Because inbred rat strains are widely used as laboratory models, knowledge of phenotypic and genetic variations between strains will be useful to obtain insight into the relationship between different strains.

Over 500 strains of inbred brown rats (Rattus norvegicus) have been developed for use as a biomedical model organism. Most of these inbred lines were derived from the colony established at the Wistar Institute in 1906 or its descendants following worldwide distribution to research and breeding centers. The geographic source of the animals that founded the Wistar colony has been lost to history; thus, we compared 25 inbred rat strains to 326 wild rats from a global diversity dataset at 32 k SNPs, and 47 mitochondrial genomes to identify the source populations. We analyzed nuclear genomic data using principal component analyses and co-ancestry heat maps, and mitogenomes using phylogenetic trees and networks. In the nuclear genome, inbred rats clustered together indicating a single geographic origin for the strains studied and showed admixed ancestral variation with wild rats in eastern Asia and western North America. The Sprague Dawley derived, Wistar derived, and Brown Norway strains each had mitogenomes from different clades which diverged between 13 and 139 kya. Thus, we posit that rats originally collected for captive breeding had high mitochondrial diversity that became fixed through genetic drift and/or artificial selection. Our results show that these important medical models share common genomic ancestry from a few source populations, and opportunities exist to create new strains with diverse genomic backgrounds to provide novel insight into the genomic basis of disease phenotypes.

We tested the hypotheses that: (1) long-term facilitation (LTF) following acute intermittent hypoxia (AIH) varies among three inbred rat strains: Fischer 344 (F344), Brown Norway (BN) and Lewis rats and (2) ventral cervical spinal levels of genes important for phrenic LTF (pLTF) vary in association with pLTF magnitude. Lewis and F344, but not BN rats exhibited significant increases in phrenic and hypoglossal burst amplitude 60min post-AIH that were significantly greater than control experiments without AIH, indicating strain differences in phrenic (98%, 56% and 20%, respectively) and hypoglossal LTF (66%, 77% and 5%, respectively). Ventral spinal 5-HT(2A) receptor mRNA and protein levels were higher in F344 and Lewis versus BN, suggesting that higher 5-HT(2A) receptor levels are associated with greater pLTF. More complex relationships were found for 5-HT(7), BDNF and TrkB mRNA. BN had higher 5-HT(7) and TrkB mRNA versus F344; BN and Lewis had higher BDNF mRNA levels versus F344. Genetic variations in serotonergic function may underlie strain differences in AIH-induced pLTF.

In an alignment of closely related genomic sequences, the existence of discordant mutation sites, which do not reflect the phylogenetic relationship of the genomes, is often observed. Although these discordant mutation sites are thought to have emerged by ancestral polymorphism or gene flow, their frequency and distribution in the genome have not yet been analyzed in detail. Using the genome sequences of all protein coding genes of 25 inbred rat strains, we analyzed the frequency and genome-wide distribution of the discordant mutation sites. From the comparison of different substrains, it was found that these loci are not substrain specific, but are common among different groups of substrains, suggesting that the discordant sites might have mainly emerged through ancestral polymorphism. It was also revealed that the discordant sites are not uniformly distributed along chromosomes, but are concentrated at certain genomic loci, such as RT1, major histocompatibility complex of rats, and olfactory receptors, indicating that genes known to be highly polymorphic tend to have more discordant sites. Our results also showed that loci with a high density of discordant sites are also rich in heterozygous variants, even though these are inbred strains.

To test the impact of genetic heterogeneity on cis- and trans-mediated mechanisms of gene expression regulation, we profiled the transcriptome of adipose tissue in 20 inbred congenic strains derived from diabetic Goto-Kakizaki (GK) rats and Brown-Norway (BN) controls, which contain well-defined blocks (1-183 Mb) of genetic polymorphisms, and in 123 genetically heterogeneous rats of an (GK × BN)F2 offspring. Within each congenic we identified 73-1351 differentially expressed genes (DEGs), only 7.7% of which mapped within the congenic blocks, and which may be regulated in cis The remainder localized outside the blocks, and therefore must be regulated in trans Most trans-regulated genes exhibited approximately twofold expression changes, consistent with monoallelic expression. Altered biological pathways were replicated between congenic strains sharing blocks of genetic polymorphisms, but polymorphisms at different loci also had redundant effects on transcription of common distant genes and pathways. We mapped 2735 expression quantitative trait loci (eQTL) in the F2 cross, including 26% predominantly cis-regulated genes, which validated DEGs in congenic strains. A hotspot of >300 eQTL in a 10 cM region of chromosome 1 was enriched in DEGs in a congenic strain. However, many DEGs among GK, BN and congenic strains did not replicate as eQTL in F2 hybrids, demonstrating distinct mechanisms of gene expression when alleles segregate in an outbred population or are fixed homozygous across the entire genome or in short genomic regions. Our analysis provides conceptual advances in our understanding of the complex architecture of genome expression and pathway regulation, and suggests a prominent impact of epistasis and monoallelic expression on gene transcription.

Post-translational protein modifications such as acetylation have significant regulatory roles in metabolic processes, but their relationship to both variation in gene expression and DNA sequence is unclear. We address this question in the Goto-Kakizaki (GK) rat inbred strain, a model of polygenic type 2 diabetes. Expression of the NAD-dependent deacetylase Sirtuin-3 is down-regulated in GK rats compared to normoglycemic Brown Norway (BN) rats. We show first that a promoter SNP causes down-regulation of Sirtuin-3 expression in GK rats. We then use mass-spectrometry to identify proteome-wide differential lysine acetylation of putative Sirtuin-3 protein targets in livers of GK and BN rats. These include many proteins in pathways connected to diabetes and metabolic syndrome. We finally sequence GK and BN liver transcriptomes and find that mRNA expression of these targets does not differ significantly between GK and BN rats, in contrast to other components of the same pathways. We conclude that physiological differences between GK and BN rats are mediated by a combination of differential protein acetylation and gene transcription and that genetic variation can modulate acetylation independently of expression.

When using rats in pain research, strain-related differences in outcomes of tests for pain and nociception are acknowledged. However, very little is known about the specific characteristics of these strain differences. In this study four phylogenetically distant inbred rat strains, i.e. Wistar Kyoto (WKY), Fawn Hooded (FH), Brown Norway (BN) and Lewis (LE), were investigated in different tests related to pain and nociception. During Pavlovian fear conditioning, the LE and WKY showed a significantly longer duration of freezing behaviour than the FH and BN. Additionally, differences in c-Fos expression in subregions of the prefrontal cortex and amygdala between rat strains during retrieval and expression of conditioned fear were found. For example, the BN did not show recruitment of the basolateral amygdala, whereas the WKY, FH and LE did. During the hot plate test, the WKY and LE showed a lower thermal threshold compared to the BN and FH. In a follow-up experiment, the two most contrasting strains regarding behaviour during the hot plate test and Pavlovian fear conditioning (i.e. FH and WKY) were selected and the hot plate test, Von Frey test and somatosensory-evoked potential (SEP) were investigated. During the Von Frey test, the WKY showed a lower mechanical threshold compared to the FH. When measuring the SEP, the FH appeared to be less reactive to increasing stimulus intensities when considering both peak amplitudes and latencies. Altogether, the combined results indicate various differences between rat strains in Pavlovian fear conditioning, nociception related behaviours and nociceptive processing. These findings demonstrate the necessity of using multiple rat strains when using tests including noxious stimuli and suggest that the choice of rat strains should be considered. When selecting a strain for a particular study it should be considered how this strain behaves during the tests used in that study.

Male animals with more conspicuous visual and acoustic signals increase their mating success, but also increase the risk of being attacked by eavesdropping predators. In rodents, males with richer sex pheromones often have higher attractiveness to females, but whether or not the males are also at higher predation risk is poorly known. Here, we used 2 laboratory inbred strains of the rat Rattus norvegicus, Brown Norway (BN) and Lewis (LEW), and wild-captured rats as odor donors to assess the relationship between the pheromone levels in male rats and attractiveness to domestic cats Felis catus. LEW rats had significantly higher levels of male pheromones (e.g., 4-heptanone, 2-heptanone, and 9-hydroxy-2-nonanone) than BN rats. Simultaneously, wild-captured male rats were selectively assigned to 2 groups (HIGH or LOW) based on pheromone content as determined by gas chromatography-mass spectrometry (GC-MS). Binary choice tests were carried out during the night in the test room. We found that cats spent more time investigating male bedding and urine of LEW rats than the counterpart of BN rats. Likewise, cats also preferred bedding and urine odor of the HIGH wild rats compared with the counterparts of LOW wild rats. Adding synthetic analogs of the 3 pheromone ketones into the urine of either BN rats or LOW wild rats significantly increased their attractiveness to cats. Our data suggest that the rats with exaggerated male pheromones more strongly attracted predators and thus as a consequence may suffer from elevated predation risk.

We have used a genetical genomic approach, in conjunction with phenotypic analysis of alcohol consumption, to identify candidate genes that predispose to varying levels of alcohol intake by HXB/BXH recombinant inbred rat strains. In addition, in two populations of humans, we assessed genetic polymorphisms associated with alcohol consumption using a custom genotyping array for 1,350 single nucleotide polymorphisms (SNPs). Our goal was to ascertain whether our approach, which relies on statistical and informatics techniques, and non-human animal models of alcohol drinking behavior, could inform interpretation of genetic association studies with human populations.

This study examines the immunological responses in rats following inhalation to titanium dioxide nanoparticles (TiO2 NPs), in naïve rats and in rats with induced allergic airway disease. The responses of two different inbred rat strains were compared: the Dark Aguoti (DA), susceptible to chronic inflammatory disorders, and the Brown Norwegian (BN), susceptible to atopic allergic inflammation. Naïve rats were exposed to an aerosol of TiO2 NPs once daily for 10 days. Another subset of rats was sensitized to the allergen ovalbumin (OVA) in order to induce airway inflammation. These sensitized rats were exposed to TiO2 NPs before and during the allergen challenge. Naïve rats exposed to TiO2 NPs developed an increase of neutrophils and lymphocytes in both rat strains. Airway hyperreactivity and production of inflammatory mediators typical of a T helper 1 type immune response were significantly increased, only in DA rats. Sensitization of the rats induced a prominent OVA-specific-IgE and IgG response in the BN rat while DA rats only showed an increased IgG response. Sensitized rats of both strains developed airway eosinophilia following allergen challenge, which declined upon exposure to TiO2 NPs. The level of neutrophils and lymphocytes increased upon exposure to TiO2 NPs in the airways of DA rats but remained unchanged in the airways of BN rats. In conclusion, the responses to TiO2 NPs were strain-dependent, indicating that genetics play a role in both immune and airway reactivity. DA rats were found to be higher responder compared to BN rats, both when it comes to responses in naïve and sensitized rats. The impact of genetically determined factors influencing the inflammatory reactions pinpoints the complexity of assessing health risks associated with nanoparticle exposures.

Genetic variation in the regulatory region of the human serotonin transporter gene (SLC6A4) has been shown to affect brain functionality and personality. However, large heterogeneity in its biological effects is observed, which is at least partially due to genetic modifiers. To gain insight into serotonin transporter (SERT)-specific genetic modifiers, we studied an intercross between the Wistar SERT-/- rat and the behaviorally and genetically divergent Brown Norway rat, and performed a QTL analysis.

Finding genetic variants that contribute to phenotypic variation is one of the main challenges of modern genetics. We used an outbred population of rats (Heterogeneous Stock, HS) in a combined sequence-based and genetic mapping analysis to identify sequence variants and genes contributing to complex traits of biomedical relevance. Here we describe the sequences of the eight inbred progenitors of the HS and the variants that segregate between them. We report the genotyping of 1,407 HS rats, and the collection from 2,006 rats of 195 phenotypic measures that are relevant to models of anxiety, type 2 diabetes, hypertension and osteoporosis. We make available haplotype dosages for the 1,407 genotyped rats, since genetic mapping in the HS is best carried out by reconstructing each HS chromosome as a mosaic of the progenitor genomes. Finally, we have deposited an R object that makes it easy to incorporate our sequence data into any genetic study of HS rats. Our genetic data are available for both Rnor3.4 and Rnor5.0 rat assemblies.

Microgravity and sleep loss lead to cognitive and learning deficits. These behavioral alterations are likely to be associated with cytomorphological changes and loss of neurons. To understand the phenomenon, we exposed rats (225-275 g) to 14 days simulated microgravity (SMg) and compared its effects on CA1 hippocampal neuronal plasticity, with that of normal cage control rats. We observed that the mean area, perimeter, synaptic cleft, and length of active zone of CA1 hippocampal neurons significantly decreased while dendritic arborization and number of spines significantly increased in SMg group as compared with controls. The mean thickness of the postsynaptic density and total dendritic length remained unaltered. The changes may be a compensatory effect induced by exposure to microgravity; however, the effects may be transient or permanent, which need further study. These findings may be useful for designing effective prevention for those, including the astronauts, exposed to microgravity. Further, subject to confirmation, we propose that SMg exposure might be useful for recovery of stroke patients.

Although laboratory rats are often considered classic nonseasonal breeders, peripubertal rats of two inbred strains, F344 and BN, have both reproductive and nonreproductive responses to short photoperiods. Unmanipulated adult rats have not been reported to have robust responses to short photoperiod alone, although several treatments can induce photoperiodic responses in adults. In this study, we tested the hypotheses that unmanipulated F344 rats retain responses to short photoperiod as adults and that they have the necessary elements for an endogenous circannual rhythm of sensitivity to short photoperiod.

The Long-Evans Agouti (LEA/Tohm) rat has recently been established as a new rat model of type 2 diabetes. The onset of diabetes mellitus was observed only in male LEA/Tohm rats; however, urinary glucose appeared before the onset of diabetes. To clarify the genetic basis of urinary glucose, we performed genetic linkage analysis using (BN × LEA) F2 intercross progeny. A recessively acting locus responsible for urinary glucose excretion (ugl) was mapped to a 7.9 Mb region of chromosome 10, which contains the cystinosin (Ctns) gene. The Ctns gene encodes the cystine transporter, which transports cystine out of the lysosome and is responsible for nephropathic cystinosis in humans. Sequence analysis identified a 13-bp deletion in the Ctns gene, leading to a truncated and loss-of-function protein, which cause cystine accumulation in various tissues. We also developed a novel congenic rat strain harboring the Ctnsugl mutation on the F344 genetic background. Phenotypic analysis of F344-Ctnsugl rats indicated that the incidence of urinary glucose was 100% in both males and females at around 40 weeks of age, and marked cystine accumulation was observed in the tissues, as well as remarkable renal lesions and cystine crystals in the lysosomes of the renal cortex. Furthermore, treatment with cysteamine depleted the cystine contents in F344-Ctnsugl rat embryonic fibroblasts. These results indicated that the F344-Ctnsugl rat provides a novel rat model of cystinosis, which allows not only a better understanding of the pathogenesis and pathophysiology of cystinosis but will also contribute to the development of new therapies.

DA (D-blood group of Palm and Agouti, also known as Dark Agouti) and F344 (Fischer) are two inbred rat strains with differences in several phenotypes, including susceptibility to autoimmune disease models and inflammatory responses. While these strains have been extensively studied, little information is available about the DA and F344 genomes, as only the Brown Norway (BN) and spontaneously hypertensive rat strains have been sequenced to date. Here we report the sequencing of the DA and F344 genomes using next-generation Illumina paired-end read technology and the first de novo assembly of a rat genome. DA and F344 were sequenced with an average depth of 32-fold, covered 98.9% of the BN reference genome, and included 97.97% of known rat ESTs. New sequences could be assigned to 59 million positions with previously unknown data in the BN reference genome. Differences between DA, F344, and BN included 19 million positions in novel scaffolds, 4.09 million single nucleotide polymorphisms (SNPs) (including 1.37 million new SNPs), 458,224 short insertions and deletions, and 58,174 structural variants. Genetic differences between DA, F344, and BN, including high-impact SNPs and short insertions and deletions affecting >2500 genes, are likely to account for most of the phenotypic variation between these strains. The new DA and F344 genome sequencing data should facilitate gene discovery efforts in rat models of human disease.

Both social environment and genetic factors are critical for smoking initiation and nicotine addiction. We reported that rats developed conditioned flavor (i.e., taste and odor) aversion to intravenously self-administered (IVSA) nicotine, and that social learning promoted nicotine IVSA with flavor cues. We thus tested the hypothesis that socially acquired nicotine IVSA is a heritable trait by using female rats of six inbred strains and six F1 hybrids. Each strain was tested for 10 daily IVSA sessions. We found that the intake of nicotine (15 and 30 μg/kg/inf) varied among these strains by 33.7-56.6-fold. The heritability of nicotine intake was estimated to be 0.54-0.65. Further, there was a strong correlation in nicotine intake (R2 = 0.85, p < 0.0001) between the two nicotine doses. Another cohort of rats was given three daily IVSA sessions followed by five sessions that tested conditioned flavor aversion. Nicotine intake was highly correlated with the extinction of the conditioned aversion (R2 = 0.58, p < 0.005). These data showed that nicotine intake in the socially acquired nicotine self-administration model is controlled by genetic factors and that the role of social learning is likely in facilitating the extinction of conditioned aversive response to nicotine.

We discovered a new cataract mutation, kfrs4, in the Kyoto Fancy Rat Stock (KFRS) background. Within 1 month of birth, all kfrs4/kfrs4 homozygotes developed cataracts, with severe opacity in the nuclei of the lens. In contrast, no opacity was observed in the kfrs4/+ heterozygotes. We continued to observe these rats until they reached 1 year of age and found that cataractogenesis did not occur in kfrs4/+ rats. To define the histological defects in the lenses of kfrs4 rats, sections of the eyes of these rats were prepared. Although the lenses of kfrs4/kfrs4 homozygotes showed severely disorganised fibres and vacuolation, the lenses of kfrs4/+ heterozygotes appeared normal and similar to those of wild-type rats. We used positional cloning to identify the kfrs4 mutation. The mutation was mapped to an approximately 9.7-Mb region on chromosome 7, which contains the Mip gene. This gene is responsible for a dominant form of cataract in humans and mice. Sequence analysis of the mutant-derived Mip gene identified a 5-bp insertion. This insertion is predicted to inactivate the MIP protein, as it produces a frameshift that results in the synthesis of 6 novel amino acid residues and a truncated protein that lacks 136 amino acids in the C-terminal region, and no MIP immunoreactivity was observed in the lens fibre cells of kfrs4/kfrs4 homozygous rats using an antibody that recognises the C- and N-terminus of MIP. In addition, the kfrs4/+ heterozygotes showed reduced expression of Mip mRNA and MIP protein and the kfrs4/kfrs4 homozygotes showed no expression in the lens. These results indicate that the kfrs4 mutation conveys a loss-of-function, which leads to functional inactivation though the degradation of Mip mRNA by an mRNA decay mechanism. Therefore, the kfrs4 rat represents the first characterised rat model with a recessive mutation in the Mip gene.

Substantia nigra (SN) is rich in dopamine (DA)-ergic and GABA-ergic neurons, which project to and receive inputs from locus coeruleus (LC) and pedunculo-pontine tegmentum (PPT) possessing REM-OFF and REM-ON neurons, respectively. Loss of DA-ergic neurons and disturbed REM sleep (REMS) are associated with Parkinson's disease, depression and REMS behavior disorder. GABA-ergic projections from SN act pre-synaptically on the noradrenaline (NA)-ergic terminals coming from the LC-REM-OFF neurons onto the REM-ON neurons in PPT and play a critical role in initiating REMS. However, it was unknown how SN neurons get activated and whether the SN-DA-ergic neurons interact with the SN-GABA-ergic neurons for REMS regulation. In freely moving chronically prepared rats, neurons in SN (bilateral) were stimulated by local microinjection of Glutamate (Glut), sparing the fibers of passage, in the presence and absence of i.p. Haloperidol (Hal, DA-antagonist). In other sets, either Hal or Bicuculine (Bic, GABA-antagonist) alone or simultaneously was microinjected bilaterally into the SN and the effects on sleep-wakefulness were recorded. We observed that Glut in SN significantly increased REMS, which was prevented by Hal. REMS was decreased and increased by Hal and Bic, respectively; while their co-injection neutralized (ineffective) the individual effects. Combining these findings with previous reports suggest that the SN-DA-ergic neurons act on the SN-GABA-ergic to regulate REMS. The results advance our understanding of the neuro-anatomo-chemical connections and pharmaco-physiological regulation of REMS in health and diseases.

Elevated plant sterol accumulation has been reported in the spontaneously hypertensive rat (SHR), the stroke-prone spontaneously hypertensive rat (SHRSP) and the Wistar-Kyoto (WKY) rat. Additionally, a blood pressure quantitative trait locus (QTL) has been mapped to rat chromosome 6 in a New Zealand genetically hypertensive rat strain (GH rat). ABCG5 and ABCG8 (encoding sterolin-1 and sterolin-2 respectively) have been shown to be responsible for causing sitosterolemia in humans. These genes are organized in a head-to-head configuration at the STSL locus on human chromosome 2p21.