An in vitro neurogenetics platform for precision disease modeling in the mouse.

The power and scope of disease modeling can be markedly enhanced through the incorporation of broad genetic diversity. The introduction of pathogenic mutations into a single inbred mouse strain sometimes fails to mimic human disease. We describe a cross-species precision disease modeling platform that exploits mouse genetic diversity to bridge cell-based modeling with whole organism analysis. We developed a universal protocol that permitted robust and reproducible neural differentiation of genetically diverse human and mouse pluripotent stem cell lines and then carried out a proof-of-concept study of the neurodevelopmental gene DYRK1A. Results in vitro reliably predicted the effects of genetic background on Dyrk1a loss-of-function phenotypes in vivo. Transcriptomic comparison of responsive and unresponsive strains identified molecular pathways conferring sensitivity or resilience to Dyrk1a1A loss and highlighted differential messenger RNA isoform usage as an important determinant of response. This cross-species strategy provides a powerful tool in the functional analysis of candidate disease variants identified through human genetic studies.

High sucrose consumption decouples intrinsic and synaptic excitability of AgRP neurons without altering body weight.

BACKGROUND/OBJECTIVE: As the obesity epidemic continues, the understanding of macronutrient influence on central nervous system function is critical for understanding diet-induced obesity and potential therapeutics, particularly in light of the increased sugar content in processed foods. Previous research showed mixed effects of sucrose feeding on body weight gain but has yet to reveal insight into the impact of sucrose on hypothalamic functioning. Here, we explore the impact of liquid sucrose feeding for 12 weeks on body weight, body composition, caloric intake, and hypothalamic AgRP neuronal function and synaptic plasticity. METHODS: Patch-clamp electrophysiology of hypothalamic AgRP neurons, metabolic phenotyping and food intake were performed on C57BL/6J mice. RESULTS: While mice given sugar-sweetened water do not gain significant weight, they do show subtle differences in body composition and caloric intake. When given sugar-sweetened water, mice show similar alterations to AgRP neuronal excitability as in high-fat diet obese models. Increased sugar consumption also primes mice for increased caloric intake and weight gain when given access to a HFD. CONCLUSIONS: Our results show that elevated sucrose consumption increased activity of AgRP neurons and altered synaptic excitability. This may contribute to obesity in mice and humans with access to more palatable (HFD) diets.

Effects of paternal high-fat diet and maternal rearing environment on the gut microbiota and behavior.

Exposing a male rat to an obesogenic high-fat diet (HFD) influences attractiveness to potential female mates, the subsequent interaction of female mates with infant offspring, and the development of stress-related behavioral and neural responses in offspring. To examine the stomach and fecal microbiome's potential roles, fecal samples from 44 offspring and stomach samples from offspring and their fathers were collected and bacterial community composition was studied by 16 small subunit ribosomal RNA (16S rRNA) gene sequencing. Paternal diet (control, high-fat), maternal housing conditions (standard or semi-naturalistic housing), and maternal care (quality of nursing and other maternal behaviors) affected the within-subjects alpha-diversity of the offspring stomach and fecal microbiomes. We provide evidence from beta-diversity analyses that paternal diet and maternal behavior induced community-wide shifts to the adult offspring gut microbiome. Additionally, we show that paternal HFD significantly altered the adult offspring Firmicutes to Bacteroidetes ratio, an indicator of obesogenic potential in the gut microbiome. Additional machine-learning analyses indicated that microbial species driving these differences converged on Bifidobacterium pseudolongum. These results suggest that differences in early-life care induced by paternal diet and maternal care significantly influence the microbiota composition of offspring through the microbiota-gut-brain axis, having implications for adult stress reactivity.

Inducing Partner Preference in Mice by Chemogenetic Stimulation of CA2 Hippocampal Subfield.

Social recognition is fundamental for social decision making and the establishment of long-lasting affiliative behaviors in behaviorally complex social groups. It is a critical step in establishing a selective preference for a social partner or group member. C57BL/6J lab mice do not form monogamous relationships, and typically do not show prolonged social preferences for familiar mice. The CA2 hippocampal subfield plays a crucial role in social memory and optogenetic stimulation of inputs to the dorsal CA2 field during a short memory acquisition period can enhance and extend social memories in mice. Here, we show that partner preference in mice can be induced by chemogenetic selective stimulation of the monosynaptic projections from the hypothalamic paraventricular nucleus (PVN) to the CA2 during the cohabitation period. Specifically, male mice spend more time in social contact, grooming and huddling with the partner compared to a novel female. Preference was not induced by prolonging the cohabitation period and allowing more time for social interactions and males to sire pups with the familiar female. These results suggest that PVN-to-CA2 projections are part of an evolutionarily conserved neural circuitry underlying the formation of social preference and may promote behavioral changes with appropriate stimulation.

Oxytocin delivered nasally or intraperitoneally reaches the brain and plasma of normal and oxytocin knockout mice.

Intranasal delivery of oxytocin (Oxt) has been identified as a potential therapeutic to target human conditions characterized by social deficits, yet the ability of this administrative route to deliver to the brain is unconfirmed. Oxt knockout (Oxt KO) and wildtype C57BL/6 J male mice received Oxt (12 µg total amount) either by nasal or intraperitoneal administration. Oxt concentrations were monitored for 2 h after administration in circulation via a jugular vein catheter and in the brain by two intracerebral microdialysis probes. Group sizes varied from 4 to 7 mice (n = 22 total). We document for the first time that Oxt applied to the nasal mucosa after nasal administration is delivered to the extracellular fluid in the brain. After nasal application, Oxt concentrations in circulation and in the extracellular fluid of the amygdala and, to an extent, the dorsal hippocampus, rose within the first 30 min and remained elevated for the subsequent hour. These findings were confirmed in an Oxt KO mouse line, establishing that the circulating and brain Oxt elevations derive from the administered dose. Interestingly, the pharmacokinetics of Oxt were slightly biased to the brain after nasal administration and to the periphery following intraperitoneal injection. No change in vasopressin levels was detected. These findings have stimulating implications for the interpretation of various behavioral and physiological effects described in animal and human studies after nasal administration of Oxt and provide the pharmacokinetics necessary to develop this drug delivery route for therapeutic purposes.

Effects of paternal high-fat diet and rearing environment on maternal investment and development of defensive responses in the offspring.

Paternal preconception risk factors (e.g. stress, diet, drug use) correlate with metabolic dysfunction in offspring, which is often comorbid with depressive and anxiety-like phenotypes. Detection of these risk factors or deleterious phenotypes informs a female about prevailing ecological demands, in addition to potential adverse environment-induced phenotypes that may be disseminated to her offspring. We examined whether a F0 male rat's prior exposure to an obesogenic high-fat diet (HFD) influences a female's attraction towards a male, subsequent mother-infant interactions and the development of defensive (emotional) responses in the F1 offspring. Females displayed less interest in the HFD exposed F0 males relative to control diet-exposed F0 males. Dams that reared F1 offspring in larger, semi-naturalistic housing provided more licking and grooming and active arched-back-nursing behavior. However, some of these effects interacted with paternal experience. F0 HFD and maternal rearing environment revealed sex-dependent, between group differences in F1 offspring wean weight, juvenile social interactions and anxiety-like behavior in adolescence. Our results show for the first time in mammals that male exposure to HFD may contribute to stable behavioral variation among females in courtship, maternal care, even when the females are not directly exposed to a HFD, and anxiety-like behavior in F1 offspring. Furthermore, when offspring were exposed to a predatory threat, hypothalamic Crf gene regulation was influenced by early housing. These results, together with our previous findings, suggest that paternal experience and maternal rearing conditions can influence maternal behavior and development of defensive responses of offspring.

Stress and the Emerging Roles of Chromatin Remodeling in Signal Integration and Stable Transmission of Reversible Phenotypes.

The influence of early life experience and degree of parental-infant attachment on emotional development in children and adolescents has been comprehensively studied. Structural and mechanistic insight into the biological foundation and maintenance of mammalian defensive systems (metabolic, immune, nervous and behavioral) is slowly advancing through the emerging field of developmental molecular (epi)genetics. Initial evidence revealed that differential nurture early in life generates stable differences in offspring hypothalamic-pituitary-adrenal (HPA) regulation, in part, through chromatin remodeling and changes in DNA methylation of specific genes expressed in the brain, revealing physical, biochemical and molecular paths for the epidemiological concept of gene-environment interactions. Herein, a primary molecular mechanism underpinning the early developmental programming and lifelong maintenance of defensive (emotional) responses in the offspring is the alteration of chromatin domains of specific genomic regions from a condensed state (heterochromatin) to a transcriptionally accessible state (euchromatin). Conversely, DNA methylation promotes the formation of heterochromatin, which is essential for gene silencing, genomic integrity and chromosome segregation. Therefore, inter-individual differences in chromatin modifications and DNA methylation marks hold great potential for assessing the impact of both early life experience and effectiveness of intervention programs-from guided psychosocial strategies focused on changing behavior to pharmacological treatments that target chromatin remodeling and DNA methylation enzymes to dietary approaches that alter cellular pools of metabolic intermediates and methyl donors to affect nutrient bioavailability and metabolism. In this review article, we discuss the potential molecular mechanism(s) of gene regulation associated with chromatin modeling and programming of endocrine (e.g., HPA and metabolic or cardiovascular) and behavioral (e.g., fearfulness, vigilance) responses to stress, including alterations in DNA methylation and the role of DNA repair machinery. From parental history (e.g., drugs, housing, illness, nutrition, socialization) to maternal-offspring exchanges of nutrition, microbiota, antibodies and stimulation, the nature of nurture provides not only mechanistic insight into how experiences propagate from external to internal variables, but also identifies a composite therapeutic target, chromatin modeling, for gestational/prenatal stress, adolescent anxiety/depression and adult-onset neuropsychiatric disease.

Effects of Paternal Predation Risk and Rearing Environment on Maternal Investment and Development of Defensive Responses in the Offspring.

Detecting past experiences with predators of a potential mate informs a female about prevailing ecological threats, in addition to stress-induced phenotypes that may be disseminated to offspring. We examined whether prior exposure of a male rat to a predator (cat) odor influences the attraction of a female toward a male, subsequent mother-infant interactions and the development of defensive (emotional) responses in the offspring. Females displayed less interest in males that had experienced predator odor. Mothers that reared young in larger, seminaturalistic housing provided more licking and grooming and active arched back-nursing behavior toward their offspring compared with dams housed in standard housing, although some effects interacted with paternal experience. Paternal predation risk and maternal rearing environment revealed sex-dependent differences in offspring wean weight, juvenile social interactions, and anxiety-like behavior in adolescence. Additionally, paternal predator experience and maternal housing independently affected variations in crf gene promoter acetylation and crf gene expression in response to an acute stressor in offspring. Our results show for the first time in mammals that variation among males in their predator encounters may contribute to stable behavioral variation among females in preference for mates and maternal care, even when the females are not directly exposed to predator threat. Furthermore, when offspring were exposed to the same threat experienced by the father, hypothalamic crf gene regulation was influenced by paternal olfactory experience and early housing. These results, together with our previous findings, suggest that paternal stress exposure and maternal rearing conditions can influence maternal behavior and the development of defensive responses in offspring.

An enhanced home cage modulates hypothalamic CRH-ir labeling in juvenile rats, with and without sub-threshold febrile convulsions.

Pre and postnatal environments can have a profound impact on offspring development. This is especially true when considering the origin of neurological diseases, including epilepsy, a relatively common and chronic neurological condition, affecting 1-2% of the population. Previously, we have used maternal stress and an enhanced home cage (EHC) in an effort to identify potential factors in the early environment that may increase the risk for experiencing seizures. First, pregnant Long-Evans rats were exposed to a predator stress (PS). Then, at birth, litters were divided into standard cage (SC) and EHC groups until postnatal Day 14 (PD14) when a model of febrile convulsions was used to determine convulsion susceptibility of the various groups. Twenty-four hours later, pup brains were processed for immunohistochemical detection of corticotrophic releasing hormone (CRH) in the paraventricular nucleus of the hypothalamus. Analysis of CRH immunoreactive (-ir) patterns revealed a buffering of CRH-ir in EHC reared offspring. Further, experiencing convulsions led to decreased CRH-ir. Our results support the concept that postnatal environmental influences affect neonatal programming and neurodevelopment of processes that could underlie seizure susceptibility, and that these effects can be modulated by EHC conditions.

Limbic system activation is affected by prenatal predator exposure and postnatal environmental enrichment and further moderated by dam and sex.

Epilepsy is a relatively common and chronic neurological condition, affecting 1-2% of the population. However, understanding of the underlying pathophysiology remains incomplete. To identify potential factors in the early environment that may increase the risk for experiencing seizures, maternal stress and environmental enrichment (EE) were utilized. Pregnant Long-Evans rats were exposed to an ethologically relevant predator stress (PS) and maternal glucocorticoid (GC) response was assessed across the exposure period. At birth, litters were divided into standard care (SC) and EE groups until postnatal day 14 (PD14) when a model of febrile convulsions was used to determine seizure susceptibility of the various groups. Pup brains were then processed for immunohistochemical detection of FosB from several structures in the limbic system as a measure of neuronal activation. Maternal PS-induced GC levels were elevated early in the exposure period, and pup birth weights, in both sexes, were lower in litters from dams exposed to PS. Seizure scores at PD14 were highly individualized and litter dependent, suggesting a dam-dependent and variable effect of controlled pre- and postnatal environmental factors. Further, analysis of FosB-immunoreactive (-ir) patterns revealed an activity dependent distribution, reflecting individual seizure susceptibility. EE had a varying effect on FosB-ir that was dependent on region. In the hippocampus FosB-ir levels were greater in the EE groups while extra-hippocampal regions showed lower levels of FosB-ir. Our results support the concept that pre- and postnatal environmental influences affect fetal programming and neurodevelopment of processes that could underlie seizure susceptibility, but that the magnitude of these effects appears to be dam- or litter-dependent.

Natural variation in gestational cortisol is associated with patterns of growth in marmoset monkeys (Callithrix geoffroyi).

High levels of prenatal cortisol have been previously reported to retard fetal growth. Although cortisol plays a pivotal role in prenatal maturation, heightened exposure to cortisol can result in lower body weights at birth, which have been shown to be associated with adult diseases like hypertension and cardiovascular disease. This study examines the relationship between natural variation in gestational cortisol and fetal and postnatal growth in marmoset monkeys. Urinary samples obtained during the mother's gestation were analyzed for cortisol. Marmoset body mass index (BMI) was measured from birth through 540 days in 30- or 60-day intervals. Multi-level modeling was used to test if marmoset growth over time was predicted by changes in gestational cortisol controlling for time, sex, litter, and litter size. The results show that offspring exposed to intra-uterine environments with elevated levels of cortisol had lower linear BMI rates of change shortly after birth than did offspring exposed to lower levels of cortisol, but exhibited a higher curvilinear growth rate during adolescence. Average daily change in gestational cortisol during the first trimester had a stronger relationship with postnatal growth than change during the third trimester. Higher exposure to cortisol during gestation does alter developmental trajectories, however there appears to be a catch-up period during later post-natal growth. These observations contribute to a larger discussion about the relationship of maternal glucocorticoids on offspring development and the possibility of an earlier vulnerable developmental window.