MicroRNA137-loaded lipid nanoparticles regulate synaptic proteins in the prefrontal cortex.

Genome-wide association studies indicate that allele variants in MIR137, the host gene of microRNA137 (miR137), confer an increased risk of schizophrenia (SCZ). Aberrant expression of miR137 and its targets, many of which regulate synaptic functioning, are also associated with an increased risk of SCZ. Thus, miR137 represents an attractive target aimed at correcting the molecular basis for synaptic dysfunction in individuals with high genetic risk for SCZ. Advancements in nanotechnology utilize lipid nanoparticles (LNPs) to transport and deliver therapeutic RNA. However, there remains a gap in using LNPs to regulate gene and protein expression in the brain. To study the delivery of nucleic acids by LNPs to the brain, we found that LNPs released miR137 cargo and inhibited target transcripts of interest in neuroblastoma cells. Biodistribution of LNPs loaded with firefly luciferase mRNA remained localized to the mouse prefrontal cortex (PFC) injection site without circulating to off-target organs. LNPs encapsulating Cre mRNA preferentially co-expressed in neuronal over microglial or astrocytic cells. Using quantitative proteomics, we found miR137 modulated glutamatergic synaptic protein networks that are commonly dysregulated in SCZ. These studies support engineering the next generation of brain-specific LNPs to deliver RNA therapeutics and improve symptoms of central nervous system disorders.

Effect of sex and autism spectrum disorder on oxytocin receptor binding and mRNA expression in the dopaminergic pars compacta of the human substantia nigra.

Oxytocin is an endogenous neuropeptide hormone that influences social behaviour and bonding in mammals. Variations in oxytocin receptor (OXTR) expression may play a role in the social deficits seen in autism spectrum disorder. Previous studies from our laboratory found a dense population of OXTR in the human substantia nigra (SN), a basal ganglia structure in the midbrain that is important in both movement and reward pathways. Here, we explore whether differences in OXTR can be identified in the dopaminergic SN pars compacta of individuals with autism. Postmortem human brain tissue specimens were processed for OXTR autoradiography from four groups: males with autism, females with autism, typically developing (TD) males and TD females. We found that females with autism had significantly lower levels of OXTR than the other groups. To examine potential gene expression differences, we performed in situ hybridization in adjacent slides to visualize and quantify OXTR mRNA as well as mRNA for tyrosine hydroxylase. We found no differences in mRNA levels for either gene across the four groups. These results suggest that a dysregulation in local OXTR protein translation or increased OXTR internalization/recycling may contribute to the differences in social symptoms seen in females with autism. This article is part of the theme issue 'Interplays between oxytocin and other neuromodulators in shaping complex social behaviours'.

Compositional variation in early-life parenting structures alters oxytocin and vasopressin 1a receptor development in prairie voles (Microtus ochrogaster).

Paternal absence can significantly alter bio-behavioural development in many biparental species. This effect has generally been demonstrated by comparing the development of offspring reared under biparental care with those reared by a single mother. However, studies employing this design conflate two significant modifications to early-life experience: removal of father-specific qualities and the general reduction of offspring-directed care. In the socially monogamous prairie vole (Microtus ochrogaster), the experience of paternal absence without substitution during development inhibits partner preference formation in adulthood, a hallmark of social monogamy, in females and males. Employing alloparents as substitutes for fathers, our previous work demonstrated that paternal absence affects pair-bond formation in female offspring via reduced quantity of care, although it affects pair-bond formation in male offspring by means of a missing paternal quality (or qualities). Here, we present evidence that paternal absence (with and without alloparental substitution) may alter the ontogeny of neural oxytocin receptor (OXTR) and/or vasopressin 1a receptor (AVPR1a) distribution in male and female prairie voles. Compared to biparentally reared controls (BPC), male offspring reared in mother only (MON) and maternal-plus-alloparental (MPA) conditions show lower densities of OXTR in the central amygdala; and MPA males show lower densities of OXTR in the caudate putamen and nucleus accumbens. Early-life experience was not associated with differences in AVPR1a density in males. However, MON and MPA females show greater densities of AVPR1a in the medial amygdala than BPC; and MPA females show greater densities of AVPR1a in the ventromedial nucleus of the hypothalamus. We also demonstrate with corticosterone concentrations that MON and MPA offspring are not differentially susceptible to a stressor (ie, social isolation) than BPC offspring. These findings suggest that paternal absence, although likely not a salient early-life stressor, has neuroendocrine consequences for offspring, some of which may affect partner preference formation.

Neural correlates of mating system diversity: oxytocin and vasopressin receptor distributions in monogamous and non-monogamous Eulemur.

Contemporary theory that emphasizes the roles of oxytocin and vasopressin in mammalian sociality has been shaped by seminal vole research that revealed interspecific variation in neuroendocrine circuitry by mating system. However, substantial challenges exist in interpreting and translating these rodent findings to other mammalian groups, including humans, making research on nonhuman primates crucial. Both monogamous and non-monogamous species exist within Eulemur, a genus of strepsirrhine primate, offering a rare opportunity to broaden a comparative perspective on oxytocin and vasopressin neurocircuitry with increased evolutionary relevance to humans. We performed oxytocin and arginine vasopressin 1a receptor autoradiography on 12 Eulemur brains from seven closely related species to (1) characterize receptor distributions across the genus, and (2) examine differences between monogamous and non-monogamous species in regions part of putative "pair-bonding circuits". We find some binding patterns across Eulemur reminiscent of olfactory-guided rodents, but others congruent with more visually oriented anthropoids, consistent with lemurs occupying an 'intermediary' evolutionary niche between haplorhine primates and other mammalian groups. We find little evidence of a "pair-bonding circuit" in Eulemur akin to those proposed in previous rodent or primate research. Mapping neuropeptide receptors in these nontraditional species questions existing assumptions and informs proposed evolutionary explanations about the biological bases of monogamy.

Developmental Fluoxetine Exposure Alters Behavior and Neuropeptide Receptors in the Prairie Vole.

Developmental exposure to selective serotonin reuptake inhibitor (SSRI) increases the risk of Autism Spectrum Disorder (ASD), however, the underlying neurobiology of this effect is not fully understood. Here we used the socially monogamous prairie vole as a translational model of developmental SSRI exposure. Paired female prairie voles (n = 20) were treated with 5 mg/kg subcutaneous fluoxetine (FLX) or saline (SAL) daily from birth of the second litter until the day of birth of the 4th litter. This design created three cohorts of FLX exposure: postnatal exposure in litter 2, both prenatal and postnatal exposure in litter 3, and prenatal exposure in litter 4. Post-weaning, subjects underwent behavioral testing to detect changes in sociality, repetitive behavior, pair-bond formation, and anxiety-like behavior. Quantitative receptor autoradiography was performed for oxytocin, vasopressin 1a, and serotonin 1a receptor density in a subset of brains. We observed increased anxiety-like behavior and reduced sociality in developmentally FLX exposed adults. FLX exposure decreased oxytocin receptor binding in the nucleus accumbens core and central amygdala, and vasopressin 1a receptor binding in the medial amygdala. FLX exposure did not affect serotonin 1A receptor binding in any areas examined. Changes to oxytocin and vasopressin receptors may underlie the behavioral changes observed and have translational implications for the mechanism of the increased risk of ASD subsequent to prenatal SSRI exposure.

Sex differences in hypothalamic-pituitary-adrenal axis regulation after chronic unpredictable stress.

INTRODUCTION: Exposure to stress, mediated through the hypothalamic-pituitary-adrenal (HPA) axis, elicits sex differences in endocrine, neurological, and behavioral responses. However, the sex-specific factors that confer resilience or vulnerability to stress and stress-associated psychiatric disorders remain largely unknown. The evident sex differences in stress-related disease prevalence suggest the underlying differences in the neurobiological underpinnings of HPA axis regulation. METHOD: Here, we used a chronic unpredictable stress (CUS) model to investigate the behavioral and biochemical responses of the HPA axis in C57BL/6 mice. Animals were tested in the open field and forced swim test to examine anxiety-like and depressive-like behaviors. Plasma corticosterone levels were measured after behavior and CUS, and glucocorticoid receptor (GR) expression and cytosolic and nuclear fractions of binding protein FKBP51 expression were taken to measure function and regulation of the stress response. RESULTS: Our results indicate increased depressive-like behavior in males and females which correlated with increased corticosterone levels following CUS. However, females displayed more anxiety-like behaviors with and without CUS. Interestingly, we found trends toward dysregulation of GR protein expression in CUS females, and an increase in the GR inhibitory protein, FKBP51, in the cytosol of CUS males but not females. CONCLUSION: These results suggest biochemical alterations to the HPA axis regulation which may elicit a glucocorticoid resistance in females after chronic stress and may contribute to the sex-biased vulnerability to stress-related psychiatric disorders.

Non-invasive Eye Tracking Methods for New World and Old World Monkeys.

Eye-tracking methods measure what humans and other animals visually attend to in the environment. In nonhuman primates, eye tracking can be used to test hypotheses about how primates process social information. This information can further our understanding of primate behavior as well as offer unique translational potential to explore causes of or treatments for altered social processing as seen in people with neurodevelopmental disorders such as autism spectrum disorder and schizophrenia. However, previous methods for collecting eye-tracking data in nonhuman primates required some form of head restraint, which limits the opportunities for research with respect to the number of or kinds of primates that can undergo an eye-tracking study. We developed a novel, noninvasive method for collecting eye tracking data that can be used both in animals that are difficult to restrain without sedation as well as animals that are of different ages and sizes as the box size can be adjusted. Using a transport box modified with a viewing window, we collected eye-tracking data in both New (Callicebus cupreus) and Old World monkeys (Macaca mulatta) across multiple developmental time points. These monkeys had the option to move around the box and avert their eyes from the screen, yet, they demonstrated a natural interest in viewing species-specific imagery with no previous habituation to the eye-tracking paradigm. Provided with opportunistic data from voluntary viewing of stimuli, we found that juveniles viewed stimuli more than other age groups, videos were viewed more than static photo imagery, and that monkeys increased their viewing time when presented with multiple eye tracking sessions. This noninvasive approach opens new opportunities to integrate eye-tracking studies into nonhuman primate research.

Effect of age and autism spectrum disorder on oxytocin receptor density in the human basal forebrain and midbrain.

The prosocial hormone oxytocin (OXT) has become a new target for research on the etiology and treatment of autism spectrum disorder (ASD), a condition characterized by deficits in social function. However, it remains unknown whether there are alterations in OXT receptor (OXTR) levels in the ASD brain. This study quantified the density of OXTR and of the structurally related vasopressin 1a receptor (AVPR1a) in postmortem brain tissue from individuals with ASD and typically developing individuals. We analyzed two regions known to contain OXTR across all primates studied to date: the nucleus basalis of Meynert (NBM), which mediates visual attention, and the superior colliculus, which controls gaze direction. In the NBM specimens, we also analyzed the neighboring ventral pallidum (VP) and the external segment of the globus pallidus. In the superior colliculus specimens, we also analyzed the adjacent periaqueductal gray. We detected dense OXTR binding in the human NBM and VP and moderate to low OXTR binding in the human globus pallidus, superior colliculus, and periaqueductal gray. AVPR1a binding was negligible across all five regions in all specimens. Compared to controls, ASD specimens exhibited significantly higher OXTR binding in the NBM and significantly lower OXTR binding in the VP, an area in the mesolimbic reward pathway. There was no effect of ASD on OXTR binding in the globus pallidus, superior colliculus, or periaqueductal gray. We also found a significant negative correlation between age and OXTR binding in the VP across all specimens. Further analysis revealed a peak in OXTR binding in the VP in early childhood of typically developing individuals, which was absent in ASD. This pattern suggests a possible early life critical period, which is lacking in ASD, where this important reward area becomes maximally sensitive to OXT binding. These results provide unique neurobiological insight into human social development and the social symptoms of ASD.

Elevated Fecal pH Indicates a Profound Change in the Breastfed Infant Gut Microbiome Due to Reduction of Bifidobacterium over the Past Century.

Historically, Bifidobacterium species were reported as abundant in the breastfed infant gut. However, recent studies in resource-rich countries show an increased abundance of taxa regarded as signatures of dysbiosis. It is unclear whether these differences are the product of genetics, geographic factors, or interventions such as formula feeding, antibiotics, and caesarean section. Fecal pH is strongly associated with Bifidobacterium abundance; thus, pH could be an indicator of its historical abundance. A review of 14 clinical studies published between 1926 and 2017, representing more than 312 healthy breastfed infants, demonstrated a change in fecal pH from 5.0 to 6.5 (adjusted r2 = 0.61). This trend of increasing infant fecal pH over the past century is consistent with current reported discrepancies in Bifidobacterium species abundance in the gut microbiome in resource-rich countries compared to that in historical reports. Our analysis showed that increased fecal pH and abundance of members of the families Enterobacteriaceae, Clostridiaceae, Peptostreptococcaceae, and Veillonellaceae are associated, indicating that loss of highly specialized Bifidobacterium species may result in dysbiosis, the implications of which are not yet fully elucidated. Critical assessment of interventions that restore this ecosystem, measured by key parameters such as ecosystem productivity, gut function, and long-term health, are necessary to understand the magnitude of this change in human biology over the past century.

Persistence of Supplemented Bifidobacterium longum subsp. infantis EVC001 in Breastfed Infants.

Attempts to alter intestinal dysbiosis via administration of probiotics have consistently shown that colonization with the administered microbes is transient. This study sought to determine whether provision of an initial course of Bifidobacterium longum subsp. infantis (B. infantis) would lead to persistent colonization of the probiotic organism in breastfed infants. Mothers intending to breastfeed were recruited and provided with lactation support. One group of mothers fed B. infantis EVC001 to their infants from day 7 to day 28 of life (n = 34), and the second group did not administer any probiotic (n = 32). Fecal samples were collected during the first 60 postnatal days in both groups. Fecal samples were assessed by 16S rRNA gene sequencing, quantitative PCR, mass spectrometry, and endotoxin measurement. B. infantis-fed infants had significantly higher populations of fecal Bifidobacteriaceae, in particular B. infantis, while EVC001 was fed, and this difference persisted more than 30 days after EVC001 supplementation ceased. Fecal milk oligosaccharides were significantly lower in B. infantis EVC001-fed infants, demonstrating higher consumption of human milk oligosaccharides by B. infantis EVC001. Concentrations of acetate and lactate were significantly higher and fecal pH was significantly lower in infants fed EVC001, demonstrating alterations in intestinal fermentation. Infants colonized by Bifidobacteriaceae at high levels had 4-fold-lower fecal endotoxin levels, consistent with observed lower levels of Gram-negative Proteobacteria and Bacteroidetes. IMPORTANCE The gut microbiome in early life plays an important role for long-term health and is shaped in large part by diet. Probiotics may contribute to improvements in health, but they have not been shown to alter the community composition of the gut microbiome. Here, we found that breastfed infants could be stably colonized at high levels by provision of B. infantis EVC001, with significant changes to the overall microbiome composition persisting more than a month later, whether the infants were born vaginally or by caesarean section. This observation is consistent with previous studies demonstrating the capacity of this subspecies to utilize human milk glycans as a nutrient and underscores the importance of pairing a probiotic organism with a specific substrate. Colonization by B. infantis EVC001 resulted in significant changes to fecal microbiome composition and was associated with improvements in fecal biochemistry. The combination of human milk and an infant-associated Bifidobacterium sp. shows, for the first time, that durable changes to the human gut microbiome are possible and are associated with improved gut function.