HR Mice Research Articles

Morphological Characteristics of Colon Tumors in Mice with Different Tolerance to Hypoxia.

In adult male C57BL/6 mice with high (HR) and low (LR) resistance to hypoxia, morphological features of colon tumors and blood parameters were evaluated 70 days after intraperitoneal injection of azoxymethane and subsequent consumption of 3 cycles of dextran sulfate sodium. On macroscopic analysis, tumors were found in the distal colon in 35% (7 of 20 animals) of HR and 31% (4 of 13 animals) of LR animals. Microscopic analysis of the distal colon revealed tumors in 75% (15 of 20 animals) of HR and 69% (9 of 13 animals) of LR mice. The tumors were presented by areas of glandular intraepithelial neoplasia and adenocarcinomas; the incidence and the area of the tumors did not differ in groups of HR and LR mice. The number of neuroendocrine and goblet cells in the distal colon mucosa in the areas of tumors was similar in the compared groups. However, in both HR and LR mice of the experimental groups, the content of goblet cells in tumors was lower and the content of endocrine cells was higher than in the corresponding control groups. In the peripheral blood, the erythrocyte count and hemoglobin content decreased in HR and LR mice of the experimental groups; the relative number of monocytes increased only in HR mice and the absolute number of lymphocytes and monocytes decreased in LR mice. Thus, 70 days after azoxymethane administration and dextran sulfate sodium consumption, the tumors in mice were presented by glandular intraepithelial neoplasia and adenocarcinomas, and their incidence and area did not differ between animals with different tolerance to hypoxia.

Lymphocyte HVEM/BTLA co-expression after critical illness demonstrates severity indiscriminate upregulation, impacting critical illness-induced immunosuppression.

The co-regulatory molecule, HVEM, can stimulate or inhibit immune function, but when co-expressed with BTLA, forms an inert complex preventing signaling. Altered HVEM or BTLA expression, separately have been associated with increased nosocomial infections in critical illness. Given that severe injury induces immunosuppression, we hypothesized that varying severity of shock and sepsis in murine models and critically ill patients would induce variable increases in HVEM/BTLA leukocyte co-expression. In this study, varying severities of murine models of critical illness were utilized to explore HVEM+BTLA+ co-expression in the thymic and splenic immune compartments, while circulating blood lymphocytes from critically ill patients were also assessed for HVEM+BTLA+ co-expression. Higher severity murine models resulted in minimal change in HVEM+BTLA+ co-expression, while the lower severity model demonstrated increased HVEM+BTLA+ co-expression on thymic and splenic CD4+ lymphocytes and splenic B220+ lymphocytes at the 48-hour time point. Patients demonstrated increased co-expression of HVEM+BTLA+ on CD3+ lymphocytes compared to controls, as well as CD3+Ki67- lymphocytes. Both L-CLP 48hr mice and critically ill patients demonstrated significant increases in TNF-α. While HVEM increased on leukocytes after critical illness in mice and patients, changes in co-expression did not relate to degree of injury severity of murine model. Rather, co-expression increases were seen at later time points in lower severity models, suggesting this mechanism evolves temporally. Increased co-expression on CD3+ lymphocytes in patients on non-proliferating cells, and associated TNF-α level increases, suggest post-critical illness co-expression does associate with developing immune suppression.

Weanling gut microbiota composition of a mouse model selectively bred for high voluntary wheel-running behavior.

We compared the fecal microbial community composition and diversity of four replicate lines of mice selectively bred for high wheel-running activity over 81 generations (HR lines) and four non-selected control lines. We performed 16S rRNA gene sequencing on fecal samples taken 24 h after weaning, identifying a total of 2074 bacterial operational taxonomic units. HR and control mice did not significantly differ for measures of alpha diversity, but HR mice had a higher relative abundance of the family Clostridiaceae. These results differ from a study of rats, where a line bred for high forced-treadmill endurance and that also ran more on wheels had lower relative abundance of Clostridiaceae, as compared with a line bred for low endurance that ran less on wheels. Within the HR and control groups, replicate lines had unique microbiomes based on unweighted UniFrac beta diversity, indicating random genetic drift and/or multiple adaptive responses to selection.

Cross-fostering selectively bred High Runner mice affects adult body mass but not voluntary exercise

While nursing, mammals progress through critical developmental periods for the cardiovascular, musculoskeletal, and central nervous systems. The suckling period in mammals is therefore especially vulnerable to environmental factors that may affect the “developmental programming” of many complex traits. As a result, various aspects of maternal behavior and physiology can influence offspring in ways that have lasting effects into adulthood. Several recent studies of animal models have shown that maternal effects can partially program adult activity behaviors, which has important implications for health and locomotor performance. Here, we used cross-fostering to test for possible maternal effects on adult wheel-running behavior (voluntary exercise), maximal aerobic capacity during forced exercise (VO2max), body mass and composition, and organ masses. Subjects were from a line of mice that has been selectively bred for ∼90 generations for high voluntary wheel-running behavior (High Runner; HR) and a non-selected Control (C) line. Adult HR mice run ∼3-fold the daily distances of C mice and have evolved other differences associated with exercise capacity, including elevated VO2max, reduced body mass and fat mass, and larger hearts. At birth, we fostered offspring to create 4 experimental groups: C pups to other C dams (in-foster), HR pups to other HR dams (in-foster), C pups to HR dams (cross-foster), HR pups to C dams (cross-foster). Thus, all pups were fostered to a different mother. Mice were weaned 3 weeks later, and adult testing began at ∼6 weeks of age. At weaning, pups raised by HR dams were smaller than those raised by C dams for both sexes and as expected, HR pups raised by HR dams weighed less than C pups raised by C dams. As adults, mice raised by HR dams continued to have reduced body masses. As expected, adult HR mice ran approximately 3-fold more than their C counterparts and females ran more than males. However, cross-fostering did not statistically affect any aspect of wheel-running behavior (distance, duration, speed). Similarly, with body mass as a covariate, HR mice had higher VO2max than C mice, and males had higher VO2max than females, but cross-fostering had no effect. With body mass as a covariate, cross-fostering had variable effects on adult organ masses in a sex-specific manner. Overall, our results indicate that development of the adult High Runner phenotype does not require rearing by an HR dam, suggesting that high adult activity in humans may be independent of high maternal activity.

Conditioned place preference for cocaine and methylphenidate in female mice from lines selectively bred for high voluntary wheel-running behavior.

Behavioral addictions can come in many forms, including overeating, gambling and overexercising. All addictions share a common mechanism involving activation of the natural reward circuit and reinforcement learning, but the extent to which motivation for natural and drug rewards share similar neurogenetic mechanisms remains unknown. A unique mouse genetic model in which four replicate lines of female mice were selectively bred (>76 generations) for high voluntary wheel running (High Runner or HR lines) alongside four non-selected control (C) lines were used to test the hypothesis that high motivation for exercise is associated with greater reward for cocaine (20 mg/kg) and methylphenidate (10 mg/kg) using the conditioned place preference (CPP) test. HR mice run ~three times as many revolutions/day as C mice, but the extent to which they have increased motivation for other rewards is unknown. Both HR and C mice displayed significant CPP for cocaine and methylphenidate, but with no statistical difference between linetypes for either drug. Taken together, results suggest that selective breeding for increased voluntary running has modified the reward circuit in the brain in a way that increases motivation for running without affecting cocaine or methylphenidate reward.

Effects of activity and selection on mouse femoral strength

Bone is a hard tissue providing mechanical support to vertebrates and is influenced by both genetic and environmental factors. Stress from mechanical loading causes micro‐fractures in bones, triggering dynamic bone remodeling by shifting the balance of absorption and formation to net formation. Prolonged inactivity can lead to bone dystrophy, which reduces bone strength. In order to assess how genetic and environmental factors can independently affect the structure of bone, we used mice from lines that have been selectively bred for high levels of voluntary wheel running (High Runner, or HR lines) that had access to cage wheels throughout their lives. Femora from HR and control mice were dissected and defleshed in order to perform material strength testing on the bone. A custom‐made rig was designed to hold the femora in place as a materials testing system (MTS) was used to perform a three‐point bending test at the mid‐diaphysis of each individual femur. We recorded morphometric data prior to testing and the modulus (stiffness) and failure stress were calculated from the MTS tests. HR mice have thicker femora, but are lighter in density, than control mice. HR mice with wheel access had longer than average femora, but stiffness and failure strength was similar among all groups.

Early Post‐Natal Maternal Effects on Voluntary Exercise, Maximal Aerobic Capacity, and Associated Traits in Mice

During the suckling period, many mammals progress through numerous developmental milestones, including critical periods of the central nervous system, musculoskeletal system, and cardiovascular system – each an important element of physical activity and exercise capacity. In a novel experimental mouse model, four replicate lines were selectively bred for ~90 generations for high voluntary wheel‐running behavior (high‐runner; HR) while four non‐selected Control (C) lines were also maintained. As a result of selection, HR mice run approximately 3‐fold the distances of C mice on a daily basis. Various other physiological and behavioral differences have also evolved in the HR lines, including higher maximal oxygen consumption (VO2max). The aim of the present study was to assess the role of the maternal environment during the birth‐weaning period in the expression of the many differences between HR and C lines using a cross‐fostering paradigm. Cross‐fostering HR mice to C dams (and vice versa) at birth and until weaning allowed us to determine the relative contribution of genetic and early environmental factors in shaping the HR phenotype. Mice were weaned three weeks after birth, housed in same‐sex cages of four, and adult testing began at approximately six weeks of age with measurement of VO2max, daily wheel running over a six‐day period, food consumption, home‐cage activity, body fat composition, and organ masses following dissection. As expected, HR mice ran approximately ~3‐fold more revolutions/day on wheels than their C counterparts. Preliminary analyses show that rearing by an HR dam has a negative effect on adult wheel running in females. This result suggests that the maternal environment provided by HR dams during the suckling period may play a role in the selection limit observed in HR lines.Support or Funding InformationNSF GRFP

Artificial Selection for Increased Voluntary Wheel Running Alters Limb Skeleton Shape and Exercise Plasticity in Mice

Individual skeletal morphology is determined by genetic effects and within‐generation phenotypic plasticity in response to mechanical loading. Changes in limb bone morphology associated with locomotor activity through ontogeny and across evolutionary time can provide insight into adaptation in locomotor systems. Here, we investigate the effects of long‐term selection for locomotor activity on the gross morphology and exercise response of the hind limb skeleton using a mouse model. A founding population of Hsd:ICR outbred mouse stock was divided into closed lines of “high runner” (HR) and control (C) animals. HR mice were bred for increased voluntary wheel‐running distance by breeding animals (n=10 pairs per generation) with the highest average wheel revolutions on days 5 and 6 of a 6‐day wheel exposure protocol. Control animals were also wheel‐tested but were randomly paired. Male HR and C mice from experimental generation 82 were randomly assigned at 5 weeks of age to be permitted or denied wheel access for 4 weeks, resulting in 4 experimental groups: C wheel (n=12), C no wheel (n=12), HR wheel (n=10), and HR no wheel (n= 10). Following wheel access, mice were euthanized and right hind limbs were micro‐CT scanned (~20 um voxel size). Three‐dimensional shapes of the os coxa, femur, and tibia were defined by digitizing 18, 23, and 18 landmarks, respectively, on each specimen. To adjust for size, individual bones were scaled to the geometric mean of all possible linear distances among landmarks. Between‐group differences were analyzed using canonical variates analysis (CVA) and localized using the Euclidean distance matrix analysis (EDMA) Form procedure. EDMA Growth procedure was used to compare the trajectories of exercise‐induced skeletal plasticity between HR and C animals. Differences ≥3% were considered substantial for both EDMA methods. Shape analyses revealed differences in morphological lability among the three limb bones examined and in subsections within each bone. More specifically, the mean hind limb skeletal morphology, especially of the pelvis and proximal femur, substantially differed between HR and C mice. Plastic bony responses to exercise also differed, most substantially in the iliac blade, ischial tuberosity, and proximal femur. Finally, throughout the hind limb, the baseline skeletal morphology of HR mice did not resemble the exercise response of C animals, indicating that genetic assimilation of plastic bone responses did not occur in this model. The results suggest regional sensitivity of the hind limb skeleton to plastic and evolved morphological change, provide further evidence of genetic influence on bone functional adaptation, and inform our understanding of the evolutionary lability of the musculoskeletal system over evolutionary timescales.Support or Funding InformationUniversity of Missouri School of Medicine, University of Missouri Dissertation Research Travel AwardThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Electrocardiograms of mice selectively bred for high levels of voluntary exercise: Effects of short-term exercise training and the mini-muscle phenotype

Changes in cardiac function that occur with exercise training have been studied in detail, but those accompanying evolved increases in the duration or intensity of physical activity are poorly understood. To address this gap, we studied electrocardiograms (ECGs) of mice from an artificial selection experiment in which four replicate lines are bred for high voluntary wheel running (HR) while four non-selected lines are maintained as controls (C). ECGs were recorded using an ECGenie (Mouse Specifics, Inc.) both before and after six days of wheel access (as used in the standard protocol to select breeders). We hypothesized that HR mice would show innate differences in ECG characteristics and that the response to training would be greater in HR mice relative to C mice because the former run more. After wheel access, in statistical analyses controlling for variation in body mass, all mice had lower heart rates, and mice from HR lines had longer PR intervals than C lines. Also after wheel access, male mice had increased heart rate variability, whereas females had decreased heart rate variability. With body mass as a covariate, six days of wheel access significantly increased ventricle mass in both HR and C males. Within the HR lines, a subset of mice known as mini-muscle individuals have a 50% reduction in hindlimb muscle mass and generally larger internal organs, including the heart ventricles. As compared with normal-muscled individuals, mini-muscle individuals had a longer QRS complex, both before and after wheel access. Some studies in other species of mammals have shown correlations between athletic performance and QRS duration. Correlations between wheel running and either heart rate or QRS duration (before wheel running) among the eight individual lines of the HR selection experiment or among 17 inbred mouse strains taken from the literature were not statistically significant. However, total revolutions and average speed were negatively correlated with PR duration among lines of the HR selection experiment for males, and duration of running was negatively correlated with PR duration among 17 inbred strains for females. We conclude that HR mice have enhanced trainability of cardiac function as compared with C mice (as indicated by their longer PR duration after wheel access), and that the mini-muscle phenotype causes cardiac changes that have been associated with increased athletic performance in previous studies of mammals.

Conditioned Place Preference of Mice Selectively Bred for High Voluntary Wheel Running

Running acts as a natural reward and shares features with other rewarding behaviors, such as eating or taking drugs of abuse. Exercise is even proposed to have addictive properties, as humans and rodents have shown signs of “withdrawal,” including anxiety and depression, after being denied exercise. Conditioned place preference (CPP) is a neuro‐behavioral test of reward and reinforcement, widely used in studies of addictive processes in rodents. In typical protocols, individuals receive a reward (e.g., cocaine injection) paired with a specific environment (usually a specific floor texture) several times and are then tested for preference of the conditioned environment as compared with a different environment in which they had not received a reward (e.g., saline injection). We studied CPP in a unique exercise model of 4 replicate lines of mice selectively bred for high voluntary wheel running (High Runner or HR lines) and 4 non‐selected control (C) lines. HR mice run ~3‐times as much as C mice on a daily basis, partly because of evolutionary changes in their reward and motivational systems. We hypothesized that HR mice would differ from C in the extent to which they show a CPP following conditioning with rewarding drugs and/or wheel running. In experiment 1, HR and C mice were tested for CPP with cocaine as the reward. Both HR and C mice were significantly conditioned by cocaine, with no statistical difference between groups in the degree of conditioning. In experiment 2, mice were tested for CPP with wheel access as the reward. Specifically, mice were granted or not granted wheel access for 10 days, and each day removed from wheels or standard cages during peak wheel running and placed in CPP chambers for 30 minutes. Preliminary results show no significant conditioning by either HR or C mice. In a recent experiment 3, mice were tested for CPP with Ritalin as the reward, following the same protocol as experiment 1. Functioning as a dopamine transporter blocker, the drug Ritalin has been shown to decrease wheel running of HR mice while increasing running by C mice. Thus, we hypothesize a significant difference in the degree of conditioning between HR and C mice with Ritalin.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Maternal exposure to Western diet affects adult body composition and voluntary wheel running in a genotype-specific manner in mice

Some human diseases, including obesity, Type II diabetes, and numerous cancers, are thought to be influenced by environments experienced in early life, including in utero. Maternal diet during the perinatal period may be especially important for adult offspring energy balance, potentially affecting both body composition and physical activity. This effect may be mediated by the genetic background of individuals, including, for example, potential “protective” mechanisms for individuals with inherently high levels of physical activity or high basal metabolic rates. To examine some of the genetic and environmental factors that influence adult activity levels, we used an ongoing selection experiment with 4 replicate lines of mice bred for high voluntary wheel running (HR) and 4 replicate, non-selected control lines (C). Dams (half HR and half C) were fed a “Western” diet (WD, high in fat and sucrose) or a standard diet (SD) from 2weeks prior to mating until their pups could feed on solid food (14days of age). We analyzed dam and litter characteristics from birth to weaning, and offspring mass and physical activity into adulthood. One male offspring from each litter received additional metabolic and behavioral tests. Maternal WD caused pups to eat solid food significantly earlier for C litters, but not for HR litters (interaction of maternal environment and genotype). With dam mass as a covariate, mean pup mass was increased by maternal WD but litter size was unaffected. HR dams had larger litters and tended to have smaller pups than C dams. Home-cage activity of juvenile focal males was increased by maternal WD. Juvenile lean mass, fat mass, and fat percent were also increased by maternal WD, but food consumption (with body mass as a covariate) was unaffected (measured only for focal males). Behavior in an elevated plus maze, often used to indicate anxiety, was unaffected by maternal WD. Maximal aerobic capacity (VO2max) was also unaffected by maternal WD, but HR had higher VO2max than C mice. Adult lean, fat, and total body masses were significantly increased by maternal WD, with greater increase for fat than for lean mass. Overall, no aspect of adult wheel running (total distance, duration, average running speed, maximum speed) or home-cage activity was statistically affected by maternal WD. However, analysis of the 8 individual lines revealed that maternal WD significantly increased wheel running in one of the 4 HR lines. On average, all groups lost fat mass after 6days of voluntary wheel running, but the absolute amount lost was greater for mice with maternal WD resulting in no effect of maternal WD on absolute or % body fat after wheel access. All groups gained lean and total body mass during wheel access, regardless of maternal WD or linetype. Measured after wheel access, circulating leptin, adiponectin, and corticosterone concentrations were unaffected by maternal WD and did not differ between HR and C mice. With body mass as a covariate, heart ventricle mass was increased by maternal WD in both HR and C mice, but fat pads, liver, spleen, and brain masses were unaffected. As found previously, HR mice had larger brains than C mice. Body mass of grand-offspring was unaffected by grand-maternal WD, but grand-offspring wheel running was significantly increased for one HR line and decreased for another HR line by grand-maternal WD. In summary, maternal Western diet had long-lasting and general effects on offspring adult morphology, but effects on adult behavior were limited and contingent on sex and genetic background.

Individual and sex differences in high and low responder phenotypes

Individual differences in responses to a novel environment are an important tool to predict predisposition to neuropsychiatric disorders. One way to examine individual differences involves classifying animals based on locomotion in a novel context. In this study we focused on individual and sex differences by categorizing female and male mice as high (HR) or low responders (LR) on the basis of open field locomotion. We then assessed whether groups differed on behavioral measures of spontaneous alternations, anxiety, depression and contextual fear conditioning. In the Y-maze, we observed no differences across HR/LR or sex on spontaneous alternations, but HR displayed more locomotion. HR male mice showed less anxiety-like behavior in the light-dark test but not the elevated plus maze. We observed no differences in the forced swim test across HR/LR, although males exhibited greater depression-like behavior overall. HR mice exhibited less contextual fear memory compared to LR regardless of sex. Principal component analyses suggested sex-specific patterns of behaviors across tests, with female responses within individual tests tending to load together. In females anxiety- and depression-like behaviors explained a large part of the variance observed across tests in our battery, whereas male behavior was primarily explained by variables related to locomotion.

Effects of activity, genetic selection and their interaction on muscle metabolic capacities and organ masses in mice.

Chronic voluntary exercise elevates total daily energy expenditure and food consumption, potentially resulting in organ compensation supporting nutrient extraction/utilization. Additionally, species with naturally higher daily energy expenditure often have larger processing organs, which may represent genetic differences and/or phenotypic plasticity. We tested for possible adaptive changes in organ masses of four replicate lines of house mice selected (37 generations) for high running (HR) compared with four non-selected control (C) lines. Females were housed with or without wheel access for 13-14 weeks beginning at 53-60 days of age. In addition to organ compensation, chronic activity may also require an elevated aerobic capacity. Therefore, we also measured hematocrit and both citrate synthase activity and myoglobin concentration in heart and gastrocnemius. Both selection (HR versus C) and activity (wheels versus no wheels) significantly affected morphological and biochemical traits. For example, with body mass as a covariate, mice from HR lines had significantly higher hematocrit and larger ventricles, with more myoglobin. Wheel access lengthened the small intestine, increased relative ventricle and kidney size, and increased skeletal muscle citrate synthase activity and myoglobin concentration. As compared with C lines, HR mice had greater training effects for ventricle mass, hematocrit, large intestine length and gastrocnemius citrate synthase activity. For ventricle and gastrocnemius citrate synthase activity, the greater training was quantitatively explainable as a result of greater wheel running (i.e. 'more pain, more gain'). For hematocrit and large intestine length, differences were not related to amount of wheel running and instead indicate inherently greater adaptive plasticity in HR lines.

Preference for Western diet coadapts in High Runner mice and affects voluntary exercise and spontaneous physical activity in a genotype-dependent manner

Do animals evolve (coadapt) to choose diets that positively affect their performance abilities? We addressed this question from a microevolutionary perspective by examining preference for Western diet (WD: high in fat and sugar, but lower in protein) versus standard rodent chow in adults of both sexes from 4 lines of mice selectively bred for high levels of voluntary wheel running (High Runner or HR lines) and 4 non-selected control (C) lines. We also assessed whether food preference or substitution affects physical activity (wheel running and/or spontaneous physical activity [SPA] in the attached home cages). In experiment 1 (generation 56), mice were given 6days of wheel acclimation (as is used routinely to pick breeders in the selection experiment) prior to a 2-day food choice trial. In experiment 2 (generation 56), 17days of wheel acclimation allowed mice to reach a stable level of daily running, followed by a 7-day food-choice trial. In experiment 3 (generation 58), mice had 6days of wheel acclimation with standard chow, after which half were switched to WD for two days. In experiment 1, WD was highly preferred by all mice, with somewhat greater preference in male C mice. In experiment 2, wheel running increased and SPA decreased continuously for the first 14days of adult wheel testing, followed by 3-day plateaus in both. During the subsequent 7-day food choice trial, HR mice of both sexes preferred WD significantly more than did C mice; moreover, wheel running increased in all groups except males from C lines, with the increase being significantly greater in HR than C, while SPA declined further in all groups. In experiment 3, the effect of being switched to WD depended on both linetype and sex. On standard chow, only HR females showed a significant change in wheel running during nights 7+8, increasing by 10%. In contrast, when switched to WD, C females (+28%), HR females (+33%), and HR males (+10%) all significantly increased their daily wheel-running distances. Our results show for the first time that dietary preferences can coadapt in response to selection on activity levels.

Acute Restraint Stress Alters Wheel-Running Behavior Immediately Following Stress and up to 20 Hours Later in House Mice.

In vertebrates, acute stressors-although short in duration-can influence physiology and behavior over a longer time course, which might have important ramifications under natural conditions. In laboratory rats, for example, acute stress has been shown to increase anxiogenic behaviors for days after a stressor. In this study, we quantified voluntary wheel-running behavior for 22 h following a restraint stress and glucocorticoid levels 24 h postrestraint. We utilized mice from four replicate lines that have been selectively bred for high voluntary wheel-running activity (HR mice) for 60 generations and their nonselected control (C) lines to examine potential interactions between exercise propensity and sensitivity to stress. Following 6 d of wheel access on a 12L∶12D photo cycle (0700-1900 hours, as during the routine selective breeding protocol), 80 mice were physically restrained for 40 min, beginning at 1400 hours, while another 80 were left undisturbed. Relative to unrestrained mice, wheel running increased for both HR and C mice during the first hour postrestraint (P < 0.0001) but did not differ 2 or 3 h postrestraint. Wheel running was also examined at four distinct phases of the photoperiod. Running in the period of 1600-1840 hours was unaffected by restraint stress and did not differ statistically between HR and C mice. During the period of peak wheel running (1920-0140 hours), restrained mice tended to run fewer revolutions (-11%; two-tailed P = 0.0733), while HR mice ran 473% more than C (P = 0.0008), with no restraint × line type interaction. Wheel running declined for all mice in the latter part of the scotophase (0140-0600 hours), restraint had no statistical effect on wheel running, but HR again ran more than C (+467%; P = 0.0122). Finally, during the start of the photophase (0720-1200 hours), restraint increased running by an average of 53% (P = 0.0443) in both line types, but HR and C mice did not differ statistically. Mice from HR lines had statistically higher plasma corticosterone concentrations than C mice, with no statistical effect of restraint and no interaction between line type and restraint. Overall, these results indicate that acute stress can affect locomotor activity (or activity patterns) for many hours, with the most prominent effect being an increase in activity during a period of typical inactivity at the start of the photophase, 15-20 h poststressor.

Antidepressant treatment differentially affects the phenotype of high and low stress reactive mice

Modelling key endophenotypes can be a powerful approach to gain insight into mechanisms underlying the aetiology and pathophysiology of neuropsychiatric disorders. Based on evidence of stress hormone system dysregulations in depression, the Stress Reactivity (SR) mouse model has been generated by a selective breeding approach for extremes in HPA axis reactivity, resulting in high (HR), intermediate (IR) and low (LR) reactive mice. The characterisation of their phenotypic alterations has highlighted many similarities of HR and LR mice with the melancholic and atypical depression, respectively. We therefore aimed to examine whether the antidepressant fluoxetine (10 mg/kg/day i.p., 4–5 weeks) can ameliorate the phenotypic characteristics of HR and LR mice in neuroendocrine functions (HPA axis basal activity, stress reactivity, negative feedback), emotional reactivity/coping-strategy (open field, forced swim tests), spatial learning/memory (Morris water-maze) and hippocampal neurogenesis. Line differences in HPA axis reactivity were maintained under fluoxetine treatment. However, we observed fluoxetine effects on glucocorticoid-induced negative feedback, stress-coping behaviours, cognitive functions and neurogenesis. Specifically, our results revealed line-dependent consequences of fluoxetine treatment: (1) an amelioration of the ‘melancholic-like’ features of HR mice (reversing the negative feedback resistance, the hyperactive coping style and the memory deficits; increasing hippocampal neurogenesis); (2) an exacerbation of the phenotypic deviations of LR mice (increasing their pronounced negative feedback and passive coping style). Thus, these findings support the predictive validity of antidepressant treatment in the HR mouse line and emphasize the translational value of the SR mouse model for the development of therapeutic strategies based on endophenotype-driven classifications.

Genetic predisposition for high stress reactivity amplifies effects of early-life adversity

A dysregulation of the hypothalamus-pituitary-adrenocortical (HPA) axis and the experience of early-life adversity are both well-established risk factors for the development of affective disorders, such as major depression. However, little is known about the interaction of these two factors in shaping endophenotypes of the disease. Here, we studied the gene-environment interaction of a genetic predisposition for HPA axis dysregulation with early-life stress (ELS), assessing the short-, as well as the long-lasting consequences on emotional behavior, neuroendocrine functions and gene expression profiles. Three mouse lines, selectively bred for either high (HR), intermediate (IR), or low (LR) HPA axis reactivity, were exposed to one week of ELS using the limited nesting and bedding material paradigm. Measurements collected during or shortly after the ELS period showed that, regardless of genetic background, ELS exposure led to impaired weight gain and altered the animals’ coping behavior under stressful conditions. However, only HR mice additionally showed significant changes in neuroendocrine stress responsiveness at a young age. Accordingly, adult HR mice also showed lasting consequences of ELS, including hyperactive stress-coping, HPA axis hyperreactivity, and gene expression changes in the Crh system, as well as downregulation of Fkbp5 in relevant brain regions. We suggest that the genetic predisposition for high stress reactivity interacts with ELS exposure by disturbing the suppression of corticosterone release during a critical period of brain development, thus exerting lasting programming effects on the HPA axis, presumably via epigenetic mechanisms. In concert, these changes lead to the emergence of important endophenotypes associated with affective disorders.

Serotonin-mediated central fatigue underlies increased endurance capacity in mice from lines selectively bred for high voluntary wheel running

Serotonin (5-hydroxytryptamine; 5-HT) is implicated in central fatigue, and 5-HT1A pharmaceuticals are known to influence locomotor endurance in both rodents and humans. We studied the effects of a 5-HT1A agonist and antagonist on both forced and voluntary exercise in the same set of mice. This cohort of mice was taken from 4 replicate lines of mice that have been selectively bred for high levels of voluntary wheel running (HR) as compared with 4 non-selected control (C) lines. HR mice run voluntarily on wheels about 3× as many revolutions per day as compared with C, and have greater endurance during forced treadmill exercise. We hypothesized that drugs targeting serotonin receptors would have differential effects on locomotor behavior of HR and C mice. Subcutaneous injections of a 5-HT1A antagonist (WAY-100,635), a combination of 5-HT1A agonist and a 5-HT1A/1B partial agonist (8-OH-DPAT+pindolol), or physiological saline were given to separate groups of male mice before the start of each of three treadmill trials. The same manipulations were used later during voluntary wheel running on three separate nights. WAY-100,635 decreased treadmill endurance in HR but not C mice (dose by linetype interaction, P=0.0014). 8-OH-DPAT+pindolol affected treadmill endurance (P<0.0001) in a dose-dependent manner, with no dose by linetype interaction. Wheel running was reduced in HR but not C mice at the highest dose of 8-OH-DPAT+pindolol (dose by linetype, P=0.0221), but was not affected by WAY-100,635 treatment. These results provide further evidence that serotonin signaling is an important determinant of performance during both forced and voluntary exercise. Although the elevated wheel running of HR mice does not appear related to alterations in serotonin signaling, their enhanced endurance capacity does. More generally, our results indicate that both forced and voluntary exercise can be affected by an intervention that acts (primarily) centrally.

Abstract 10854: Ghrelin Promotes Functional Angiogenesis in a Mouse Model Of Critical Limb Ischemia Through Activation of Pro-angiogenic MicroRNAs

Background: Current therapeutic strategies for the treatment of critical limb ischemia (CLI) have only limited success. Recent in vitro evidence in the literature, using cell lines, proposes that the peptide hormone ghrelin may have angiogenic properties. Objective: To investigate if ghrelin could promote post-ischemic angiogenesis in a mouse model of CLI and identify the mechanistic pathway(s) that underpin ghrelin’s pro-angiogenic properties. Methods and Results: CLI was induced in male CD1 mice by femoral artery ligation. Animals were then randomized to receive either vehicle or ghrelin (Ghr,150μg/kg/sc) for 14 consecutive days. Subsequently, Synchrotron Radiation microangiography was used to assess hindlimb perfusion. Ischemia alone stimulated the generation of new microvessels typically having an ID &lt; 80 μm, however, the magnitude of ‘angiogenesis’ was significantly augmented in ghrelin-treated mice (13.9 ± 1.3 vessels in CLI+Ghr mice vs. 7.6 ± 0.9 vessels in CLI+Vehicle mice, P &lt; 0.05). Moreover, the newly formed vessels of only the ghrelin-treated CLI mice were both structurally and functionally normal; evident by robust endothelium-dependent vasodilatory responses to acetylcholine. Histological analysis showed marked increase in the number of arterioles in Ghrelin treated group (81±8/mm 2 in vehicle treated group vs. 134±11/mm 2 in Ghrelin treated group, P&lt;0.01, n=5), confirming the angiography data. Molecular analysis revealed that ghrelin’s angiogenic properties were linked to activation of pro-survival Akt/VEGF/Bcl-2 signaling cascade, thus reducing the apoptotic cell death and subsequent fibrosis. Furtherstill, ghrelin treatment activated pro-angiogenic (miR-126, -132 and -27a) and anti-fibrotic (miR-30a) microRNAs while inhibiting anti-angiogenic (miR-92a and -206) microRNAs. Further, in vitro knockdown of miR-126 and miR-132 in human umblical vein endothelial cells markedly attenuated the angiogenic potential of ghrelin, as evaluated by decreased proliferation and tube forming capacity (P&lt;0.05). Conclusion: Ghrelin therapy significantly restored the blood flow to the ischemic hind limb by accelerating the formation of new functional arterioles through activation of pro-angiogenic microRNAs.

Mice selected for extremes in stress reactivity reveal key endophenotypes of major depression: A translational approach

Summary Clear evidence has linked dysregulated hypothalamus–pituitary–adrenocortical (HPA) axis function to the aetiology and pathophysiology of major depression (MD), as observed in the majority of patients. Increased stress reactivity and hyperactivity of the HPA axis seem characteristic for psychotic/melancholic depression, while the atypical subtype of depression has been connected with the opposing phenotypes. However, the underlying molecular-genetic mechanisms are poorly understood. In the present study, mouse lines selectively bred for extremes in stress reactivity (SR), i.e. presenting high (HR) or low (LR) corticosterone secretion in response to stressors, were used to characterise the molecular alterations on all levels of the HPA axis. Results were contrasted with clinical phenotypes of MD patients from the Munich Antidepressant Response Signature project, stratified according to their cortisol response in the Dex/CRH test. Distinct differences between HR and LR mice were found in the expression of HPA axis-related genes in the adrenals, pituitary and selected brain areas. Moreover, HR animals presented an enhanced adrenal sensitivity, increased stress-induced neuronal activation in the PVN and an overshooting Dex/CRH test response, whereas LR animals showed a blunted response in these paradigms. Interestingly, analogous neuroendocrine, morphometric, psychopathological and behavioural differences were observed between the respective high and low HPA axis responder groups of MD patients. Our findings suggests that (i) the SR mouse model can serve as a valuable tool to elucidate HPA axis-related mechanisms underlying affective disorders and (ii) a stratification of MD patients according to their HPA axis-related neuroendocrine function should be considered for clinical research and treatment.