What Have We Learned (or Expect to) From Analysis of Murine Genetic Models Related to Substance Use Disorders?

The tremendous public health problem created by substance use disorders (SUDs) presents a major opportunity for mouse genetics. Inbred mouse strains exhibit substantial and heritable differences in their responses to drugs of abuse (DOA) and in many of the behaviors associated with susceptibility to SUD. Therefore, genetic discoveries emerging from analysis of murine genetic models can provide critically needed insight into the neurobiological effects of DOA, and they can reveal how genetic factors affect susceptibility drug addiction. There are already indications, emerging from our prior analyses of murine genetic models of responses related to SUDs that mouse genetic models of SUD can provide actionable information, which can lead to new approaches for alleviating SUDs. Lastly, we consider the features of murine genetic models that enable causative genetic factors to be successfully identified; and the methodologies that facilitate genetic discovery.

Working memory and pattern separation in founder strains of the BXD recombinant inbred mouse panel

Working memory and pattern separation are fundamental cognitive abilities which, when impaired, significantly diminish quality of life. Discovering genetic mechanisms underlying innate and disease-induced variation in these cognitive abilities is a critical step towards treatments for common and devastating neurodegenerative conditions such as Alzheimer's disease. In this regard, the trial-unique nonmatching-to-location assay (TUNL) is a touchscreen operant conditioning procedure allowing simultaneous quantification of working memory and pattern separation in mice and rats. In the present study, we used the TUNL assay to quantify these cognitive abilities in C57BL/6J and DBA/2J mice. These strains are the founders of the BXD recombinant inbred mouse panel which enables discovery of genetic mechanisms underlying phenotypic variation. TUNL testing revealed that pattern separation was significantly influenced by mouse strain, whereas working memory was not. Moreover, horizontal distance and vertical distance between choice-phase stimuli had dissociable effects on TUNL performance. These findings provide novel data on mouse strain differences in pattern separation and support previous findings of equivalent working memory performance in C57BL/6J and DBA/2J mice. Although working memory of the BXD founder strains was equivalent in this study, working memory of BXD strains may be divergent because of transgressive segregation. Collectively, data presented here indicate that pattern separation is heritable in the mouse and that the BXD panel can be used to identify mechanisms underlying variation in pattern separation.

Mapping of novel loci involved in lung and colon tumor susceptibility by the use of genetically selected mouse strains

Two non-inbred mouse lines, phenotypically selected for maximal (AIRmin) and minimal (AIRmax) acute inflammatory response, show differential susceptibility/resistance to the development of several chemically-induced tumor types. An intercross pedigree of these mice was generated and treated with the chemical carcinogen dimethylhydrazine, which induces lung and intestinal tumors. Genome wide high-density genotyping with the Restriction Site-Associated DNA genotyping (2B-RAD) technique was used to map genetic loci modulating individual genetic susceptibility to both lung and intestinal cancer. Our results evidence new common quantitative trait loci (QTL) for those phenotypes and provide an improved understanding of the relationship between genomic variation and individual genetic predisposition to tumorigenesis in different organs.

Harmonizing labeling and analytical strategies to obtain protein turnover rates in intact adult animals.

Changes in the abundance of individual proteins in the proteome can be elicited by modulation of protein synthesis (the rate of input of newly synthesized proteins into the protein pool) or degradation (the rate of removal of protein molecules from the pool). A full understanding of proteome changes therefore requires a definition of the roles of these two processes in proteostasis, collectively known as protein turnover. Because protein turnover occurs even in the absence of overt changes in pool abundance, turnover measurements necessitate monitoring the flux of stable isotope labeled precursors through the protein pool such as labeled amino acids or metabolic precursors such as ammonium chloride or heavy water. In cells in culture, the ability to manipulate precursor pools by rapid medium changes is simple, but for more complex systems such as intact animals, the approach becomes more convoluted. Individual methods bring specific complications, and the suitability of different methods has not been comprehensively explored. In this study we compare the turnover rates of proteins across four mouse tissues, obtained from the same inbred mouse strain maintained under identical husbandry conditions, measured using either [13C6]lysine or [2H2]O as the labeling precursor. We show that for long-lived proteins, the two approaches yield essentially identical measures of the first order rate constant for degradation. For short-lived proteins, there is a need to compensate for the slower equilibration of lysine through the precursor pools. We evaluate different approaches to provide that compensation. We conclude that both labels are suitable, but careful determination of precursor enrichment kinetics in amino acid labeling is critical and has a considerable influence on the numerical values of the derived protein turnover rates.

A Combined Adjuvant TF–Al Consisting of TFPR1 and Aluminum Hydroxide Augments Strong Humoral and Cellular Immune Responses in Both C57BL/6 and BALB/c Mice

TFPR1 is a novel adjuvant for protein and peptide antigens, which has been demonstrated in BALB/c mice in our previous studies; however, its adjuvanticity in mice with different genetic backgrounds remains unknown, and its adjuvanticity needs to be improved to fit the requirements for various vaccines. In this study, we first compared the adjuvanticity of TFPR1 in two commonly used inbred mouse strains, BALB/c and C57BL/6 mice, in vitro and in vivo, and demonstrated that TFPR1 activated TLR2 to exert its immune activity in vivo. Next, to prove the feasibility of TFPR1 acting as a major component of combined adjuvants, we prepared a combined adjuvant, TF–Al, by formulating TFPR1 and alum at a certain ratio and compared its adjuvanticity with that of TFPR1 and alum alone using OVA and recombinant HBsAg as model antigens in both BALB/c and C57BL/6 mice. Results showed that TFPR1 acts as an effective vaccine adjuvant in both BALB/c mice and C57BL/6 mice, and further demonstrated the role of TLR2 in the adjuvanticity of TFPR1 in vivo. In addition, we obtained a novel combined adjuvant, TF–Al, based on TFPR1, which can augment antibody and cellular immune responses in mice with different genetic backgrounds, suggesting its promise for vaccine development in the future.

Integrative analysis reveals mouse strain-dependent responses to acute ozone exposure associated with airway macrophage transcriptional activity

Acute ozone (O3) exposure is associated with multiple adverse cardiorespiratory outcomes, the severity of which varies across individuals in human populations and inbred mouse strains. However, molecular determinants of response, including susceptibility biomarkers that distinguish who will develop severe injury and inflammation, are not well characterized. We and others have demonstrated that airway macrophages (AMs) are an important resident immune cell type that are functionally and transcriptionally responsive to O3 inhalation. Here, we sought to explore influences of strain, exposure, and strain-by-O3 exposure interactions on AM gene expression and identify transcriptional correlates of O3-induced inflammation and injury across 6 mouse strains, including 5 Collaborative Cross (CC) strains. We exposed adult mice of both sexes to filtered air (FA) or 2 ppm O3 for 3 hours, and measured inflammatory and injury parameters 21 hours later. Mice exposed to O3 developed airway neutrophilia and lung injury with strain-dependent severity. In AMs, we identified a common core O3 response signature across all strains, as well as a set of genes exhibiting strain-by-O3 exposure interactions. In particular, a prominent gene expression contrast emerged between a low- (CC017/Unc) and high-responding (CC003/Unc) strain, as reflected by cellular inflammation and injury. Further inspection indicated that differences in their baseline gene expression and chromatin accessibility profiles likely contributes to their divergent post-O3 exposure transcriptional responses. Together, these results suggest that aspects of O3-induced respiratory responses are mediated through altered AM transcriptional signatures, and further confirms the importance of gene-environment interactions in mediating differential responsiveness to environmental agents.

Cocaine-induced locomotor activation differs across six sets of inbred mouse substrains

Cocaine use disorders (CUD) are devastating for affected individuals and impose a significant burden on society, but there are currently no FDA-approved therapies. The development of novel and effective treatments has been hindered by substantial gaps in our knowledge about the etiology of these disorders. The risk for developing a CUD is influenced by genetics, the environment and complex interactions between the two. Identifying specific genes and environmental risk factors that increase CUD risk would provide an avenue for the development of novel treatments. Rodent models of addiction-relevant behaviors have been a valuable tool for studying the genetics of response to drugs of abuse. Traditional genetic mapping using genetically and phenotypically divergent inbred mice has been successful in identifying numerous chromosomal regions that influence addiction-relevant behaviors, but these strategies rarely result in identification of the causal gene or genetic variant. To overcome this challenge, reduced complexity crosses (RCC) between closely related inbred mouse substrains have been proposed as a method for rapidly identifying and validating functional variants. The RCC approach is dependent on identifying phenotypic differences between substrains. To date, however, the study of addiction-relevant behaviors has been limited to very few sets of substrains, mostly comprising the C57BL/6 lineage. The present study expands upon the current literature to assess cocaine-induced locomotor activation in 20 inbred mouse substrains representing six inbred strain lineages (A/J, BALB/c, FVB/N, C3H/He, DBA/2 and NOD) that were either bred in-house or supplied directly by a commercial vendor. To our knowledge, we are the first to identify significant differences in cocaine-induced locomotor response in several of these inbred substrains. The identification of substrain differences allows for the initiation of RCC populations to more rapidly identify specific genetic variants associated with acute cocaine response. The observation of behavioral profiles that differ between mice generated in-house and those that are vendor-supplied also presents an opportunity to investigate the influence of environmental factors on cocaine-induced locomotor activity.

Gait performance of adolescent mice assessed by the CatWalk XT depends on age, strain and sex and correlates with speed and body weight

The automatization of behavioral tests assessing motor activity in rodent models is important for providing robust and reproducible results and evaluating new therapeutics. The CatWalk system is an observer-independent, automated and computerized technique for the assessment of gait performance in rodents. This method has previously been used in adult rodent models of CNS-based movement disorders such as Parkinson’s and Huntington’s diseases. As motor and gait abnormalities in neuropsychiatric disorders are observed during infancy and adolescence, it became important to validate the CatWalk XT in the gait analysis of adolescent mice and unravel factors that may cause variations in gait performance. Three adolescent wild-type inbred mouse strains, C57BL/6N, DBA/2 and FVB/N, were tested using the CatWalk XT (Version 10.6) for suitable detection settings to characterize several gait parameters at P32 and P42. The same detection settings being suitable for C57BL/6N and DBA/2 mice allowed a direct comparison between the two strains. On the other hand, due to their increased body weight and size, FVB/N mice required different detection settings. The CatWalk XT reliably measured the temporal, spatial, and interlimb coordination parameters in the investigated strains during adolescence. Additionally, significant effects of sex, development, speed and body weight within each strain confirmed the sensitivity of motor and gait functions to these factors. The CatWalk gait analysis of rodents during adolescence, taking the effect of age, strain, sex, speed and body weight into consideration, will decrease intra-laboratory discrepancies and increase the face validity of rodent models of neuropsychiatric disorders.

Impact of C57BL/6J and SV-129 Mouse Strain Differences on Ischemia-Induced Postnatal Angiogenesis and the Associated Leukocyte Infiltration in a Murine Hindlimb Model of Ischemia

Strain-related differences in arteriogenesis in inbred mouse strains have already been studied excessively. However, these analyses missed evaluating the mouse strain-related differences in ischemia-induced angiogenic capacities. With the present study, we wanted to shed light on the different angiogenic potentials and the associated leukocyte infiltration of C57BL/6J and SV-129 mice to facilitate the comparison of angiogenesis-related analyses between these strains. For the induction of angiogenesis, we ligated the femoral artery in 8–12-week-old male C57BL/6J and SV-129 mice and performed (immuno-) histological analyses on the ischemic gastrocnemius muscles collected 24 h or 7 days after ligation. As evidenced by hematoxylin and eosin staining, C57BL/6J mice showed reduced tissue damage but displayed an increased capillary-to-muscle fiber ratio and an elevated number of proliferating capillaries (CD31+/BrdU+ cells) compared to SV-129 mice, thus showing improved angiogenesis. Regarding the associated leukocyte infiltration, we found increased numbers of neutrophils (MPO+ cells), NETs (MPO+/CitH3+/DAPI+), and macrophages (CD68+ cells) in SV-129 mice, whereas macrophage polarization (MRC1- vs. MRC1+) and total leukocyte infiltration (CD45+ cells) did not differ between the mouse strains. In summary, we show increased ischemia-induced angiogenic capacities in C57BL/6J mice compared to SV-129 mice, with the latter showing aggravated tissue damage, inflammation, and impaired angiogenesis.