The Quinpirole Hypolocomotive Effects are Strain and Route of Administration Dependent in SHR and SLA16 Isogenic Rats.

The SHR and SLA16 inbred strains present behavioral differences in anxiety/emotionality that could be under the influence of dopaminergic neurotransmission. In order to investigate the role of D2 receptors in modulating such differences, an agonist (quinpirole) and an antagonist (haloperidol) of this receptor were administered, either via systemic injection (IP), or microinjected into the ventral area of the hippocampus (vHIP). Quinpirole and haloperidol IP decreased locomotor activity, only in SLA16 rats in the open-field (OF), and in both strains in the elevated plus-maze (EPM). Quinpirole also increased the preference for the aversive areas of the EPM. Quinpirole vHIP decreased locomotor activity in both strains. Haloperidol vHIP did not elicit behavioural changes and no differences in the levels of D2 receptors and of dopamine transporter in the hippocampus were found. Results indicate that systemic activation/blocking of D2 receptors caused a strain-dependent hypolocomotion, whereas activation of D2 receptors in the vHIP, but not D2 receptor antagonism, regardless of dose, decreased general locomotor activity in the two strains. Therefore, we suggest that genomic differences in the chromosome 4 can influence the locomotor activity regulated by the D2 dopaminergic receptor, especially in the vHIP.

Genetic Relatedness of WNIN and WNIN/Ob with Major Rat Strains in Biomedical Research.

WNIN (Wistar/NIN) is an inbred rat strain maintained at National Institute of Nutrition (NIN) for more than 90 years, and WNIN/Ob is an obese mutant originated from it. To determine their genetic relatedness with major rat strains in biomedical research, they were genotyped at various marker loci. The recently identified markers for albino and hooded mutations which clustered all the known albino rats into a single lineage also included WNIN and WNIN/Ob rats. Genotyping using microsatellite DNA markers and phylogenetic analysis with 49 different rat strains suggested that WNIN shares a common ancestor with many Wistar originated strains. Fst estimates and Fischer's exact test suggest that WNIN rats differed significantly from all other strains tested. WNIN/Ob though shows hyper-leptinemia, like Zucker fatty rat, did not share the Zucker fatty rat mutation. The above analyses suggest WNIN as a highly differentiated rat strain and WNIN/Ob a novel obese mutant evolved from it.

The transmembrane protein meckelin (MKS3) is mutated in Meckel-Gruber syndrome and the wpk rat.

Meckel-Gruber syndrome is a severe autosomal, recessively inherited disorder characterized by bilateral renal cystic dysplasia, developmental defects of the central nervous system (most commonly occipital encephalocele), hepatic ductal dysplasia and cysts and polydactyly. MKS is genetically heterogeneous, with three loci mapped: MKS1, 17q21-24 (ref. 4); MKS2, 11q13 (ref. 5) and MKS3 (ref. 6). We have refined MKS3 mapping to a 12.67-Mb interval (8q21.13-q22.1) that is syntenic to the Wpk locus in rat, which is a model with polycystic kidney disease, agenesis of the corpus callosum and hydrocephalus. Positional cloning of the Wpk gene suggested a MKS3 candidate gene, TMEM67, for which we identified pathogenic mutations for five MKS3-linked consanguineous families. MKS3 is a previously uncharacterized, evolutionarily conserved gene that is expressed at moderate levels in fetal brain, liver and kidney but has widespread, low levels of expression. It encodes a 995-amino acid seven-transmembrane receptor protein of unknown function that we have called meckelin.

Modelling the genetic contribution to mental illness: a timely end for the psychiatric rodent?

Rodent models are a key factor in the process of translating psychiatric genetics and genomics findings, allowing us to shed light on how risk-genes confer changes in neurobiology by merging different types of data across fields, from behavioural neuroscience to the burgeoning omics (e.g. genomics, epigenomics, proteomics, etc.). Moreover, they also provide an indispensable first step for drug discovery. However, recent evidence from both clinical and genetic studies highlights possible limitations in the current methods for classifying psychiatric illness, as both symptomology and underlying genetic risk are found to increasingly overlap across disorder diagnoses. Meanwhile, integration of data from animal models across disorders is currently limited. Here, we argue that behavioural neuroscience is in danger of missing informative data because of the practice of trying to 'diagnose' an animal model with a psychiatric illness. What is needed is a shift in emphasis, from seeking to ally an animal model to a specific disorder, to one focused on a more systematic assessment of the neurobiological and behavioural outcomes of any given genetic or environmental manipulation.

Generation of leptin-deficient Lepmkyo/Lepmkyo rats and identification of leptin-responsive genes in the liver.

Leptin is one of the key molecules in maintaining energy homeostasis. Although genetically leptin-deficient Lep(ob)/Lep(ob) mice have greatly contributed to elucidating leptin physiology, the use of more than one species can improve the accuracy of analysis results. Using the N-ethyl-N-nitrosourea mutagenesis method, we generated a leptin-deficient Lep(mkyo)/Lep(mkyo) rat that had a nonsense mutation (Q92X) in leptin gene. Lep(mkyo)/Lep(mkyo) rats showed obese phenotypes including severe fatty liver, which were comparable to Lep(ob)/Lep(ob) mice. To identify genes that respond to leptin in the liver, we performed microarray analysis with Lep(mkyo)/Lep(mkyo) rats and Lep(ob)/Lep(ob) mice. We sorted out genes whose expression levels in the liver of Lep(mkyo)/Lep(mkyo) rats were changed from wild-type (WT) rats and were reversed toward WT rats by leptin administration. In this analysis, livers were sampled for 6 h, a relatively short time after leptin administration to avoid the secondary effect of metabolic changes such as improvement of fatty liver. We did the same procedure in Lep(ob)/Lep(ob) mice and selected genes whose expression patterns were common in rat and mouse. We verified their gene expressions by real-time quantitative PCR. Finally, we identified eight genes that primarily respond to leptin in the liver commonly in rat and mouse. These genes might be important for the effect of leptin in the liver.

Chromosomal mapping of genetic loci associated with non-insulin dependent diabetes in the GK rat.

Goto-Kakizaki (GK) rats are a well characterized model for non-insulin dependent diabetes mellitus (NIDDM). We have used a combination of physiological and genetic studies to identify quantitative trait loci (QTLs) responsible for the control of glucose homeostasis and insulin secretion in a F2 cohort bred from spontaneously diabetic GK rats. The genetic dissection of NIDDM allowed us to map up to six independently segregating loci predisposing to hyperglycaemia, glucose intolerance or altered insulin secretion, and a seventh locus implicated in body weight. QTLs implicated in glucose tolerance and adiposity map to the same region of rat chromosome 1, and may indicate the influence of a single locus. Our study demonstrates that distinct combinations of genetic loci are responsible for different physiological characteristics associated with the diabetic phenotype in the GK rat, and it constitutes an important step for directing the search for the genetic factors involved in human NIDDM.

Renal disease susceptibility and hypertension are under independent genetic control in the fawn-hooded rat.

Hypertension, diabetes and hyperlipidemia are risk factors for life-threatening complications such as end-stage renal disease, coronary artery disease and stroke. Why some patients develop complications is unclear, but only susceptibility genes may be involved. To test this notion, we studied crosses involving the fawn-hooded rat, an animal model of hypertension that develops chronic renal failure. Here, we report the localization of two genes, Rf-1 and Rf-2, responsible for about half of the genetic variation in key indices of renal impairment. In addition, we localize a gene, Bpfh-1, responsible for about 26% of the genetic variation in blood pressure. Rf-1 strongly affects the risk of renal impairment, but has no significant effect on blood pressure. Our results show that susceptibility to a complication of hypertension is under at least partially independent genetic control from susceptibility to hypertension itself.

Genetic analysis of non-insulin dependent diabetes mellitus in the GK rat.

Non-insulin dependent diabetes mellitus (NIDDM) is a major public health problem, but its aetiology remains poorly understood. We have performed a comprehensive study of the genetic basis of diabetes in the Goto-Kakizaki (GK) rat, the most widely used animal model of non-obese NIDDM. The genetic dissection of NIDDM using this model has allowed us to map three independent loci involved in the disease. In addition, we identify a major factor affecting body weight, but not glucose tolerance, on chromosome 7 and map a further 10 regions that are suggestive for linkage. We conclude that NIDDM is polygenic and fasting hyperglycaemia and postprandial hyperglycaemia clearly have distinct genetic bases.

Deficient sensorimotor gating induced by selective breeding in rats is improved by entopeduncular nucleus lesions.

Deficient prepulse inhibition (PPI) of startle reflects disturbed sensorimotor gating found in certain neuropsychiatric disorders, such as Tourette's syndrome, ADHD, Huntington's and schizophrenia. We here tested, whether lesions of the entopeduncular nucleus (EPN) would improve a PPI-deficit induced by selective breeding. Rats with breeding induced high and low expression of PPI were stereotaxically microinjected with ibotenate (0.2 microg in 0.3 microl phosphate buffered saline) or vehicle into the EPN and two weeks later tested for PPI of the acoustic startle response (ASR) and motor activity. Lesions of the EPN counteracted the breeding-induced PPI-deficit and reduced ASR in the PPI low group without affecting their motor activity. In the PPI high group EPN lesions did not affect PPI, ASR, and motor activity. This work indicates an important role of the EPN in the modulation of sensorimotor gating. Additionally, PPI low rats may provide a non-pharmacological model that can be used to develop new therapeutic strategies for neuropsychiatric disorders.

Arginine vasopressin gene regulation in the homozygous Brattleboro rat.

The Brattleboro rat, which has an autosomally recessive form of diabetes insipidus, has been reported to have a marked defect in the regulation of arginine vasopressin (AVP) gene expression. However, it is not known whether this is a primary genetic defect or occurs secondary to the urinary water losses which occur in the absence of circulating AVP in the Brattleboro rat. This present study was therefore undertaken to study AVP gene regulation in the Brattleboro rat after chronic AVP treatment by osmotic minipump for 2 wk. In Brattleboro rats without AVP treatment, neither urinary osmolality (Uosm) nor hypothalamic AVP mRNA was significantly changed after 24 h of fluid deprivation (Uosm, 413 +/- 33 to 588 +/- 44, NS; AVP mRNA, 39.33 +/- 2.95 to 46.39 +/- 2.71 pg/micrograms total RNA, NS). In contrast, when Brattleboro rats were treated with AVP for 2 wk, the regulation of AVP gene occurred in response to 24 h of fluid deprivation. In these studies, hypothalamic AVP mRNA was significantly increased compared with the Brattleboro rats still receiving AVP with free access of water (28.9 +/- 3.5 vs. 65.0 +/- 3.3 pg/micrograms total RNA, P less than 0.001). Further studies in Long-Evans rats demonstrate a similar response to a comparable degree of fluid deprivation as Uosm and AVP mRNA were significantly increased after 72 h of fluid deprivation (Uosm, 1,505 +/- 186 to 5,460 +/- 560 mosmol/kg, P less than 0.001; AVP mRNA, 31.7 +/- 3.9 to 77.5 +/- 4.6 pg/micrograms total RNA, P less than 0.001). These results indicate that AVP-replaced homozygous Brattleboro rats can regulate AVP gene expression normally in response to fluid deprivation. This finding indicates that the defect in AVP gene regulation in the Brattleboro rat not receiving AVP replacement is a secondary phenomenon rather than a primary genetic defect.

Downregulation of Ke 6, a novel gene encoded within the major histocompatibility complex, in murine polycystic kidney disease.

Polycystic kidney disease (PKD) is characterized by progressive enlargement of the kidneys due to numerous expanding cysts ultimately leading to renal failure. We have identified a gene, Ke 6, located within the H-2K/tw5 region on mouse chromosome 17, which is downregulated in two distinct murine models of heritable PKD. Ke 6 is a member of the short-chain alcohol dehydrogenase family and possess remarkable amino acid sequence conservation with several bacterial proteins with oxidoreductase function. The Ke 6 gene gives rise to two transcripts--a 1-kb Ke 6a mRNA which is abundant in kidney and liver tissue and a 1.4-kb Ke 6b mRNA which is found at a moderate level in spleen tissue. We report here the complete nucleotide sequence of Ke 6a cDNA and the expression of the Ke 6 gene in murine models of PKD. The Ke 6 gene may be intimately involved in the manifestation of these cystic kidney diseases.

Production of knockout rats using ENU mutagenesis and a yeast-based screening assay.

The rat is a widely used model in biomedical research and is often the preferred rodent model in many areas of physiological and pathobiological research. Although many genetic tools are available for the rat, methods to produce gene-disrupted knockout rats are greatly needed. In this study, we developed protocols for creating N-ethyl-N-nitrosourea (ENU)-induced germline mutations in several rat strains. F1 preweanling pups from mutagenized Sprague Dawley (SD) male rats were then screened for functional mutations in Brca1 and Brca2 using a yeast gap-repair, ADE2-reporter truncation assay. We produced knockout rats for each of these two breast cancer suppressor genes.

Phenotypic characterization of spontaneously mutated rats showing lethal dwarfism and epilepsy.

We have characterized the phenotype of spontaneously mutated rats, found during experimental inbreeding in a closed colony of Wistar Imamichi rats. Mutant rats showed severe dwarfism, short lifespan (early postnatal lethality), and high incidence of epileptic seizures. Mutant rats showed growth retardation after 3 d of age, and at 21 d their weight was about 56% that of normal rats. Most mutant rats died without reaching maturity, and 95% of the mutant rats had an ataxic gait. About 34% of the dwarf rats experienced epileptic seizures, most of which started as 'wild running' convulsions, progressing to generalized tonic-clonic convulsions. At age 28 d, the relative weight of the testes was significantly lower, and the relative weight of the brain was significantly higher, in mutant than in normal rats. Histologically, increased apoptotic germ cells, lack of spermatocytes, and immature Leydig cells were found in the mutant testes, and extracellular vacuoles of various sizes were present in the hippocampus and amygdala of the mutant brain. Mutant rats had significantly increased concentrations of plasma urea nitrogen, creatinine, and inorganic phosphate, as well as decreased concentrations of plasma growth hormone. Hereditary analysis showed that the defects were inherited as a single recessive trait. We have named the hypothetically mutated gene as lde (lethal dwarfism with epilepsy).

A set of highly informative rat simple sequence length polymorphism (SSLP) markers and genetically defined rat strains.

BACKGROUND: The National Bio Resource Project for the Rat in Japan (NBRP-Rat) is focusing on collecting, preserving and distributing various rat strains, including spontaneous mutant, transgenic, congenic, and recombinant inbred (RI) strains. To evaluate their value as models of human diseases, we are characterizing them using 109 phenotypic parameters, such as clinical measurements, internal anatomy, metabolic parameters, and behavioral tests, as part of the Rat Phenome Project. Here, we report on a set of 357 simple sequence length polymorphism (SSLP) markers and 122 rat strains, which were genotyped by the marker set. RESULTS: The SSLP markers were selected according to their distribution patterns throughout the whole rat genome with an average spacing of 7.59 Mb. The average number of informative markers between all possible pairs of strains was 259 (72.5% of 357 markers), showing their high degree of polymorphism. From the genetic profile of these rat inbred strains, we constructed a rat family tree to clarify their genetic background. CONCLUSION: These highly informative SSLP markers as well as genetically and phenotypically defined rat strains are useful for designing experiments for quantitative trait loci (QTL) analysis and to choose strategies for developing new genetic resources. The data and resources are freely available at the NBRP-Rat web site 1.

Beneficial effect of intravenous bolus of corticotropin-releasing factor on glucose intolerance of genetically obese (fa/fa) rats.

The effect of an ovine corticotropin-releasing factor (oCRF) bolus administered intravenously at the onset of glucose ingestion during oral glucose tolerance tests (OGTTs) was evaluated in conscious lean (FA/FA) and genetically obese (fa/fa) rats. When the amount of oCRF was purposely small to not stimulate the hypothalamo-pituitary-adrenal (HPA) axis, it normalized the glucose intolerance of genetically obese rats as tested during OGTTs and decreased their insulin output, whereas it had no effect in lean rats. In obese rats, plasma xylose levels measured after the ingestion of a xylose load were unaltered by the intravenous oCRF bolus, indicating that the beneficial effect of oCRF on glucose intolerance of fa/fa rats was unlikely to be dependent on glucose absorption. When the intravenous bolus of oCRF was doubled at the onset of OGTTs, it stimulated the HPA axis and produced a worsening of glucose intolerance in obese rats together with an increase in their insulin response. Again, it had no effect in lean rats. The abnormal intravenous glucose tolerance of obese rats was unaffected by the administration of an oCRF bolus: This is in keeping with previous data showing that bypassing the oral cavity fails to elicit several sensory reflexes that markedly influence subsequent glucose clearance. It has been suggested that obese rats may have deficient oropharyngeal reflexes that could be reactivated by the oCRF bolus, thereby being responsible for the normalization of their impaired OGTT, which lies in the hepatic glucose production process.

Delayed insulin transport across endothelium in insulin-resistant JCR:LA-cp rats.

Capillary endothelial cells are thought to limit the transport of insulin across the endothelium, resulting in attenuated insulin action at target sites. Whether endothelial insulin transport is altered in dysglycemic insulin-resistant states is not clear and was therefore investigated in the JCR:LA-cp corpulent male rat, which exhibits the metabolic syndrome of obesity, insulin resistance, hyperlipidemia, and hyperinsulinemia. Lean littermates that did not develop these alterations served as controls. Animals of both groups were normotensive (mean arterial pressure 136+/-2 mmHg). Hearts from obese and lean rats aged 7 (n = 6) or 18 (n = 8) weeks were perfused in vitro at 10 ml/min per gram wet wt over 51 min with Krebs-Henseleit buffer containing 0.1 or 0.5 U human insulin/l (equivalent to 0.6 and 3 nmol/l). Interstitial fluid was collected using a validated method, and interstitial insulin was determined with a radioimmunoassay. At 0.1 U/l, insulin transfer velocity was similar in both experimental groups (half-times of transfer: 11+/-0.2 min in obese and 18+/-4 min in lean rats; NS), but at 0.5 U/l, the respective half-times were 7+/-1 min in lean and 13+/-2 min in obese rats (P < 0.05). The steady-state level of insulin in the interstitium was 34+/-1% of the vascular level at 0.1 U/l and reached the vascular level (102+/-2%) at 0.5 U/l in both lean and obese rats. In rats aged 18 weeks, the half-times of insulin transfer were 31+/-2 and 14+/-l min in obese rats and 10+/-0.3 and 7+/-0.3 min in lean rats (P < 0.05). Again, interstitial steady-state levels were similar in both groups. Finally, postprandial insulin dynamics were simulated over a period of 120 min with a peak concentration of 0.8 U/l in rats aged 27 weeks (n = 4). The maximal interstitial level was 0.38+/-0.02 U/l in lean rats and 0.24+/-0.02 U/l in obese rats (P < 0.05), and a similar difference was noted throughout insulin infusion (areas under the transudate concentration-time curves: 17 and 11 U/min per 1, respectively). These data show, for the first time in a genetic animal model of insulin resistance, that transfer of insulin across the endothelium is substantially delayed in obese insulin-resistant rats and that it likely contributes to the postprandial alterations of glucose metabolism observed in the metabolic syndrome.

A new strain of rat for functional analysis of PINA.

Long Evans cinnamon (LEC) rat is an animal model for human Wilson disease (WD) due to a deletion in Atp7b, the copper transporter defective in WD patients. Previously, we have demonstrated presence of an alternative product termed PIneal Night-specific ATPase (PINA) generated by an intronic promoter in Atp7b gene. Analysis of LEC rat in this study demonstrates that PINA is absent in the LEC pineal establishing its usefulness for investigating PINA function. Studies of the LEC pineal, however, revealed an additional defect in serotonin N-acetyltransferase (NAT), the key enzyme in melatonin production. Linkage studies confirm that the NAT phenotype is entirely independent of PINA mutation in the pineal gland of LEC rats, and sequence analysis demonstrates that NAT defect is due to a point mutation in NAT coding region. In addition, we demonstrate that the cinnamon coat color of the LEC rat is unlinked to PINA and NAT deficiencies in these animals. To facilitate further functional analysis of PINA in pineal physiology, we crossed LEC rats with PVG rats that are wildtype for PINA, NAT and coat color, and obtained rats that are defective only in PINA/Atp7b locus (termed LPP rats) and normal for NAT activity and coat color. Furthermore, we have identified the deletion breakpoint of Atp7b gene in LPP rats, which allows simplified genotyping of mutant animals. The separation of PINA mutation from both NAT and coat color mutations in the new LPP rats will permit better functional studies of PINA in pineal circadian physiology.

Structural differences in the renin gene of Dahl salt-sensitive and salt-resistant rats.

Genomic libraries in lambda EMBL4 phage were constructed from both inbred Dahl salt-hypertension-sensitive (S) and inbred Dahl salt-hypertension-resistant (R) rats. Overlapping clones containing the renin genes were isolated from these libraries by screening with a renin cDNA probe. Clones were characterized by a combination of restriction mapping and Southern blot analysis. The results showed that the S-rat renin gene is remarkably different from the R-rat renin gene. The major differences are 1) a 1.2-kilobase (kb) insertion in the first intron of the S-gene which accounts for most of the restriction fragment length polymorphisms found in the renin genes between S and R strains, such as those generated with Bg/II [2.7 kb (S)/1.5 kb (R)], EcoRI [6.4 kb (S)/5.2 kb (R)], and HindIII [9.6 kb (S)/8.4 kb (R)]; 2) an additional HindIII site located at the 3' end of the R-gene which accounts for another HindIII restriction fragment length polymorphisms [25 kb (S)/22 kb, 3.4 kb (R)]; 3) two SmaI sites at the 5' flanking region of the first exon of the S-gene, whereas there is only one SmaI site in the corresponding region of the R-gene; and 4) three AvaI sites in the first intron of the S-gene in contrast to two AvaI sites in the same region of the R-gene These differences in the renin genes of Dahl rats might affect renin gene expression, which could account for the known strain differences in plasma and tissue renin activities. These structural studies provide a basis for genetic investigation into the relationship of the renin gene to blood pressure in Dahl rats.

Brain and liver insulin binding is decreased in Zucker rats carrying the 'fa' gene.

Insulin binding was measured in membrane particles prepared from the liver and several brain regions of 4-month-old female Zucker fa/fa (obese), Fa/fa (heterozygous), and Fa/Fa (lean) rats. High affinity insulin binding was decreased in the olfactory bulb of fatty (0.23 pmol bound/mg protein) and heterozygous (0.16 pmol/mg) rats compared with that in the lean controls (0.64 pmol/mg). Total binding was not changed in the cerebral cortex or hypothalamus. High affinity insulin binding was also decreased in the liver of both fatty (0.44 +/- 0.22 pmol/mg; P less than 0.01) and heterozygous (0.75 +/- 0.35 pmol/mg) animals compared with that in the lean rats (2.10 +/- 1.55 pmol/mg). This decreased binding is probably not due to down-regulation of receptors in the heterozygous rats, as they do not exhibit the hyperinsulinemia observed in the fatty rats. Rather, our findings suggest that there is a gene-related alteration in insulin binding in the Zucker rat, as low binding was observed in rats carrying either one (Fa/fa) or two (fa/fa) doses of the gene. We postulate that this central defect in insulin binding may contribute to inadequate perception of a central insulin feedback signal and to the hyperphagia observed in the obese rats.

Growth hormone secretion in genetic large (LL) and small (SS) rats.

We investigated whether genetic selection for growth influences pituitary GH secretion in two strains of rats, LL (large) and SS (small). Animals were bled every 15 min for 6 h via an indwelling atrial Silastic catheter, and GH levels were determined by RIA. LL and SS males displayed a low frequency, high amplitude episodic pattern of GH secretion, with surges of GH occurring at 3- to 4-h intervals, separated by trough periods of approximately 60-120 min. In contrast, LL females showed a high frequency, low amplitude pattern of GH secretion, with GH pulses occurring every 1-2 h. The number of GH pulses in SS females was lower than that in LL females. SS males and SS females displayed lower peak amplitudes and lower baseline levels and, therefore, lower mean plasma GH levels compared to LL animals. The anterior pituitary GH content was not significantly different in LL and SS animals of either sex. Thus, the reduction of GH levels in SS animals is most likely the result of reduced release of GH-releasing factor from the hypothalamus or an attenuated pituitary sensitivity to GH-releasing factor.