, graduate-level physiology course. We covered the major physiological systems, with each system preceded by an overview of its evolution/ontogeny to provide a broader perspective. Lectures provided a framework for integration by giving examples of how each system interacted with systems that preceded and followed. In lieu of a final exam, the course culminated in capstone presentations by small groups to promote student-centered learning of integrative physiology. At the beginning of the semester, students were assigned to groups; each group chose from predetermined topics. This allowed them to accumulate information throughout the semester and required them to attend to lecture content to assess how the material applied to their topic, thereby facilitating learning and retention. Faculty were deliberate in choosing material that was presented in each system, and material that was strategically omitted, establishing "gaps" that students filled in their capstone presentations. The final week was dedicated to student preparation for their presentations, which promoted peer-to-peer teaching and problem solving by the group, assisted by faculty as necessary. Capstone presentations demonstrated students' mastery of basic physiological principles and their ability to integrate among physiological systems, and they rated capstone presentations highly in helping with integration and critical thinking. Thus students showed a better understanding of systems physiology and the importance of integration across systems in normal function and in responding to homeostatic challenges.

Chronic exposure to inorganic mercury alters stress responses in male prairie voles (Microtus ochrogaster).

Male, but not female, prairie voles that experience chronic exposure to inorganic mercury display aberrant social behavior - avoiding unfamiliar conspecifics rather than approaching them. The mechanisms that underlie such behavioral changes are unknown, but likely involve the hypothalamus-pituitary-adrenal (HPA) axis. We tested this hypothesis by providing voles of both sexes with mercury chloride in their drinking water for ten weeks and then staging same-sex dyadic encounters after which plasma was assayed for corticosterone as an index of HPA activity. Consistent with sex-specific behavioral responses previously reported, mercury-treated males had lower plasma corticosterone after social encounters than did similarly-treated females or males that consumed normal drinking water. The results suggest that mercury-treated males may be less inclined toward social engagement with conspecifics due to reduced HPA activity.

Postnatal maturation of the intestinal epithelial barrier in prairie voles.

Intestinal epithelium develops during gestation and continues to mature post-natally into a selective barrier that will protect the individual while still allowing passage of nutrients. Until fully mature, the risk of translocation of microorganisms, toxins or antigens into the sub-epithelial tissue is high and could result in pathologies with life-altering consequences, or even premature death. Because of their monogamous mating system, prairie voles are an emerging model for studying the role of the intestinal microbiota in modulating social behavior via the microbiota-gut-brain-behavior axis. However, knowledge about the voles' intestinal barrier maturation is lacking. Understanding the maturation of the intestine epithelial barrier can complement the extensive behavioral literature for future studies involving the vole gut-brain axis. In this study, we characterized intestinal barrier function by demonstrating that two-week-old prairie voles have high paracellular absorption of FITC-dextran molecules prior to markedly decreased permeability at three weeks of age. In light of the fundamental role of tight junctions in maintaining epithelial integrity regulating intestinal permeability, we examined tight junction gene expression profiles. Transmission electron microscopy was used to visualize tight junction structure. Our results provide a timeline for intestinal barrier maturation and point to tight junction proteins involved in this process in prairie voles.

Fecal microbiota in the female prairie vole (Microtus ochrogaster).

We examined the fecal microbiota of female prairie voles. This species is socially and, likely, sexually monogamous, and thus serves as a valuable model in which to examine the interaction between the microbiota-gut-brain axis and social behavior. At present, little is known about the gastrointestinal microbiota of prairie voles; therefore, we performed a first characterization of the fecal microbiota using 16S rRNA gene amplicon sequencing. Semiconductor sequencing technology on an Ion Torrent PGM platform was used to assess the composition of fecal microbiotas from twelve female prairie voles. Following quality filtering, 1,017,756 sequencing reads were classified from phylum to genus level. At the phylum level, Firmicutes, Bacteroidetes, and Saccharibacteria were the predominant taxa, while the Bacteriodales, Erysipelotrichaceae, Ruminococcaceae, and Lachnospiraceae contributed the most dominant microbial groups and genera. Microbial community membership was most similar between vole sibling pairs, but consideration of taxon abundances weakened these associations. The interdependence of host factors such as genetics and behavior with the gastrointestinal microbiota is likely to be particularly pronounced in prairie voles. Our pilot characterization of the prairie vole intestinal microbiota revealed a microbial community composition remarkably consistent with the monogastric alimentary system of these rodents and their diet rich in complex plant carbohydrates. The highly social nature of these animals poses specific challenges to microbiome analyses that nonetheless are valuable for advancing research on the microbiota-gut-brain-behavior axis. Our study provides an important basis for future microbiome research in this emerging model organism for studying social behavior.

Early developmental exposure to inorganic mercury does not alter affiliative behavior of adult prairie voles (Microtus ochrogaster).

Mercury chloride exposure through drinking water in adult male prairie voles (Microtus ochrogaster) has been shown to alter their social behavior. Here, we examined the potential disruption of adult social behavior in prairie voles that were exposed to 60 ppm mercury during early development. We used a cross-fostering approach to test the effects of mercury exposure: (1) from conception until birth; (ii) from birth until weaning; and (iii) from conception until weaning, on adult affiliative behavior. Untreated and mercury-treated voles were given the option of remaining in an empty cage or affiliating with a same-sex conspecific in a 3-h choice test. We found that early developmental mercury exposure had little if any effect on the reproductive success of breeder pairs or on affiliative behavior by either sex when subjects were tested as adults. These results suggest that, at least in the context of the behavior tested, the effects of early developmental exposure to mercury do not permanently alter adult prairie vole affiliative behavior, or do so in a way that is too subtle to be detected using the current testing paradigm.

Cardiovascular control is associated with pair-bond success in male prairie voles.

Social support structures reduce mortality and morbidity in humans, but the mechanisms underlying these reductions are not fully understood. The prevailing hypothesis is that social support buffers stress and reduces allostatic load, thereby increasing longevity. However, the possibility that affiliative social interactions confer health benefits independent of stress buffering is understudied. We examined autonomic function in prairie voles - arguably the premier species for modeling human social affiliation - to assess the possibility that the formation of strong social bonds alters autonomic function and contributes to health benefits. We examined cardiovascular measures in male prairie voles before and after two weeks of cohabitation with a female, during a partner preference test, and during social isolation. There were strong correlations between social contact and heart rate (HR) and heart rate variability (HRV), the latter being an index of autonomic nervous system function. Males that successfully pair-bonded with their partners displayed higher HRV prior to pairing than did unsuccessful males, suggesting higher basal parasympathetic tone in the successful males. HRV increased further still when pair-bonded males huddled quietly with their mates during the partner preference test. Non-pair-bonded males not only had lower baseline parasympathetic activity, but showed a further decrease after pairing. HR increased and HRV decreased during social isolation only in pair-bonded males. Since differences in HRV are thought to reflect the relative influences of the parasympathetic and sympathetic nervous systems on cardiac function, these results suggest that autonomic balance may contribute to social bonding and thus to its health benefits.

Male prairie voles display cardiovascular dipping associated with an ultradian activity cycle.

Mammals typically display alternating active and resting phases and, in most species, these rhythms follow a circadian pattern. The active and resting phases often are accompanied by corresponding physiological changes. In humans, blood pressure decreases during the resting phase of the activity cycle, and the magnitude of that "nocturnal dipping" has been used to stratify patients according to the risk for cardiovascular disease. However, in contrast to most mammals, prairie voles (Microtus ochrogaster) have periods of activity and rest that follow an ultradian rhythm with period lengths significantly <24h. While rhythmic changes in blood pressure across a circadian activity cycle have been well-documented, blood pressure patterns in species that display ultradian rhythms in activity are less well-studied. In the current study, we implanted pressure-sensitive radiotelemetry devices in male prairie voles and recorded activity, mean arterial pressure (MAP), and heart rate (HR) continuously for 3days. Visualization of the ultradian rhythms was enhanced using a 1h running average to filter the dataset. Positive correlations were found between activity and MAP and between activity and HR. During the inactive period of the ultradian cycle, blood pressure decreased by about 15%, which parallels the nocturnal dipping pattern seen in healthy humans. Further, the duration of inactivity did not affect any of the cardiovascular measures, so the differences in blood pressure values between the active and inactive periods are likely driven by ultradian oscillations in hormones and autonomic function. Finally, specific behavioral patterns also were examined. Both the instrumented animal and his non-instrumented cagemate appeared to show synchronized activity patterns, with both animals displaying sleep-like behavior for more than 90% of the inactive period. We propose that the prairie vole ultradian rhythm in blood pressure is an analogue for circadian blood pressure variability and can be used to study the long-term effects of commonly prescribed drugs on blood pressure dipping.

Female-Paced Mating does not Affect Pair-Bond Expression by Male Prairie Voles (Microtus ochrogaster).

Prairie vole males typically display robust preferences for affiliation with their respective mates that indicate the expression of a pair-bond. However, it recently has been shown that the strength of a male vole's pair-bond can differ depending on the reproductive status of his mate. In the present study, we examined the possibility that female-controlled pacing of the mating sequence could alter males' affiliative behaviors in a partner-preference test by affecting reproductive success. We expected an earlier onset of mating and thus earlier onset of pregnancy would occur if females controlled the pace of mating, in turn, reinforcing males' preference for their familiar mates vs for a stranger. We found that female-pacing did not affect latency to mating, mating duration, or any of our other measures of social or mating behaviors. Further, female paced-mating did not alter reproductive success as indicated by litter size. We conclude that female-paced mating in prairie voles does not impact the formation, consolidation and/or expression of a pair-bond, either directly or indirectly, by their male partners.

Lactobacilli with probiotic potential in the prairie vole (Microtus ochrogaster).

BACKGROUND: Recent research suggests integration of the intestinal microbiota in gut-brain communication which could lead to new approaches to treat neurological disorders. The highly social prairie voles are an excellent model system to study the effects of environmental factors on social behavior. For future studies on the role of probiotics in ameliorating disorders with social withdrawal symptoms, we report the characterization of intestinal Lactobacillus isolates with probiotic potential from voles. METHODS AND RESULTS: 30 bacterial strains were isolated from the vole intestine and found to be distinct but closely related to Lactobacillus johnsonii using 16S rRNA gene sequencing and Random Amplification of Polymorphic DNA fingerprinting. In vitro characterizations including acid and bile tolerance, antimicrobial effects, antibiotic susceptibility, and adherence to intestinal epithelial cells were performed to assess the probiotic potential of selected strains. Since previous studies revealed that mercury ingestion triggers social deficits in voles, mercury resistance of the probiotic candidates was evaluated which could be an important factor in preventing/treating these behavioral changes. CONCLUSIONS: This study demonstrates that lactobacilli with probiotic potential are present in the vole intestine. The Lactobacillus isolates identified in this study will provide a basis for the investigation of probiotic effects in the vole behavioral model system.

Does paedomorphosis contribute to prairie vole monogamy?

We examined skull morphology in prairie voles (Microtus ochrogaster) and meadow voles (M. pennsylvanicus), two closely related species with fundamentally different mating systems, to test the hypothesis that paedomorphosis contributes to the evolution of monogamous mating systems. Using several skull measurements, we found that the overall length:width ratio of meadow vole skulls was greater than that of prairie voles suggesting that meadow vole have longer narrower skulls. We then examined which aspects of skull morphology differed between the species and found that the ratio difference was attributable primarily to longer snout length in meadow voles. Finally, we compared adult morphology in both species to that of pups and found the prairie vole, a monogamous species, displays a more juvenile-like skull morphology than does the meadow vole, a promiscuous species. These results suggest that monogamous vole species retain more juvenile-like morphology than do promiscuous species, and thus possibly retain juvenile-like behaviors that may contribute to a monogamous mating system.

Regional differences in serotonin content in the nucleus of the solitary tract of male rats after hypovolemia produced by polyethylene glycol.

Serotonin (5-HT) has been implicated in centrally mediated compensatory responses to volume loss in rats. Accordingly, we hypothesized that slowly developing, non-hypotensive hypovolemia increases serotonin in the hindbrain nucleus of the solitary tract (NTS). We produced volume loss in adult male rats by administering hyperoncotic polyethylene glycol (PEG) and then assessed 5-HT levels in the NTS using measurements of tissue 5-HT content or 5-HT immunohistochemistry. The results show selective increases of 5-HT in the caudal NTS after PEG treatment, but no change in the primary 5-HT metabolite, 5-HIAA. Moreover, the intensity of 5-HT immunolabeled fibers in the caudal NTS was increased after PEG treatment. These findings suggest that, after PEG-induced hypovolemia, 5-HT accumulates in neural elements in the caudal NTS. We propose that this accumulation is attributable to an initial release of 5-HT that then acts at presynaptic autoreceptors to inhibit subsequent 5-HT release.

Dendritic arbor of neurons in the hypothalamic ventromedial nucleus in female prairie voles (Microtus ochrogaster).

Female mating behavior in rats is associated with hormone-induced changes in the dendritic arbor of neurons in the ventromedial nucleus of the hypothalamus (VMH), particularly the ventrolateral portion. Regulation of mating behavior in female prairie voles differs substantially from that in rats; therefore, we examined the dendritic morphology of VMH neurons in this species. Sexually naïve adult female prairie voles were housed with a male to activate the females' reproductive endocrine system. Following 48 h of cohabitation, females were tested for evidence of reproductive activation by assessing the level of male sexual interest, after which their brains were processed using Golgi impregnation, which allowed ventrolateral VMH neurons to be visualized and analyzed. Dendritic arborization in the female prairie vole VMH neurons was strikingly similar to that of female rats. The key difference was that in the prairie voles the long primary dendrites extended considerably further than those observed in rats. Although most female voles paired with males exhibited sexual activation, some females did not. These two groups displayed specific differences in their VMH dendrites. In particular, the long primary dendrites were longer in the reproductively active females compared with those in the non-activated females. Overall, dendrite lengths were positively correlated with plasma estradiol levels in females exposed to males, but not in unpaired females. Although causal relationships between the neuroendocrine events, dendrite length, and the outward, behavioral manifestation of reproductive activation cannot be determined from this study, these results suggest an association between ventrolateral VMH dendrite morphology and female mating behavior in prairie voles, akin to what has been observed in female rats.

α-Synuclein potentiates interleukin-1ß-induced CXCL10 expression in human A172 astrocytoma cells.

Neuroinflammation and neuronal degeneration observed in Parkinson's disease (PD) has been attributed in part to glial-mediated events. Increased expression of proinflammatory cytokines and abnormal accumulation of the neuronal protein, α-synuclein in the brain are also characteristic of PD. While increasing evidence suggests that astrocytes contribute to neuroinflammation and dopaminergic neuronal degeneration associated with PD, there remains much to learn about these astroglial-mediated events. Therefore, we investigated the in vitro effects of interleukin-1ß (IL-1ß) and α-synuclein on astroglial expression of interferon-γ inducible protein-10 (CXCL10), a proinflammatory and neurotoxic chemokine. IL-1ß-induced CXCL10 protein expression was potentiated by co-exposure to α-synuclein. α-Synuclein did not significantly affect IL-1ß-induced CXCL10 mRNA expression, but did mediate increased CXCL10 mRNA stability, which may explain, in part, the increased levels of secreted CXCL10 protein. Future investigations are warranted to more fully define the mechanism by which α-synuclein enhances IL-1ß-induced astroglial CXCL10 expression. These findings highlight the importance of α-synuclein in modulating inflammatory events in astroglia. These events may be particularly relevant to the pathology of CNS disorders involving α-synuclein accumulation, including PD and HIV-1 associated dementia.

Social bonding decreases the rewarding properties of amphetamine through a dopamine D1 receptor-mediated mechanism.

Although the protective effects of social bonds on drug use/abuse have been well documented, we know little about the underlying neural mechanisms. Using the prairie vole (Microtus ochrogaster)--a socially monogamous rodent that forms long-term pair bonds after mating--we demonstrate that amphetamine (AMPH) conditioning induced a conditioned place preference (CPP) in sexually naive (SN), but not pair-bonded (PB), males. Although AMPH treatment induced a similar magnitude of dopamine release in the nucleus accumbens (NAcc) of SN and PB males, it had differential effects on NAcc D1 receptor (D1R) binding. Specifically, AMPH treatment increased D1R binding in SN, but decreased D1R binding in PB males. NAcc D1R, but not D2 receptor, antagonism blocked AMPH-induced CPP in SN males and NAcc D1R activation before AMPH conditioning enabled AMPH-induced CPP in PB males. Together, our data demonstrate that pair-bonding experience decreases the rewarding properties of AMPH through a D1R-mediated mechanism.

Does fertility trump monogamy?

Monogamous animals face an interesting dilemma: if and when to terminate a nonproductive relationship. To address this issue, we asked whether reproductive compatibility is a criterion for maintaining a monogamous pair-bond between mates. Prairie voles (Microtus ochrogaster), are small rodents that form long-term, monogamous pair-bonds evidenced by a strong preference for their familiar partners, and thus are an excellent model animal in which to study mate-fidelity. Accordingly, we examined partner-preferences by male prairie voles and related their behavior to the reproductive status of their mates. We found that, when given a choice between their familiar female partner and a stranger, male prairie voles differ in their responses depending on the pregnancy status of the partner. Males that were paired with females for two weeks displayed mate-fidelity only when mating had been initiated within ~48 hours after pairing. In contrast, males whose mates experienced a delay in the onset of sexual receptivity displayed non-selective affiliative behavior. We also found that, in addition to being appropriately timed, mating must be successful. Males that mated with ovariectomized females for whom pregnancy was precluded did not display mate-fidelity even though mating was initiated within the proper timeframe. These observations of mate-fidelity only when the partner's pregnancy was sufficiently advanced relative to the duration of the pairing suggest that mating must be both successful and timely. Thus, reproductive compatibility can influence mate-fidelity.

Female prairie vole mate-choice is affected by the males' birth litter composition.

Experimental testing and retrospective examination of breeding records were used to examine the influence of sex composition and/or size of males' birth litters on female mate-choice. Sexually naïve female prairie voles (Microtus ochrogaster) avoided males derived from all-male litters, but showed no preference for, or aversion to, males from single-male litters or from more typical mixed-sex litters. Examination of the pregnancy status of females after two weeks of pairing with a male allowed us to estimate the probabilites of a pups' intrauterine position relative to siblings for various litter sizes. The typical prairie vole pup derived from a mixed-sex litter comprised of 4.4 pups, and had a 13% chance of being isolated from siblings in utero and a 22% chance of being between siblings in utero. Pups from single-sex litters tended to be larger at weaning than did pups from mixed-sex litters; however, male size did not influence female choice behavior. These results suggest that some aspect of the perinatal experience of prairie vole pups from single sex litters can influence social interactions later in life.