Behavioral assessment of intermittent wheel running and individual housing in mice in the laboratory

Physical cage enrichment—exercise devices for rodents in the laboratory—often includes running wheels. This study compared responses of mice in enriched physical and social conditions and in standard social conditions to wheel running, individual housing, and open-field test. The study divided into 6 groups, 48 female BALB/c mice group housed in enriched and standard conditions. On alternate days, the study exposed 2 groups to individual running wheel cages. It intermittently separated from their cage mates and housed individually 2 groups with no running wheels; 2 control groups remained in enriched or standard condition cages. There were no significant differences between enriched and standard group housed mice in alternate days' wheel running. Over time, enriched, group housed mice ran less. Both groups responded similarly to individual housing. In open-field test, mice exposed to individual housing without running wheel moved more and faster than wheel running and home cage control mice. They have lower body weights than group housed and wheel running mice. Intermittent withdrawal of individual housing affects the animals more than other commodities. Wheel running normalizes some effects of intermittent separation from the enriched, social home cage.     

Training Nonhuman Primates Using Positive Reinforcement Techniques

Physical cage enrichment—exercise devices for rodents in the laboratory—often includes running wheels. This study compared responses of mice in enriched physical and social conditions and in standard social conditions to wheel running, individual housing, and open-field test. The study divided into 6 groups, 48 female BALB/c mice group housed in enriched and standard conditions. On alternate days, the study exposed 2 groups to individual running wheel cages. It intermittently separated from their cage mates and housed individually 2 groups with no running wheels; 2 control groups remained in enriched or standard condition cages. There were no significant differences between enriched and standard group housed mice in alternate days' wheel running. Over time, enriched, group housed mice ran less. Both groups responded similarly to individual housing. In open-field test, mice exposed to individual housing without running wheel moved more and faster than wheel running and home cage control mice. They have lower body weights than group housed and wheel running mice. Intermittent withdrawal of individual housing affects the animals more than other commodities. Wheel running normalizes some effects of intermittent separation from the enriched, social home cage.     

Phenotypic differences in the GnRH neuronal system of deer mice Peromyscus maniculatus under a natural short photoperiod

The neural mechanisms by which short photoperiod induces gonadal regression among seasonally breeding mammals are not well understood. One hypothesis suggests that the proximate cause of seasonal gonadal regression is a photoperiod-induced modification in GnRH secretion. This hypothesis is indirectly supported by our recent findings using immunocytochemistry which identified specific photoperiod-induced adjustments in the number and morphology of GnRH containing neurones between reproductively competent and reproductively regressed laboratory housed male deer mice. Herein, we report that the GnRH neuronal system is similarly affected in reproductively responsive and nonresponsive wild male deer mice Peromyscus maniculatus exposed to a natural short photoperiod. The distribution of immunoreactive (IR)-GnRH neurones was nearly identical in field caught animals and those housed under artificial photoperiod in the laboratory. Compared with reproductively nonresponsive males, reproductively responsive mice from the field population possessed a greater total number of IR-GnRH neurones, a greater number of IR-GnRH neurones within the lateral hypothalamus, and a greater proportion of bipolar IR-GnRH neurones. Each of these distributional and morphological characters was consistent with our findings in laboratory housed male deer mice exposed to an artificial short photoperiod. Taken together, these data underscore the validity of using an artificial photoperiod to evaluate seasonal adjustments in reproductive function in the laboratory.     

Differential reproductive response to short photoperiod in deer mice: role of melatonin

Summary Inhibitory photoperiod differentially effects reproduction in deer mice (Peromyscus maniculatus nebrascensis). Pituitary-testicular function is arrested in about one-third of short-day exposed males (reproductively responsive mice), while an equal number remain fertile (reproductively nonresponsive mice). Both phenotypes are found in natural populations and their disparate reproductive responses have a genetic basis. To assess whether this difference is attributable to a prepineal/pineal or post-pineal mechanism, we compared spermatogenic responses of known and unknown phenotype to exogenous melatonin. Melatonin significantly reduced mean sperm number in long-day housed mice of unknown phenotype. But, individual responses ranged from azoospermia to normal spermatogenesis, and this range was not significantly different from that previously recorded for short-day exposed mice. Reproductively nonresponsive males were unaffected by melatonin administration when housed under long or short daylength. In contrast, melatonin significantly suppressed sperm production in reproductively responsive males housed under long photoperiod, but had no additional suppressive effect in short-day housed mice with regressed testes. These data demonstrate that melatonin is only effective in eliciting testicular regression in reproductively responsive males. Taken together, these results suggest that differential testicular response to photoperiod are caused by a post-pineal mechanism.Abbreviations LD long day - SD short day - 16L:8D 16 h light, 8 h dark - 8L:16D 8 h light, 16 h dark     

Insulin Receptor Signaling in the GnRH Neuron Plays a Role in the Abnormal GnRH Pulsatility of Obese Female Mice

Infertility associated with obesity is characterized by abnormal hormone release from reproductive tissues in the hypothalamus, pituitary, and ovary. These tissues maintain insulin sensitivity upon peripheral insulin resistance. Insulin receptor signaling may play a role in the dysregulation of gonadotropin-releasing hormone (GnRH) secretion in obesity, but the interdependence of hormone secretion in the reproductive axis and the multi-hormone and tissue dysfunction in obesity hinders investigations of putative contributing factors to the disrupted GnRH secretion. To determine the role of GnRH insulin receptor signaling in the dysregulation of GnRH secretion in obesity, we created murine models of diet-induced obesity (DIO) with and without intact insulin signaling in the GnRH neuron. Obese control female mice were infertile with higher luteinizing hormone levels and higher GnRH pulse amplitude and total pulsatile secretion compared to lean control mice. In contrast, DIO mice with a GnRH specific knockout of insulin receptor had improved fertility, luteinizing hormone levels approaching lean mice, and GnRH pulse amplitude and total secretion similar to lean mice. Pituitary responsiveness was similar between genotypes. These results suggest that in the obese state, insulin receptor signaling in GnRH neurons increases GnRH pulsatile secretion and consequent LH secretion, contributing to reproductive dysfunction.     

Behaviour of house mice artificially selected for high levels of voluntary wheel running

We have developed a novel model to study the correlated evolution of behavioural and morphophysiological traits in response to selection for increased locomotor activity. We used selective breeding to increase levels of voluntary wheel running in four replicate lines of laboratory house mice, Mus domesticus, with four random-bred lines maintained as controls. The experiment presented here tested for correlated behavioural responses in the wheel-cage complex, with wheels either free to rotate or locked (environmental factor). After 13 generations, mice from selected lines ran 2.2 times as many revolutions/day as controls on days 5 and 6 of initial exposure to wheels (10 826 versus 4890 revolutions/day, corresponding to 12.1 and 5.5 km/day, respectively). This increase was caused primarily by mice from selected lines running faster, not more minutes per day. Focal-animal observations confirmed that the increase in revolutions/day involved more actual running (or climbing in locked wheels), not an increase in coasting (or hanging). Not surprisingly, access to free versus locked wheels had several effects on behaviour, including total time spent in wheels, sniffing and biting. However, few behaviours showed statistically significant differences between the selected and control lines. Selection did not increase the total time spent in wheels (either free or locked), the frequency of nonlocomotor activities performed in the wheels, nor the amount of locomotor activity in cages attached to the wheels; as well, selection did not decrease the amount of time spent sleeping. Thus, wheel running is, at the genetic level, a largely independent axis of behaviour. Moreover, the genetic architecture of overall wheel running and its components seem conducive to increasing total distance moved without unduly increasing energy or time-related costs. The selection experiment also offers a new approach to study the proximate mechanisms of wheel-running behaviour itself. For example, frequencies of sniffing and wire biting were reduced in selected females but not males. This result suggests that motivation or function of wheel running may differ between the sexes. Copyright 1999 The Association for the Study of Animal Behaviour.     

Adult female prairie voles and meadow voles do not suppress reproduction in their daughters

Reproductive suppression of young females by conspecific females has been reported from laboratory studies on several species of rodents, including the prairie vole, Microtus ochrogaster, but not meadow voles, M. pennsylvanicus. We exposed female prairie voles and meadow voles to two treatments: a mother and one 23-26-day-old daughter paired with a strange male and a 23-26-day-old daughter paired with a different strange male. We found no differences in the proportion of daughters breeding or the time to sexual maturation for daughters raised in the two treatments for either species. Thus, we have no indication that mothers had any adverse effect on reproductive efforts of their daughters. These results differ from previous studies that concluded young female prairie voles were reproductively suppressed by female relatives. The difference between our and previous studies on reproductive suppression is that we examined breeding in young females rather than proximate measures of growth and reproductive development. We question the evolutionary significance of reproductive suppression among related female microtine rodents, especially in that it has not been documented from field populations.     

Response to selection for photoperiod responsiveness on the density and location of mature GnRH-releasing neurons

Natural variation in neuroendocrine traits is poorly understood, despite the importance of variation in brain function and evolution. Most rodents in the temperate zones inhibit reproduction and other nonessential functions in short winter photoperiods, but some have little or no reproductive response. We tested whether genetic variability in reproductive seasonality is related to individual differences in the neuronal function of the gonadotropin-releasing hormone network, as assessed by the number and location of mature gonadotropin-releasing hormone-secreting neurons under inhibitory and excitatory photoperiods. The experiments used lines of Peromyscus leucopus previously developed by selection from a wild population. One line contained individuals reproductively inhibited by short photoperiod, and the other line contained individuals nonresponsive to short photoperiod. Expression of mature gonadotropin-releasing hormone (GnRH) immunoreactivity in the brain was detected using SMI-41 antibody in the single-labeled avidin-biotin-peroxidase-complex method. Nonresponsive mice had 50% more immunoreactive GnRH neurons than reproductively inhibited mice in both short- and long-day photoperiods. The greatest differences were in the anterior hypothalamus and preoptic areas. In contrast, we detected no significant within-lines differences in the number or location of immunoreactive GnRH neurons between photoperiod treatments. Our data indicate that high levels of genetic variation in a single wild population for a specific neuronal trait are related to phenotypic variation in a life history trait, i.e., winter reproduction. Variation in GnRH neuronal activity may underlie some of the natural reproductive and life history variation observed in wild populations of P. leucopus. Similar genetic variation in neuronal traits may be present in humans and other species.     

Response of Sod-2 enzyme activity to selection for high voluntary wheel running

The objective of this study was to examine the correlated response of anti-oxidant enzyme activity to selective breeding for increased voluntary wheel running in house mice. Activity of liver superoxide dismutase-2 (Sod-2), a free radical scavenger, was measured in four groups of mice. 'Active' individuals were housed in cages with attached wheels for 8 weeks beginning at weaning; 'sedentary' individuals were housed in cages with attached wheels that were prevented from rotating. Both of these treatments were applied to male and female mice from generation 14 of a replicated artificial selection experiment, which is composed of four lines selected for high wheel running and four randomly bred lines that serve as controls. In females, Sod-2 activity was significantly lower in selected vs control animals, regardless of presence/absence of a free-turning wheel. This difference suggests a trade-off between early-age voluntary wheel-running activity and Sod-2 activity. In males, Sod-2 activity was significantly affected by an interaction between selection group and activity group, with males from selected lines having lower Sod-2 activity relative to control males only in the sedentary treatment. These negative correlated responses of Sod-2 activity to selection on wheel running are discussed in the context of antagonistic pleiotropy models of aging and with respect to potential effects on lifespan.     

Plasma adiponectin is increased in mice selectively bred for high wheel-running activity, but not by wheel running per sé.

Mice selectively bred for high wheel-running activity (S) have decreased fat content compared to mice from randomly bred control (C) lines. We explored whether this difference was associated with alterations in levels of circulating hormones involved in regulation of food intake and energy balance, and whether alterations were caused by the presence of a running wheel. Plasma levels of leptin, adiponectin, and corticosterone as well as body composition were analyzed in male S mice housed with (+) and without (-) access to running wheels at ages of 10 and 18 months. These levels were compared to those found in C+ mice. Plasma corticosterone did not differ among groups. While plasma leptin levels tended to be lower in S+ mice as compared to S- or C+ mice, these differences were largely attributable to differences in fat content. Adiponectin levels were increased in S mice (+60%) compared to C mice, irrespective of wheel access. High levels of this hormone may be a trait co-segregated in mice bred for high wheel-running activity.     

Nocturnal to Diurnal Switches with Spontaneous Suppression of Wheel-Running Behavior in a Subterranean Rodent

Several rodent species that are diurnal in the field become nocturnal in the lab. It has been suggested that the use of running-wheels in the lab might contribute to this timing switch. This proposition is based on studies that indicate feed-back of vigorous wheel-running on the period and phase of circadian clocks that time daily activity rhythms. Tuco-tucos (Ctenomys aff. knighti) are subterranean rodents that are diurnal in the field but are robustly nocturnal in laboratory, with or without access to running wheels. We assessed their energy metabolism by continuously and simultaneously monitoring rates of oxygen consumption, body temperature, general motor and wheel running activity for several days in the presence and absence of wheels. Surprisingly, some individuals spontaneously suppressed running-wheel activity and switched to diurnality in the respirometry chamber, whereas the remaining animals continued to be nocturnal even after wheel removal. This is the first report of timing switches that occur with spontaneous wheel-running suppression and which are not replicated by removal of the wheel.     

Wheel running in the wild

The importance of exercise for health and neurogenesis is becoming increasingly clear. Wheel running is often used in the laboratory for triggering enhanced activity levels, despite the common objection that this behaviour is an artefact of captivity and merely signifies neurosis or stereotypy. If wheel running is indeed caused by captive housing, wild mice are not expected to use a running wheel in nature. This however, to our knowledge, has never been tested. Here, we show that when running wheels are placed in nature, they are frequently used by wild mice, also when no extrinsic reward is provided. Bout lengths of running wheel behaviour in the wild match those for captive mice. This finding falsifies one criterion for stereotypic behaviour, and suggests that running wheel activity is an elective behaviour. In a time when lifestyle in general and lack of exercise in particular are a major cause of disease in the modern world, research into physical activity is of utmost importance. Our findings may help alleviate the main concern regarding the use of running wheels in research on exercise.     

Photoperiod and temperature interact to affect the GnRH neuronal system of male prairie voles (Microtus ochrogaster)

Individuals of numerous species limit energy expenditure during winter by inhibiting reproduction and other nonessential functions. To time these adaptations appropriately with the annual cycle, animals rely on environmental cues that predict, well in advance, the onset of winter. The most commonly studied environmental factor that animals use to time reproduction is photoperiod. Rodents housed in short photoperiods in the laboratory or in naturally declining day lengths exhibit pronounced alterations in reproductive function concomitant with alterations in the hypothalamic gonadotropin-releasing hormone neuronal system. Because animals in their natural environment use factors in addition to photoperiod to time reproduction, the present study sought to determine the independent effects of photoperiod and temperature, as well as the interaction between these factors, on reproductive parameters and the GnRH neuronal system. Male prairie voles were housed in either long (LD 16:8) or short (LD 8:16) day lengths for 10 weeks. Animals in each photoperiod were further subdivided into groups housed in either mild (i.e., 20 degrees C) or low (i.e., 8 degrees C) temperatures. As shown with immunohistochemistry, voles that underwent gonadal regression in response to short photoperiods and long-day voles housed in low temperatures (and maintained large gonads) exhibit higher GnRH-immunoreactive (GnRH-ir) neuron numbers in the preoptic area/anterior hypothalamus (POA/AH) relative to all other groups. In addition, voles that underwent gonadal regression in response to both short days and low temperatures did not exhibit an increase in GnRH-ir neuron numbers compared to long-day, mild-temperature controls. These data suggest that photoperiod and temperature interact to influence reproductive function potentially by alterations of the GnRH neuronal system.     

Voluntary exercise delays monogenetic obesity and overcomes reproductive dysfunction of the melanocortin-4 receptor knockout mouse

The melanocortin system is involved in hypothalamic regulation of energy homeostasis. The melanocortin-4 receptor (MC4R) has been linked to both obesity and reproductive dysfunction. Deletion of the MC4R from the mouse genome has resulted in phenotypes including adult onset obesity, hyperphagia, and difficulty in reproducing when homozygote parents are bred. Additionally, polymorphisms of the human MC4R have been identified in morbidly obese children and adults. Herein, we have identified that voluntary exercise, provided via the presence of a running wheel, impedes the monogenetic obesity (at 20 weeks of age running wheel housed body weight=31+/-1.8 g versus conventionally housed body weight=41+/-2.3 g, a 25% decrease in body weight p<0.01), hyperphagia (average cumulative food intake is not statistically different than wild type mice housed in running wheel cages), and reproductive dysfunction phenotypes associated with the MC4R knockout mice housed by conventional means. These data demonstrate the novel finding that voluntary exercise at a young age may hinder genetically induced obesity.     

Effects of early‐life exposure to Western diet and wheel access on metabolic syndrome profiles in mice bred for high voluntary exercise

Experimental studies manipulating diet and exercise have shown varying effects on metabolic syndrome components in both humans and rodents. To examine the potential interactive effects of diet, exercise and genetic background, we studied mice from four replicate lines bred (52 generations) for high voluntary wheel running (HR lines) and four unselected control lines (C). At weaning, animals were housed for 60 days with or without wheels and fed either a standard chow or Western diet (WD, 42% kcal from fat). Four serial (three juvenile and one adult) blood samples were taken to measure fasting total cholesterol (TC), high‐density lipoprotein cholesterol (HDL‐C), triglycerides and glucose. Western diet was obesogenic for all mice, even after accounting for the amount of wheel running and kilojoules consumed. Western diet significantly raised glucose as well as TC and HDL‐C concentrations. At the level of individual variation (repeatability), there was a modest correlation (r = 0.3–0.5) of blood lipids over time, which was reduced with wheel access and/or WD. Neither genetic selection history nor wheel access had a statistically significant effect on blood lipids. However, HR and C mice had divergent ontogenetic trajectories for body mass and caloric intake. HR mice also had lower adiposity, an effect that was dependent on wheel access. The environmental factors of diet and wheel access had pronounced effects on body mass, food consumption and fasting glucose concentrations, interacting with each other and/or with genetic strain. These data underscore the importance (and often unpredictable nature) of genotype‐by‐environment and environment‐by‐environment interactions when studying body weight regulation.     

Social interactions unmask sex differences in humoral immunity in voles

Sex differences in immune function are well established among laboratory rodents, with males typically having lower immunity than females. This sex difference may reflect the suppressive effects of testosterone on immune function. Because polygynous males generally have higher circulating testosterone concentrations than monogamous males, sex differences in immune function are hypothesized to be more pronounced among polygynous as compared to monogamous species. Sex differences in immune function have not been consistently observed among individually housed Microtus in the laboratory; thus, social interactions are hypothesized to be necessary for the expression of sex differences in immune function. We assessed the effect of differential housing conditions on humoral immunity and steroid hormone concentrations in polygynous meadow voles Microtus pennsylvanicus, and monogamous prairie voles M. ochrogaster. We examined humoral immunity by immunizing voles with keyhole limpet haemocyanin (KLH) and measuring antibody production 5, 10, 15 and 30 days postimmunization. Overall, meadow voles mounted higher anti-KLH immunoglobulin (Ig)M and IgG responses than prairie voles, regardless of the housing condition. Sex differences in antibody production were only observed among meadow voles housed in pairs, in which females had higher anti-KLH IgM and IgG responses than males. Sex differences in antibody production were not observed among prairie voles or meadow voles housed individually. Sex and species differences in circulating oestradiol, testosterone, and corticosterone concentrations were not related to differences in humoral immunity. These data suggest that sex differences in immune function are more pronounced among polygynous species than monogamous species, but may be context dependent. Copyright 1999 The Association for the Study of Animal Behaviour.     

The thalamic intergeniculate leaflet mediates locomotor activity-induced reversal of phenotype in photoperiod nonresponsive Siberian hamsters

The role of the intergeniculate leaflet of the thalamus (IGL) in photoperiod responsiveness was examined in a laboratory-selected line of photoperiod nonresponsive (NR) Siberian hamsters. NR hamsters fail to exhibit typical winter-type responses (i.e., gonadal regression and development of winter-type pelage) when exposed to short day lengths (e.g., 10 h of light/day). Earlier studies revealed that NR hamsters will exhibit winter-type responses when exposed to short photoperiod if they are given free access to a running wheel. The present study tested the hypothesis that this locomotor activity-induced reversal of phenotype is dependent on the IGL. Male NR hamsters underwent destruction of the IGL prior to being housed in short day lengths in cages equipped with running wheels. Activity rhythms were monitored for 8 weeks, after which time pelage response and paired testes weights were obtained. In contrast to sham-operated NR animals given access to running wheels, IGL-ablated animals showed no increase in the duration of nocturnal running wheel activity and became active later in the night than sham-lesioned animals. Lesioned animals also failed to exhibit the typical short photoperiod-induced gonadal regression and pelage molt. The results implicate the IGL in the mechanism by which running wheel activity can influence photoperiodic responses.     

Behavioral feedback regulation of circadian rhythm phase angle in light-dark entrained mice

Induced and spontaneous wheel running can alter the phase and period (tau) of circadian rhythms in rodents. The relationship between spontaneous running and the phase angle (psi) of entrainment to 24-h light-dark (LD) cycles was evaluated in C57BL/6j mice. With a wheel freely available, psi was significantly correlated with the absolute (r = 0.32) and relative (r = 0.44) amount of activity during the first 2 h of the activity period. When wheels were locked during the first half of the night in LD and then unlocked in constant dark (DD), mice exhibited a delayed psi and lengthened tau compared with mice that had wheels locked during the second half of the night. In DD, tau correlated negatively with total daily activity. To evaluate if wheel running modulates the phase-resetting actions of LD, phase shifts to light pulses were measured at two time points in DD, when daily activity levels differed by 40%. Phase delays to light were 56% greater when activity levels were lower. However, in a counterbalanced follow-up experiment, phase advances and delays to light pulses were not affected by the availability of wheels, although an effect of time in DD was replicated. Spontaneous activity can regulate psi and tau without altering the response of the pacemaker to light.     

Patterns of wheel running are related to Fos expression in neuropeptide-Y-containing neurons in the intergeniculate leaflet of Arvicanthis niloticus

A variety of nonphotic influences on circadian rhythms have been documented in mammals. In hamsters, one such influence, running in a novel wheel, is mediated in part by the pathway extending from neuropeptide-Y (NPY)-containing cells within the intergeniculate leaflet (IGL) of the thalamus to the hypothalamic suprachiasmatic nucleus (SCN). Arvicanthis niloticus is a species in which all individuals are diurnal with respect to general activity and body temperature when they are housed without a running wheel, but access to a running wheel induces a subset of individuals to become nocturnal. In the first study, the authors evaluated the possibility that nocturnal and diurnal patterns of wheel running in Arvicanthis are correlated with differences in IGL function. Adult male Arvicanthis housed in a 12:12 light-dark (LD) cycle were monitored in wheels, classified as nocturnal or diurnal, and then perfused either 4 h after lights-on or 4 h after lights-off. Sections through the intergeniculate leaflet were processed for immunohistochemical labeling of Fos and NPY. The percentage of NPY cells that expressed Fos was significantly influenced by an interaction between time of day and phenotype such that it rose from night to day in diurnal animals, and from day to night in nocturnal animals. In the second experiment, the authors established that running in a wheel actually induces Fos in the IGL of Arvicanthis. Specifically, the proportion of NPY cells expressing Fos was increased by access to wheels in nocturnal animals at night and in diurnal animals during the day. In the third experiment, the authors established that lesions of the IGL eliminate NPY fibers within the SCN, suggesting that these IGL cells project to the SCN in this species as has been established in other rodents. Together, these data demonstrate a clear difference in NPY cell function in nocturnal and diurnal Arvicanthis that appears to be caused, at least in part, by the differences in their wheel-running patterns, and that NPY cells within the IGL project to the SCN in Arvicanthis.