Nociceptin attenuates the escalation of oxycodone self-administration by normalizing CeA-GABA transmission in highly addicted rats.

Approximately 25% of patients who are prescribed opioids for chronic pain misuse them, and 5 to 10% develop an opioid use disorder. Although the neurobiological target of opioids is well known, the molecular mechanisms that are responsible for the development of addiction-like behaviors in some but not all individuals are poorly known. To address this issue, we used a unique outbred rat population (heterogeneous stock) that better models the behavioral and genetic diversity that is found in humans. We characterized individual differences in addiction-like behaviors using an addiction index that incorporates the key criteria of opioid use disorder: escalated intake, highly motivated responding, and hyperalgesia. Using in vitro electrophysiological recordings in the central nucleus of the amygdala (CeA), we found that rats with high addiction-like behaviors (HA) exhibited a significant increase in γ-aminobutyric acid (GABA) transmission compared with rats with low addiction-like behaviors (LA) and naive rats. The superfusion of CeA slices with nociceptin/orphanin FQ peptide (N/OFQ; 500 nM), an endogenous opioid-like peptide, normalized GABA transmission in HA rats. Intra-CeA levels of N/OFQ were lower in HA rats than in LA rats. Intra-CeA infusions of N/OFQ (1 μg per site) reversed the escalation of oxycodone self-administration in HA rats but not in LA rats. These results demonstrate that the downregulation of N/OFQ levels in the CeA may be responsible for hyper-GABAergic tone in the CeA that is observed in individuals who develop addiction-like behaviors. Based on these results, we hypothesize that small molecules that target the N/OFQ system might be useful for the treatment of opioid use disorder

Context and strain-dependent behavioral response to stress

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Comparative Microbiome Signatures and Short-Chain Fatty Acids in Mouse, Rat, Non-human Primate, and Human Feces

Gut microbiome plays a fundamental role in several aspects of host health and diseases. There has been an exponential surge in the use of animal models that can mimic different phenotypes of the human intestinal ecosystem. However, data on host species-specific signatures of gut microbiome and its metabolites like short-chain fatty acids (SCFAs; i.e., acetate, propionate, and butyrate) and lactate in these models and their similarities/differences from humans remain limited, due to high variability in protocols and analyses. Here, we analyze the fecal microbiota composition and the fecal levels of SCFAs and lactate in three of the most-widely used animal models, i.e., mice, rats, and non-human primates (NHPs) and compare them with human subjects, using data generated on a single platform with same protocols. The data show several species-specific similarities and differences in the gut microbiota and fecal organic acids between these species groups. Based on β-diversity, the gut microbiota in humans seems to be closer to NHPs than to mice and rats; however, among rodents, mice microbiota appears to be closer to humans than rats. The phylum-level analyses demonstrate higher Firmicutes–Bacteroidetes ratio in humans and NHPs vs. mice and rats. Human microbiota is dominated by Bacteroides followed by Ruminococcaceae and Clostridiales. Mouse gut is predominated by members of the family S24-7 followed by those from the order Clostridiales, whereas rats and NHPs have higher abundance of Prevotella compared with mice and humans. Also, fecal levels of lactate are higher in mice and rats vs. NHPs and humans, while acetate is highest in human feces. These data of host species-specific gut microbiota signatures in some of the most widely used animal models in context to the human microbiota might reflect disparities in host factors, e.g., diets, genetic origin, gender and age, and hence call for prospective studies investigating the features of gut microbiome in such animal models by controlling for these host elements

Sex-dependent associations between addiction-related behaviors and the microbiome in outbred rats.

BackgroundMultiple factors contribute to the etiology of addiction, including genetics, sex, and a number of addiction-related behavioral traits. One behavioral trait where individuals assign incentive salience to food stimuli ("sign-trackers", ST) are more impulsive compared to those that do not ("goal-trackers", GT), as well as more sensitive to drugs and drug stimuli. Furthermore, this GT/ST phenotype predicts differences in other behavioral measures. Recent studies have implicated the gut microbiota as a key regulator of brain and behavior, and have shown that many microbiota-associated changes occur in a sex-dependent manner. However, few studies have examined how the microbiome might influence addiction-related behaviors. To this end, we sought to determine if gut microbiome composition was correlated with addiction-related behaviors determined by the GT/ST phenotype.MethodsOutbred male (N=101) and female (N=101) heterogeneous stock rats underwent a series of behavioral tests measuring impulsivity, attention, reward-learning, incentive salience, and locomotor response. Cecal microbiome composition was estimated using 16S rRNA gene amplicon sequencing. Behavior and microbiome were characterized and correlated with behavioral phenotypes. Robust sex differences were observed in both behavior and microbiome; further analyses were conducted within sex using the pre-established goal/sign-tracking (GT/ST) phenotype and partial least squares differential analysis (PLS-DA) clustered behavioral phenotype.ResultsOverall microbiome composition was not associated to the GT/ST phenotype. However, microbial alpha diversity was significantly decreased in female STs. On the other hand, a measure of impulsivity had many significant correlations to microbiome in both males and females. Several measures of impulsivity were correlated with the genus Barnesiella in females. Female STs had notable correlations between microbiome and attentional deficient. In both males and females, many measures were correlated with the bacterial families Ruminocococcaceae and Lachnospiraceae.ConclusionsThese data demonstrate correlations between several addiction-related behaviors and the microbiome specific to sex

Identification of a Novel Gene for Diabetic Traits in Rats, Mice, and Humans

The genetic basis of type 2 diabetes remains incompletely defined despite the use of multiple genetic strategies. Multiparental populations such as heterogeneous stocks (HS) facilitate gene discovery by allowing fine mapping to only a few megabases, significantly decreasing the number of potential candidate genes compared to traditional mapping strategies. In the present work, we employed expression and sequence analysis in HS rats (Rattus norvegicus) to identify Tpcn2 as a likely causal gene underlying a 3.1-Mb locus for glucose and insulin levels. Global gene expression analysis on liver identified Tpcn2 as the only gene in the region that is differentially expressed between HS rats with glucose intolerance and those with normal glucose regulation. Tpcn2 also maps as a cis-regulating expression QTL and is negatively correlated with fasting glucose levels. We used founder sequence to identify variants within this region and assessed association between 18 variants and diabetic traits by conducting a mixed-model analysis, accounting for the complex family structure of the HS. We found that two variants were significantly associated with fasting glucose levels, including a nonsynonymous coding variant within Tpcn2. Studies in Tpcn2 knockout mice demonstrated a significant decrease in fasting glucose levels and insulin response to a glucose challenge relative to those in wild-type mice. Finally, we identified variants within Tpcn2 that are associated with fasting insulin in humans. These studies indicate that Tpcn2 is a likely causal gene that may play a role in human diabetes and demonstrate the utility of multiparental populations for positionally cloning genes within complex loci

Sept8/SEPTIN8 involvement in cellular structure and kidney damage is identified by genetic mapping and a novel human tubule hypoxic model.

Chronic kidney disease (CKD), which can ultimately progress to kidney failure, is influenced by genetics and the environment. Genes identified in human genome wide association studies (GWAS) explain only a small proportion of the heritable variation and lack functional validation, indicating the need for additional model systems. Outbred heterogeneous stock (HS) rats have been used for genetic fine-mapping of complex traits, but have not previously been used for CKD traits. We performed GWAS for urinary protein excretion (UPE) and CKD related serum biochemistries in 245 male HS rats. Quantitative trait loci (QTL) were identified using a linear mixed effect model that tested for association with imputed genotypes. Candidate genes were identified using bioinformatics tools and targeted RNAseq followed by testing in a novel in vitro model of human tubule, hypoxia-induced damage. We identified two QTL for UPE and five for serum biochemistries. Protein modeling identified a missense variant within Septin 8 (Sept8) as a candidate for UPE. Sept8/SEPTIN8 expression increased in HS rats with elevated UPE and tubulointerstitial injury and in the in vitro hypoxia model. SEPTIN8 is detected within proximal tubule cells in human kidney samples and localizes with acetyl-alpha tubulin in the culture system. After hypoxia, SEPTIN8 staining becomes diffuse and appears to relocalize with actin. These data suggest a role of SEPTIN8 in cellular organization and structure in response to environmental stress. This study demonstrates that integration of a rat genetic model with an environmentally induced tubule damage system identifies Sept8/SEPTIN8 and informs novel aspects of the complex gene by environmental interactions contributing to CKD risk

Reversing age: Dual species measurement of epigenetic age with a single clock

Young blood plasma is known to confer beneficial effects on various organs in mice. However, it was not known whether young plasma rejuvenates cells and tissues at the epigenetic level; whether it alters the epigenetic clock, which is a highly-accurate molecular biomarker of aging. To address this question, we developed and validated six different epigenetic clocks for rat tissues that are based on DNA methylation values derived from n=593 tissue samples. As indicated by their respective names, the rat pan-tissue clock can be applied to DNA methylation profiles from all rat tissues, while the rat brain-, liver-, and blood clocks apply to the corresponding tissue types. We also developed two epigenetic clocks that apply to both human and rat tissues by adding n=850 human tissue samples to the training data. We employed these six clocks to investigate the rejuvenation effects of a plasma fraction treatment in different rat tissues. The treatment more than halved the epigenetic ages of blood, heart, and liver tissue. A less pronounced, but statistically significant, rejuvenation effect could be observed in the hypothalamus. The treatment was accompanied by progressive improvement in the function of these organs as ascertained through numerous biochemical/physiological biomarkers and behavioral responses to assess cognitive functions. Cellular senescence, which is not associated with epigenetic aging, was also considerably reduced in vital organs. Overall, this study demonstrates that a plasma-derived treatment markedly reverses aging according to epigenetic clocks and benchmark biomarkers of aging.Fil: Horvath, Steve. University of California at Los Angeles; Estados UnidosFil: Singh, Kavita. NMIMS University; IndiaFil: Raj, Ken. Public Health England; Reino UnidoFil: Khairnar, Shraddha. NMIMS University; IndiaFil: Sanghav, Akshay. Nugenics Research Pvt Ltd; IndiaFil: Shrivastava, Agnivesh. Nugenics Research Pvt Ltd; IndiaFil: Zoller, Joseph A.. University of California at Los Angeles; Estados UnidosFil: Li, Caesar Z.. University of California at Los Angeles; Estados UnidosFil: Hereñú, Claudia Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Farmacología Experimental de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Instituto de Farmacología Experimental de Córdoba; ArgentinaFil: Canatelli Mallat, Martina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner"; ArgentinaFil: Lehmann, Marianne. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner"; ArgentinaFil: Solberg Woods, Leah C.. Wake Forest University School of Medicine; Estados UnidosFil: Garcia Martinez, Angel. University of Tennessee; Estados UnidosFil: Wang, Tengfei. University of Tennessee; Estados UnidosFil: Chiavellini, Priscila. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner"; ArgentinaFil: Levine, Andrew J.. University of California at Los Angeles; Estados UnidosFil: Chen, Hao. University of Tennessee; Estados UnidosFil: Goya, Rodolfo Gustavo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner"; ArgentinaFil: Katcher, Harold L.. Nugenics Research Pvt Ltd; Indi

Genetic Mapping of Multiple Metabolic Traits Identifies Novel Genes for Adiposity, Lipids and Insulin Secretory Capacity in Outbred Rats

Despite the successes of human genome-wide association studies, the causal genes underlying most metabolic traits remain unclear. We used outbred heterogeneous stock (HS) rats, coupled with expression data and mediation analysis, to identify quantitative trait loci (QTLs) and candidate gene mediators for adiposity, glucose tolerance, serum lipids, and other metabolic traits. Physiological traits were measured in 1519 male HS rats, with liver and adipose transcriptomes measured in over 410 rats. Genotypes were imputed from low coverage whole genome sequence. Linear mixed models were used to detect physiological and expression QTLs (pQTLs and eQTLs, respectively), employing both SNP- and haplotype-based models for pQTL mapping. Genes with cis-eQTLs that overlapped pQTLs were assessed as causal candidates through mediation analysis. We identified 14 SNP-based pQTLs and 19 haplotype-based pQTLs, of which 10 were in common. Using mediation, we identified the following genes as candidate mediators of pQTLs: Grk5 for a fat pad weight pQTL on Chr1, Krtcap3 for fat pad weight and serum lipids pQTLs on Chr6, Ilrun for a fat pad weight pQTL on Chr20 and Rfx6 for a whole pancreatic insulin content pQTL on Chr20. Furthermore, we verified Grk5 and Ktrcap3 using gene knock-down/out models, thereby shedding light on novel regulators of obesity

Sex-dependent associations between addiction-related behaviors and the microbiome in outbred rats

Background: Multiple factors contribute to the etiology of addiction, including genetics, sex, and a number of addiction-related behavioral traits. One behavioral trait where individuals assign incentive salience to food stimuli (â sign-trackersâ , ST) are more impulsive compared to those that do not (â goal-trackersâ , GT), as well as more sensitive to drugs and drug stimuli. Furthermore, this GT/ST phenotype predicts differences in other behavioral measures. Recent studies have implicated the gut microbiota as a key regulator of brain and behavior, and have shown that many microbiota-associated changes occur in a sex-dependent manner. However, few studies have examined how the microbiome might influence addiction-related behaviors. To this end, we sought to determine if gut microbiome composition was correlated with addiction-related behaviors determined by the GT/ST phenotype. Methods: Outbred male (N=101) and female (N=101) heterogeneous stock rats underwent a series of behavioral tests measuring impulsivity, attention, reward-learning, incentive salience, and locomotor response. Cecal microbiome composition was estimated using 16S rRNA gene amplicon sequencing. Behavior and microbiome were characterized and correlated with behavioral phenotypes. Robust sex differences were observed in both behavior and microbiome; further analyses were conducted within sex using the pre-established goal/sign-tracking (GT/ST) phenotype and partial least squares differential analysis (PLS-DA) clustered behavioral phenotype. Results: Overall microbiome composition was not associated to the GT/ST phenotype. However, microbial alpha diversity was significantly decreased in female STs. On the other hand, a measure of impulsivity had many significant correlations to microbiome in both males and females. Several measures of impulsivity were correlated with the genus Barnesiella in females. Female STs had notable correlations between microbiome and attentional deficient. In both males and females, many measures were correlated with the bacterial families Ruminocococcaceae and Lachnospiraceae. Conclusions: These data demonstrate correlations between several addiction-related behaviors and the microbiome specific to sex