Anthrax lethal toxin (LT) is a bipartite protease-containing toxin and a key virulence determinant of Bacillus anthracis. In mice, LT causes the rapid lysis of macrophages isolated from certain ...inbred strains, but the correlation between murine macrophage sensitivity and mouse strain susceptibility to toxin challenge is poor. In rats, LT induces a rapid death in as little as 37 minutes through unknown mechanisms. We used a recombinant inbred (RI) rat panel of 19 strains generated from LT-sensitive and LT-resistant progenitors to map LT sensitivity in rats to a locus on chromosome 10 that includes the inflammasome NOD-like receptor (NLR) sensor, Nlrp1. This gene is the closest rat homolog of mouse Nlrp1b, which was previously shown to control murine macrophage sensitivity to LT. An absolute correlation between in vitro macrophage sensitivity to LT-induced lysis and animal susceptibility to the toxin was found for the 19 RI strains and 12 additional rat strains. Sequencing Nlrp1 from these strains identified five polymorphic alleles. Polymorphisms within the N-terminal 100 amino acids of the Nlrp1 protein were perfectly correlated with LT sensitivity. These data suggest that toxin-mediated lethality in rats as well as macrophage sensitivity in this animal model are controlled by a single locus on chromosome 10 that is likely to be the inflammasome NLR sensor, Nlrp1.
Toxoplasma gondii is an intracellular parasite that infects a wide range of warm-blooded species. Rats vary in their susceptibility to this parasite. The Toxo1 locus conferring Toxoplasma resistance ...in rats was previously mapped to a region of chromosome 10 containing Nlrp1. This gene encodes an inflammasome sensor controlling macrophage sensitivity to anthrax lethal toxin (LT) induced rapid cell death (pyroptosis). We show here that rat strain differences in Toxoplasma infected macrophage sensitivity to pyroptosis, IL-1β/IL-18 processing, and inhibition of parasite proliferation are perfectly correlated with NLRP1 sequence, while inversely correlated with sensitivity to anthrax LT-induced cell death. Using recombinant inbred rats, SNP analyses and whole transcriptome gene expression studies, we narrowed the candidate genes for control of Toxoplasma-mediated rat macrophage pyroptosis to four genes, one of which was Nlrp1. Knockdown of Nlrp1 in pyroptosis-sensitive macrophages resulted in higher parasite replication and protection from cell death. Reciprocally, overexpression of the NLRP1 variant from Toxoplasma-sensitive macrophages in pyroptosis-resistant cells led to sensitization of these resistant macrophages. Our findings reveal Toxoplasma as a novel activator of the NLRP1 inflammasome in rat macrophages.
A quantitative genetic approach, which involves correlation of transcriptional networks with the phenotype in a recombinant inbred (RI) population and in selectively bred lines of rats, and ...determination of coinciding quantitative trait loci for gene expression and the trait of interest, has been applied in the present study. In this analysis, a novel approach was used that combined DNA‐Seq data, data from brain exon array analysis of HXB/BXH RI rat strains and six pairs of rat lines selectively bred for high and low alcohol preference, and RNA‐Seq data (including rat brain transcriptome reconstruction) to quantify transcript expression levels, generate co‐expression modules and identify biological functions that contribute to the predisposition of consuming varying amounts of alcohol. A gene co‐expression module was identified in the RI rat strains that contained both annotated and unannotated transcripts expressed in the brain, and was associated with alcohol consumption in the RI panel. This module was found to be enriched with differentially expressed genes from the selected lines of rats. The candidate genes within the module and differentially expressed genes between high and low drinking selected lines were associated with glia (microglia and astrocytes) and could be categorized as being related to immune function, energy metabolism and calcium homeostasis, as well as glial–neuronal communication. The results of the present study show that there are multiple combinations of genetic factors that can produce the same phenotypic outcome. Although no single gene accounts for predisposition to a particular level of alcohol consumption in every animal model, coordinated differential expression of subsets of genes in the identified pathways produce similar phenotypic outcomes.
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.
In the HXB/BXH recombinant inbred (RI) rats, correlation analysis of brain gene expression levels with alcohol consumption in a two-bottle choice paradigm, and filtering based on behavioral and gene expression quantitative trait locus (QTL) analyses, generated a list of candidate genes. A literature-based, functional analysis of the interactions of the products of these candidate genes defined pathways linked to presynaptic GABA release, activation of dopamine neurons, and postsynaptic GABA receptor trafficking, in brain regions including the hypothalamus, ventral tegmentum and amygdala. The analysis also implicated energy metabolism and caloric intake control as potential influences on alcohol consumption by the recombinant inbred rats. In the human populations, polymorphisms in genes associated with GABA synthesis and GABA receptors, as well as genes related to dopaminergic transmission, were associated with alcohol consumption.
Our results emphasize the importance of the signaling pathways identified using the non-human animal models, rather than single gene products, in identifying factors responsible for complex traits such as alcohol consumption. The results suggest cross-species similarities in pathways that influence predisposition to consume alcohol by rats and humans. The importance of a well-defined phenotype is also illustrated. Our results also suggest that different genetic factors predispose alcohol dependence versus the phenotype of alcohol consumption.
Background
Two features of alcohol addiction that have been widely studied in animal models are relapse drinking following periods of alcohol abstinence and the escalation of alcohol consumption ...after chronic continuous or intermittent alcohol exposure. The genetic contribution to these phenotypes has not been systematically investigated.
Methods
HXB/BXH recombinant inbred (RI) rat strains were given access to alcohol sequentially as follows: alcohol (10%) as the only fluid for 1 week; alcohol (10%) and water in a 2‐bottle choice paradigm for 7 weeks (“pre‐alcohol deprivation effect ADE alcohol consumption”); 2 weeks of access to water only (alcohol deprivation); and 2 weeks of reaccess to 10% alcohol and water (“post‐ADE alcohol consumption”). The periods of deprivation and reaccess to alcohol were repeated 3 times. The ADE was defined as the amount of alcohol consumed in the first 24 hours after deprivation minus the average daily amount of alcohol consumed in the week prior to deprivation. Heritability of the phenotypes was determined by analysis of variance, and quantitative trait loci (QTLs) were identified.
Results
All strains showed increased alcohol consumption, compared to the predeprivation period, in the first 24 hours after each deprivation (ADE). Broad‐sense heritability of the ADEs was low (ADE1, 9.1%; ADE2, 26.2%; ADE3, 16.3%). Alcohol consumption levels were relatively stable over weeks 2 to 7. Post‐ADE alcohol consumption levels consistently increased in some strains and were decreased or unchanged in others. Heritability of pre‐ and post‐ADE alcohol consumption was high and increased over time (week 2, 38.5%; week 7, 51.1%; week 11, 56.8%; week 15, 63.3%). QTLs for pre‐ and post‐ADE alcohol consumption were similar, but the strength of the QTL association with the phenotype decreased over time.
Conclusions
In the HXB/BXH RI rat strains, genotypic variance does not account for a large proportion of phenotypic variance in the ADE phenotype (low heritability), suggesting a role of environmental factors. In contrast, a large proportion of the variance across the RI strains in pre‐ and post‐ADE alcohol consumption is due to genetically determined variance (high heritability).
Epidemiological studies suggest that prenatal malnutrition increases the risk of developing schizophrenia. Animal models indicate that prenatal protein deprivation (PPD) affects many aspects of adult ...brain function. We tested the hypothesis that PPD in rats would alter prepulse inhibition (PPI), which is an operational measure of sensorimotor gating that is deficient in schizophrenia patients. Additionally, we examined dopaminergic and glutaminergic receptor binding in the striatum and hippocampus, which have been suggested to play a role in the etiology of schizophrenia. Rat dams were fed normal (25%) or low (6%) protein diets beginning 5 weeks prior to, and throughout pregnancy. The pups were tested at postnatal days (PND) 35 and 56 for PPI. Striatal and hippocampal NMDA receptor, and striatal dopamine receptor binding were quantified post-mortem in a subset of these rats. Female rats exposed to PPD had reduced levels of PPI at PND 56, but not PND 35, suggesting the emergence of a sensorimotor gating deficit in early adulthood. Striatal NMDA receptor binding was increased in PPD females. A decrease in initial startle response (SR) was also observed in all PPD rats relative to control rats. These results suggest that PPD causes age- and sex-dependent decreases in PPI and increases in NMDA receptor binding. This animal model may be useful for the investigation of neurodevelopmental changes that are associated with schizophrenia in humans.
Prepulse inhibition (PPI) of the acoustic startle response is altered by manipulations that affect brain monoamine neurotransmission. Corticotropin-releasing factor (CRF), a neurotransmitter that is ...released during stress, and CRF receptors are expressed in areas of the brain which contribute to PPI, and central administration of CRF changes extracellular concentrations of the monoamines. Therefore, CRF is in a position to alter PPI, either by causing the release of other neurotransmitters, or by direct effects at CRF receptors.
The present experiments were conducted to test the hypothesis that intracerebroventricular (ICV) administration of CRF would decrease PPI in rats. Additionally, these experiments were used to examine whether CRF results in differential changes in PPI in rat strains that show high and low basal PPI, and whether CRF-induced grooming behavior and increased startle amplitude are also strain-dependent.
Male Wistar-Kyoto (WKY) rats inbred in our colony in La Jolla, WKY rats obtained from Charles River, and Brown Norway (BN) rats from Harlan, Sprague-Dawley were tested for grooming behavior, PPI and startle amplitude following ICV infusion of either CRF (1.0-3.0 microg) or saline.
CRF significantly decreased PPI in both BN rats, which show relatively little PPI in the basal condition and, in WKY rats. The amplitude of the acoustic startle response was increased in WKY rats only and, only by the 3.0 microg dose of CRF. CRF increased grooming behavior in the La Jolla colony WKY and BN rats. However, within the time frame during which the rats were being observed, CRF failed to significantly increase grooming in Charles River WKY rats.
CRF diminished PPI of the acoustic startle response in rats that show high (WKY) and low (BN) basal PPI. This effect does not appear to be dependant on CRF-induced changes in startle amplitude. The results suggest the possibility that stress-induced exacerbation of symptoms in schizophrenia, which is characterized by deficient PPI, may be CRF-dependent.
Amplitude and habituation of the acoustic startle response were assessed in four recombinant inbred (RI) rat strains. One group from each strain underwent repeated restraint stress, the last session ...of which was 24
h before startle testing while, a second group from each strain was not stressed prior to testing. Additionally, prepulse inhibition of the acoustic startle response, and anxiety behavior in the elevated plus-maze were assessed in separate, non-stressed groups of each strain. In the non-stressed condition, these RI strains differed significantly from each other on all behaviors measured. In the two RI strains that showed the greatest habituation of the startle response, repeated stress resulted in significantly lower acoustic startle amplitude than that seen in non-stressed controls of those strains. In the strains showing low levels of habituation, repeated stressed increased the level. Neither genotype-dependent levels of startle amplitude, prepulse inhibition of the startle response, nor anxiety in the plus-maze were closely related to the effect of stress on either startle amplitude or habituation. The results suggest that genotype-dependent habituation of the startle response may be important in determining whether stress will alter startle amplitude.
Models of human disease have long been used to understand the basic pathophysiology of disease and to facilitate the discovery of new therapeutics. However, as long as models have been used there ...have been debates about the utility of these models and their ability to mimic clinical disease at the phenotypic level. The application of genetic studies to both humans and model systems allows for a new paradigm, whereby a novel comparative genomics strategy combined with phenotypic correlates can be used to bridge between clinical relevance and model utility. This study presents a comparative genomic map for "candidate hypertension loci in humans" based on translating QTLs between rat and human, predicting 26 chromosomal regions in the human genome that are very likely to harbor hypertension genes. The predictive power appears robust, as several of these regions have also been implicated in mouse, suggesting that these regions represent primary targets for the development of SNPs for linkage disequilibrium testing in humans and/or provide a means to select specific models for additional functional studies and the development of new therapeutics.
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