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Pharmacology, Biochemistry and Behavior 101 (2012) 528–537 Contents lists available at SciVerse ScienceDirect Pharmacology, Biochemistry and Behavior Sex differences in cannabinoid receptor-1 (CB1) pharmacology in mice selectivelybred for high voluntary wheel-running behavior Brooke K. Keeney a, Thomas H. Meek a, Kevin M. Middleton a,b, Loana F. Holness a, Theodore Garland, Jr. a,⁎a University of California, Riverside, Riverside, CA, 92521, USAb California State University San Bernardino, San Bernardino, CA, 92407, USA The endocannabinoid system (ECS) is involved in regulation of various physiological functions, including Received 15 June 2011 locomotion, antinociception, emotional states, and motivated behaviors. The ECS has been implicated in Received in revised form 7 February 2012 regulation of voluntary wheel running in mice via actions at the cannabinoid receptor-1 (CB1). Previously, Accepted 26 February 2012 we showed that four replicate lines of mice bred for high levels of voluntary wheel running (high-runner Available online 1 March 2012 or HR lines) sex-specifically (females only) decreased running in response to antagonism of the CB1 receptor,as compared with four unselected Control lines. Here, we administered a CB1 receptor agonist, WIN 55,212-2 (WIN). We predicted that if CB1 activation is involved in the regulation of voluntary wheel running, then HR mice would show a greater response to CB1 agonism. Following our previous protocols, mice from generation 53 were acclimated to running wheels for 24 days, then received, in random order, either an intra-peritoneal Experimental evolution injection of vehicle or a low (0.5 mg/kg), medium (1 mg/kg) or high dosage (3 mg/kg) of WIN. Each mouse received an injection and then experienced two nights without injections, for a total period of 12 days.
Locomotor activity Response to WIN was quantified as wheel revolutions, time spent running, and average running speed in Locomotor performance the 10–120 min immediately following injection. Injection decreased wheel revolutions in all mice, but male HR mice decreased their running to a greater degree relative to Controls in response to the high dose Selective breeding of WIN over the entire period analyzed, whereas HR females showed a differential response relative to Sex differencesVoluntary exercise Controls only in the latter 70–120 min post-injection. These results, in conjunction with our previous study, show that (a) aspects of endocannabinoid signaling have diverged in four lines of mice bred for highlevels of voluntary exercise and (b) male and female HR mice differ from one another in CB1 signaling as itrelates to wheel running.
2012 Elsevier Inc. All rights reserved.
HR mice of both sexes ran at least 70% more revolutions/day thantheir Control counterparts (Swallow et al., 1998). The divergence Understanding the control of voluntary behavior is one of the between HR and Control lines eventually reached a plateau at a differ- greatest challenges for neurobiology. In particular, knowledge of how ential of approximately +170% (Kolb et al., 2010; Rhodes et al., 2000; the brain motivates imperative, yet potentially costly behaviors – such Swallow et al., 2009). Concomitant with increases in voluntary wheel as voluntary exercise – is of great relevance to an increasingly inactive running, HR mice have also undergone a shift toward increased levels human population (Garland et al., 2011a). The literature reflects an of spontaneous physical activity (SPA) in cages when wheels are almost endless array of approaches to study the neurobiology of absent (Malisch et al., 2009; Rhodes et al., 2001). Likewise, when exercise; we have chosen to use a long-term selection experiment running wheels were locked, HR mice spent more time climbing in that targets high levels of voluntary wheel running in mice (Garland the locked wheels, apparently trying to run (Koteja et al., 1999).
et al., 2011b; Rhodes et al., 2005; Swallow et al., 2009).
In addition to changes in locomotor behavior (see also Girard et al., Within this experiment, four independent, genetically closed lines 2001), the selective breeding regimen has led to changes in capacities of house mice (Mus domesticus) have been selectively bred since 1993 for aerobic exercise (Kolb et al., 2010; Meek et al., 2009), and in various (>60 generations) on the basis of their voluntary wheel running on lower-level morphological and physiological traits that may affect days 5 and 6 of a 6-day trial (High Runner lines, HR), in parallel endurance capacity (Garland, 2003). For example, HR mice exhibit with four unselected Control lines. After 10 generations of selection, reduced total body mass (Swallow et al., 1999), reduced body fat(Meek et al., 2010; Swallow et al., 2001; Vaanholt et al., 2008), moresymmetrical hind limb bones (Garland and Freeman, 2005), higher ⁎ Corresponding author at: Department of Biology, University of California, Riverside, circulating corticosterone (Girard and Garland, 2002; Malisch et al., Riverside, CA, 92521, USA.
E-mail address: tgarland@ucr.edu (T. Garland,).
2008) and adiponectin levels (Vaanholt et al., 2007), as well as 0091-3057/$ – see front matter 2012 Elsevier Inc. All rights reserved.
doi:10.1016/j.pbb.2012.02.017 Author's personal copy
B.K. Keeney et al. / Pharmacology, Biochemistry and Behavior 101 (2012) 528–537 increased plasticity of some traits in response to wheel access (Gomes receptor was blocked, female HR mice decreased running to a greater et al., 2009).
degree than male HR mice or those from Control lines. Although that Although it is currently not well understood to what degree study was the first to use pharmacology on both sexes of HR mice to voluntary exercise can be considered a classical motivated behavior demonstrate possible differences in neural correlates of wheel running, (Garland et al., 2011a), several studies in HR mice have documented we have long known that male and female HR mice have responded divergences in the neural systems traditionally thought to regulate differently to selective breeding for high wheel running (Garland et al., motivation and reward (see also Bronikowski et al., 2004; Belke and 2011b; Keeney et al., 2008). Specifically, female HR mice have evolved Garland, 2007). For instance, studies found linetype wheel-running their higher daily running distances almost entirely by increasing the differences in response to drugs that affect the D1 receptor system speed at which they run, whereas males have shown increases in both (but not in the D2 receptor, serotonergic or opioidergic systems) (Li et the speed and duration of wheel activity (Garland, 2003; Girard et al., al., 2004; Rhodes et al., 2001, 2003, 2005; Rhodes et al., 2001; Rhodes 2001; Keeney et al., 2008; Koteja and Garland, 2001; Rezende et al., and Garland, 2003). In addition, Fos immunohistochemistry showed 2009; Rhodes et al., 2000; Swallow et al., 1998, 1999).
HR mice to have a greater proportional increase in activity in some It is well known that males and females of many species may brain regions implicated in reward and motivation when wheel access accomplish a given behavior in different ways, often as a result of is blocked, consistent with a state of withdrawal (Rhodes et al., 2003).
the influence of either androgens or hormones of the estrous cycle. It Mathes et al. (2010) hypothesized that many of these differences reflect is not definitively known to what extent such behaviors as voluntary an overall dysregulation of dopaminergic signaling.
exercise differ by sex, nor how these putative differences manifest in Alongside alterations in dopaminergic and reward signaling, we the brain (see Lightfoot, 2008 for review). The HR and their Control hypothesized that HR mice differ from Controls in their response to lines offer a unique system in which to explore the neurobiological drugs that act upon one of the major receptors (cannabinoid receptor- underpinnings of an "exerciser" phenotype, as well as how these 1; CB1) of the endocannabinoid system (ECS) (Keeney et al., 2008). The systems may differ between the sexes. Given the literature, and our ECS is a complex modulatory system, primarily composed of cannabinoid previous results for this system, we believe that the CB1 receptor is receptors, their endogenous ligands (endocannabinoids), and proteins important in the neural control of voluntary exercise in general, and involved in the synthesis and modification of endocannabinoids.
specifically to the evolution of the HR phenotype. The role of CB1 Although the role of central cannabinoid signaling as mediated by the transmission in sex-specific voluntary exercise is not yet clear; nor is CB1 receptor is not fully understood, cannabinoids likely have a natural it clear if stimulation of this receptor would have similar results by role in antinociception, memory, the perception of natural rewards, and sex or by linetype (HR or Control). To further address these questions, the regulation of complex locomotor outputs (particularly those paired we administered a CB1 receptor agonist (WIN 55,212-2) to HR and with rewarding stimuli) (De Chiara et al., 2010; Iversen, 2003).
Control mice of both sexes and observed their subsequent wheel Much evidence suggests a relationship between endocannabinoid running (at the time of peak nightly activity). We predicted that if, signaling and physical activity (see Fuss and Gass, 2010 for review).
as indicated by our previous study (Keeney et al., 2008), there are Recent discussion has highlighted the tight involvement of central sex-specific differences in ECS physiology that underlie high levels of endocannabinoid activity with the expression of motor behavior (El voluntary wheel running, then male and female HR mice will differ Manira and Kyriakatos, 2010), and in particular, voluntary running from their Control counterparts in their wheel-running response to (Chaouloff et al., 2011). Specific to rodent systems, it has been CB1 agonism.
suggested that the ECS may regulate wheel-running behavior(Chaouloff et al., 2011; Dubreucq et al., 2010). To that end, Hill et al.
(2010) have shown that wheel running, a form of voluntary exercise in rodents (Garland et al., 2011a), increases both CB1 signaling andthe concentration of anandamide within the hippocampal formation of rats. Likewise, mouse synaptic responses to HU210, a selectivecannabinoid CB1 receptor agonist, were greatly potentiated following The subjects of study were male and female mice (M. domesticus), 7 or 15 days of wheel access (De Chiara et al., 2010). Similarly, two originally derived from Hsd:ICR stock (Harlan Sprague Dawley, weeks of voluntary wheel access was found to sensitize CB1 Indianapolis, Indiana, USA). As discussed in detail elsewhere receptor-mediated inhibition of striatal GABAergic transmission in (Swallow et al., 1998), four lines were designated for selection for mice (Rossi et al., 2009). CB1 knockout mice showed less voluntary high voluntary wheel running on days 5 and 6 of 6-day period of wheel running over a period of 6 weeks as compared with their wheel access (High Runner or HR lines), while four additional lines wildtype counterparts (Dubreucq et al., 2010); however, they did were maintained without selective breeding to serve as controls for not differ statistically in locomotion in an activity cage, exploration random genetic effects, including drift (Control lines). In brief, the in an open field, or immobility time in the forced swim test. Similarly, general selection protocol is as follows. Following birth, mice are Chaouloff et al. (2011) found that male mice lacking CB1 receptors weighed, toe-clipped for individual identification, and weaned at display decreased voluntary running when housed with a running 21 days of age. Mice are then housed four/cage/sex by line. At wheel for several weeks when compared to wild-type littermates 6–8 weeks of age, mice are housed individually in cages with wheel (attributable to a decrease in the time spent running). In humans, (1.12 m circumference) access for a 6-day period. Wheel revolutions parallels between the psychotropic effects of traditional cannabinoid are recorded daily in 1-minute intervals by a photocell counter drugs and the positive feelings associated with sustained, endurance- attached to the wheel. Revolutions are compiled via customized type exercise has led some to hypothesize that endocannabinoids may software (San Diego Instruments, San Diego, California, USA). Following be involved, at least in part, with a so-called "runner's high" sensation wheel-testing, breeders are selected for the next generation. In the HR that may help to motivate exercise behaviors (Dietrich and McDaniel, lines, a male and a female mouse from each family are selected for 2004). Consistent with this hypothesis, Sparling et al. (2003) showed having the highest total revolutions during days 5 and 6 of the wheel- that anandamide, the major endogenous ligand of the ECS, is increased running trial. In the Control lines, a male and a female mouse from in the circulation following exercise in trained male college students.
each family are chosen without regard to wheel revolutions. Breeders In the context of our system, we have previously shown that HR are then randomly paired within each line, with the exception that mice have a differential and sex-specific wheel-running response to sibling pairs are not allowed. Throughout the selection process, and in intraperitoneal (i.p.) injection of a selective CB1 antagonist (SR141716; all studies described here, mice are maintained on a standard 12-h Rimonabant) (Keeney et al., 2008). When transmission at the CB1 light/dark cycle, with ad lib access to water and food.
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B.K. Keeney et al. / Pharmacology, Biochemistry and Behavior 101 (2012) 528–537 Following our previous protocol (Keeney et al., 2008), mice for the 2.3. Statistical analysis current study were chosen from among those that underwent theroutine 6-day wheel-running trial. Our sample excluded both the During the course of experimentation, a total of 4 males and 1 highest and lowest runners from each family. Of the remaining female were eliminated from the sample because of death before or mice, one male or female was chosen from each family for a total during experimentation (3 males), or because they were observed sample size of 96 (48 males and 48 females), equally representing to exhibit twirling behavior (running in rapid, small, stereotypic all 8 independently breeding lines (4 HR, 4 Control). Mice were circles) in their cages (1 male and 1 female). Individuals with injection allowed 24 days of acclimation to wheels prior to drug testing.
problems, wheel malfunction or injury were excluded from analysis While with wheel access, mice were maintained on a 12:12 photoperiod on a night-by-night basis. Thus, 44 males and 47 females were statis- with lights on at 03:00 h and lights off at 15:00 h. Placement of mice tically analyzed (total N = 91).
with wheels was randomized with respect to sex and line, and experi- Statistical analyses were performed using SAS version 9.1 (SAS menters were blind to sex, line, and linetype (HR or Control). Animal Institute, Cary, NC, USA). Analyses were first conducted separately procedures were in accordance with University guidelines and with by sex. The primary grouping factors were linetype (HR vs. Control) the National Institutes of Health Guide for the Care and Use of Laboratory and dose, with replicate line as a random effect nested within linetype.
Individual was the factor for repeated measures, and we assumedcompound symmetry of the residual covariance matrix in SASProcedure Mixed. In this mixed-model analysis of covariance, the 2.2. Drug protocol degrees of freedom for testing the effect of linetype, relative to line,are always 1 and 6. For dose and the dose *linetype interaction (tested WIN 55,212-2 (WIN) was obtained from BIOMOL International, LP relative to the dose *line [linetype] effect), degrees of freedom are 2 (Enzo Life Sciences International, Inc., Plymouth Meeting, PA), and and 12. This interaction term is of chief interest because, if statistically then dissolved in a vehicle solution of DMSO (20% final volume), significant, it indicates a differential response of the HR and Control Tween-80 (10% final volume), and physiological saline (70% final lines to the drug dose. Wheel-freeness (a measure of how long each volume). Vehicle solutions were added in the order listed, with vigorous wheel rotates following acceleration to a constant velocity) was vortexing between steps. This vehicle has been used for the delivery of recorded four days prior to injections and was included as a covariate WIN in both rats (French, 1997; Hoffman et al., 2003) and mice in statistical analyses, as was individual age. After inspection of the (Kochman et al., 2006). This vehicle solution does not by itself influence residuals from the statistical models, all wheel-running traits were open-field locomotor behavior in mice (Gerdeman et al., 2008), nor transformed by raising to the 0.6 power in order to reduce skewness.
extinguish nightly wheel running in mice from the HR and Control For analyses of proportional responses, all values were log10- lines (Keeney et al., 2008).
transformed prior to analyses.
All drug solutions were prepared fresh immediately prior to use.
On the 25th night of wheel-access, mice were divided into three batches to minimize the length of any disturbance during the activeperiod. Batches were randomized by line and sex. At two hours 3.1. Baseline wheel running and effects of vehicle injection after lights-off (17:00 h), during typical peak wheel-running activity(Girard et al., 2001; Girard and Garland, 2002; Malisch et al., 2008; For generation 53, 553 mice representative of all eight lines Rhodes et al., 2003), a single batch received treatments. The total underwent the standard 6-day wheel test (N= 272 females, 281 injection period for a batch was roughly two hours (17:00 h–19:00 h).
males). Females from the HR lines (10,004 ±913 rev/day; least- Each mouse in a batch received one of four treatments (vehicle squares mean ±standard error) ran 3.01-fold more than Control females injection; low WIN [0.5 mg/kg]; medium WIN [1 mg/kg]; high WIN (3323 ± 387 rev/day) on days 5 + 6 (p=0.0005). HR males (7126 ± [3 mg/kg]) via i.p. injections. Doses were chosen based upon review of 490 rev/day) ran 2.7-fold more than Control males (2636 ±493 rev/ the literature, with specific attention to Patel and Hillard (2006).
day) on days 5+ 6 (p=0.0007).
Injection volumes were adjusted for dose and body-mass of the animal.
Results were similar for the subset of males and females used in Over the entire experimentation period, injection volumes ranged from the present experiment (47 males and 47 females from the present 0.104 to 0.242 mL for body masses that ranged from 20.7 g to 48.4 g.
study underwent wheel testing; one male and female did not, but Following the design of Li et al. (2004) and Keeney et al. (2008), were included as replacements at a later date due to unexpected each mouse received one treatment per three-night period until mortality). Females from the HR lines (8749 ± 843 rev/day) ran 2.92- every individual had been injected with every treatment (12 nights fold more than Control females (2995 ± 490 rev/day) on days 5 + 6 total), with 72 h between each injection to avoid carryover effects. Six (p= 0.0011). For males, HR ran 6599 (±549) as compared with 2680 individuals from batch one were initially tested at a higher maximum (±392) revolutions/day for Control, yielding a 2.46-fold differential dose level (10 mg/kg WIN) but did not display any wheel running (or (p= 0.0011). As expected, Online Supplemental Figure A shows that activity at all) over the entire testing period. This dose was subsequently HR mice ran significantly more than C mice in the three days prior to abandoned, and these individuals received an extra night of injections, injections. Likewise, females of both linetypes (HR and C) ran more such that they received all four doses comparable to the rest of the than males of both linetypes.
sample, making 13 total nights of injections for this subset.
Fig. 1 shows the average wheel-running during the 30 min prior to Following Keeney et al. (2008), the acute locomotor response to injections (an hour and forty five minutes past lights off), as well as in treatment was measured as the total number of wheel revolutions each 10-minute interval during the first 10–130 min post-injection.
in the period from 10 to 70 min, and also 70 to 120 min post- Prior to injections, females from the HR lines (273 ± 18 rev/10 min) injection (not analyzed in our previous study). These time points are were running 2.95-fold more than Control females (94 ± 18) consistent with the known time course of WIN in mice (Spina et al., (p= 0.022). For males, HR ran 252 (±11 rev, p = 0.0001) as compared 1998), as well as injection effects on HR wheel-running (Keeney et with 95 (±11 rev, p b 0.0001) revolutions/10 min for Control, yielding a al., 2008; Li et al., 2004). In addition to wheel revolutions, we also 2.65-fold differential (p b 0.0001). Following vehicle injection, a analyzed the number of 1-minute intervals with at least one revolution repeated-measures ANCOVA for the difference between the average (time spent running), the average running speed (revolutions/minute), wheel-running during the 30 min prior to injection and the average and the maximum running speed (revolutions in the single highest wheel-running during the first 10–70 min following injection (with 1-minute interval) for the same time periods.
covariates of age and wheel-freeness) shows that both female HR


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B.K. Keeney et al. / Pharmacology, Biochemistry and Behavior 101 (2012) 528–537 Fig. 1. Wheel running revolutions in 10-min bins during intraperitoneal WIN 55,212-2 injections (high WIN (3 mg/kg); medium WIN (1 mg/kg); low WIN (0.5 mg/kg)). (First10-min period after injection is omitted.) Values at −15 min are pooled revolutions in the 30-min period before injections. Values are simple means and standard errors. Pointsare centered on the 5-min mid-point (i.e. the point for the 11–20 min bin is located at 15 min), but have been offset slightly for clarity. WIN 55,212-2 reduced wheel running acutelyin all mice, but for males (lower panel) the reduction was significantly greater for High Runner (HR) lines than for Control lines at the highest dose (Table 1, p for linetype × doseinteraction = 0.0004; Online Supplemental Table B).
(−86±13 rev/10 min, p=0.0007) and Control mice (−36±14, analyzed in our previous study). For female mice in the first p = 0.0420) significantly decrease their wheel running, with the 10–70 min post-injection, the reduction in wheel revolutions decrease being significantly greater for HR females (p=0.0424). The depended on both dose and linetype. Likewise, there was a significant trends were similar for males, with HR decreasing by 86 rev/10 min effect of both dose and linetype on the average and maximum speed, (±17, p=0.0025) and Controls decreasing by 32 rev/10 min (±17 rev, but only a statistically significant effect of dose for the amount of time p=0.0975), and the effect of linetype marginally nonsignificant spent running. Results were similar 70–120 min post-injection for (p=0.0674). These decreases may be the result of a natural trend for females, in that there was a significant effect of both dose and linetype decreasing wheel running over the course of the night (peak running is on total revolutions run, as well as the average and maximum running typically reached around 2 h following lights out, e.g., see Girard and speed. However, unlike the first 10–70 min, in this period there was a Garland, 2002; Malisch et al., 2009; Rhodes et al., 2001) and/or an effect significant effect of both dose and linetype on the time spent running, of the vehicle injections per se. Without a control group that received no as well as a dose by linetype interaction for both the total revolutions injections, we cannot separate these possibilities. For proportional and average running speed. Thus, for up to 70 min, the wheel running responses (average running 10–70 min following injection divided by of females from both linetypes is depressed in a similar fashion for all average running during the 30 min prior to injections), we observed no doses. However, inspection of Fig. 1 indicates that during the final difference between HR and Control lines for either females (p= 70–120 min HR females receiving the 1 mg/kg dose gradually 0.5013) or males (p=0.3212).
resumed running to values near vehicle levels, while wheel runningof Control females given this dose remained depressed.
3.2. Drug response For males, wheel revolutions, as well as average and maximum speed, depended on dose and linetype (p b 0.05 for all) for all time Table 1 shows results of the repeated-measures ANCOVAs for the periods studied (Fig. 2), with higher doses generally depressing first 10–70 (corresponding to our previous study: Keeney et al., wheel running more for both linetypes. Similar to females, only 2008) and also for 70–120 min post-injection (which was not dose had a significant effect on the time spent running, both in the Author's personal copy
B.K. Keeney et al. / Pharmacology, Biochemistry and Behavior 101 (2012) 528–537 Table 1Repeated-measures analyses (SAS procedure mixed) of wheel running (binned in 10-min intervals) during 10–70 and 70–120 min following injections with vehicle, low, mediumor high dose of WIN 55,212-2 in males and females.
Trait and transform used F for interaction P for interaction Females (10–70 min)Revolutions 0.6 Average speed (rpm) 0.6 Maximum speed (rpm) 0.6 Females (70–120 min)Revolutions 0.6 Average speed (rpm) 0.6 Maximum speed (rpm) 0.6 Males (10–70 min)Revolutions 0.6 Average speed (rpm) 0.6 Maximum speed (rpm) 0.6 Males (70–120 min)Revolutions 0.6 Average speed (rpm) 0.6 Maximum speed (rpm) 0.6 Time denotes number of 1-minute intervals with at least one revolution; average speed is revolutions/time; maximum speed is revolutions in the single highest 1-minute interval.
Degrees of freedom are 2 and 12 for dose, 1 and 6 for linetype, and 2 and 12 for the dose *linetype interaction. All p-values are for 2-tailed tests. All analyses also included age and wheel freeness as covariates (results not shown).
first 10–70 min and in the latter 70–120 min post-injection. Unlike behavioral endpoints (i.e., similar factorial increase in voluntary results for females, the reduced wheel running in males was signifi- wheel running) via at least partially separate mechanisms (Garland cantly greater for HR lines than for Controls for total revolutions et al., 2011b).
run, time spent running, as well as average and maximum speed It is important to note that, as expected from numerous previous (p b 0.05 for all dose by linetype interactions) in the first 10–70 min studies (e.g., Garland et al., 2011b; Keeney et al., 2008), HR mice of post-injection. In addition, males also showed a significant dose by both sexes differ substantially from Control-line mice in baseline run- linetype interaction for the total revolutions run and for all measures ning. In the present study, HR mice ran approximately three-fold of speed in the 70–120 min post-injection (p b 0.05 for all).
more than Control under baseline conditions (e.g., see Fig. 1), a typicaldifferential. Given this large difference in baseline running, it is possible that HR and Control mice are operating under considerably differentphysiological regimens during times of peak nightly running, when Results from the present study show that agonism of the CB1 the present study was conducted. For example, in a given night of receptor (via i.p. injection of WIN) decreases wheel running in all wheel running, HR mice could voluntarily approach performances at groups analyzed. However, male mice from the HR lines decreased or near their maximal aerobic speed (i.e., almost at their maximal rate their wheel running to a greater degree in response to WIN, as of oxygen consumption), unlike the nightly running of typical Control compared with males from the non-selected Control lines, for both mice (Girard et al., 2001; Rezende et al., 2005, 2009). Therefore, even time periods studied (10–70 and 70–120 min post-injection) nominal increases or decreases in total wheel running during the active (Table 1, Fig. 2). In contrast, female HR mice had a differential period could have different physiological consequences for an HR decrease in wheel running in response to WIN only during the latter mouse as compared with a Control mouse. These and other differences time period (70–120 min post-injection). These results complement between the HR and Control lines (e.g., differences in body fat: Meek et a prior study, in which we found that female HR mice from generation al., 2010; Swallow et al., 2001; Vaanholt et al., 2008) may also influence 48 showed altered responsiveness to a selective CB1 receptor antagonist the pharmacokinetics of WIN. In many cases, exercise is known to alter (SR141716; Rimonabant) as compared with females from the four non- the metabolism, absorption, and excretion of drugs (van Baak, 1990), selected Control lines, while HR males did not differ from their Control and it is certainly possible that the elevated activity levels of HR mice counterparts (Keeney et al., 2008). Both the previous and current studies (or of females relative to males) can affect the dynamics of WIN show that HR mice differ from Control mice in the magnitude of the wheel-running response of one sex or the other to the highest dose of It is of interest that seemingly opposing treatments (Rimonabant CB1 antagonist or agonist, respectively. In both studies (and sexes), blocks CB1 receptors, while WIN stimulates them) had negative this reduction in wheel-running is primarily caused by a decrease in effects on wheel running in both HR and Control mice. This is consistent the speed of running, with only HR males during the first 10–70 min of with other studies of HR mice, which show that to date there has not WIN injection showing a statistical reduction in the amount of time been a pharmacological agent that has significantly increased wheel spent running.
running in HR mice (Rhodes et al., 2001; Rhodes et al., 2005; Rhodes These results suggest that over the course of selective breeding, and Garland, 2003; Li et al., 2004). Indeed, the only substance that has HR mice have evolved to utilize CB1 signaling in a different way significantly increased wheel running in HR mice has been a high-fat than Control-line mice during the performance of voluntary wheel (Western) diet (Meek et al., 2010), which may have had effects on running (i.e., voluntary exercise: Garland et al., 2011a). Furthermore, both exercise abilities and motivational aspects of wheel running.
HR mice have done this in a sex-specific manner. Put differently, our Despite the direction of the effect of pharmacological manipulation of results show that male and female HR mice have evolved to similar the CB1 receptor, there is a sex-specific differential in wheel running


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B.K. Keeney et al. / Pharmacology, Biochemistry and Behavior 101 (2012) 528–537 between HR and Control mice. Similar to Rhodes and Garland (2003), of selection. In general, the increased daily running distance of HR we believe that this differential is of prime interest. If the CB1 receptor mice is accomplished mainly by speed in female HR mice, but by is important to the performance of HR wheel running, then we might both speed and duration of running (to a lesser degree than females) predict that pharmacological manipulations that perturb the specific in HR males (Garland, 2003; Girard et al., 2001; Keeney et al., 2008; function of the receptor, regardless of the type of perturbation (stimula- Koteja and Garland, 2001; Rezende et al., 2009; Rhodes et al., 2000; tion or blockade), could potentially interfere with such a specific, and Swallow et al., 1998, 1999). In recent generations HR males (seem- ingly at a selection limit) can run for as many minutes per day asHR females (Garland et al., 2011b; Rezende et al., 2009). The rodent 4.1. Sex differences literature supports a generalized trend for higher levels of locomotoractivity in female rodents. This trend seems particularly true for Several studies have documented robust sex differences in the wheel running activity (see Lightfoot, 2008 for review). For instance, evolution of high levels of voluntary wheel running over the course Eikelboom and Mills (1988) find that female rats run more than Fig. 2. Least squares means and standard errors from repeated-measures analyses of revolutions, minutes spent running, and revolutions per minute during 10–70 min followinginjection (A,B,C) and 70–120 min following injection (D,E,F) for males and females after injection of the CB1 receptor agonist WIN 55,212-2. Results show a dose by linetype inter-action in the first 10–70 min for total revolutions, time spent running, and average speed of running for males only. During the latter 70–120 min, there is a dose by linetype in-teraction for males and females for total revolutions, as well as average speed of running. Significance levels are presented in Table 1; least squares means and standard errorsare presented in Online Supplemental Table A.


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B.K. Keeney et al. / Pharmacology, Biochemistry and Behavior 101 (2012) 528–537 Fig. 2 (continued).
males, at a higher speed. Likewise, Konhilas et al. (2004) found that by pharmacologically blocking CB1 receptors, thus implicating a female mice of two different strains ran more than male mice of their modulatory role of estrogen on the ECS. Similarly, although THC and respective strain, both at higher speeds and for a longer duration.
other cannabinoid agonists are antinociceptive regardless of sex, these Indeed, Field and Pellis (2008) find significant, overarching differences effects seem to be stronger in female rats than males (Cohn et al., in the ways male and female rats organize their movements across a 1972; for review and other species, see Craft, 2005; Fattore et al., 2008; wide variety of motor tasks, and suggest that these sex differences in Tseng and Craft, 2001).
movement are not a byproduct of dimorphisms in body size or shape, Although it seems evident that female sex hormones can affect but rather a result of neural differences.
aspects of ECS functioning, there is also evidence that suggests In the context of our understanding of the voluntary wheel running male-specific ECS dynamics. For example, Reich et al. (2009) found of HR mice, it is likely that some of these neural sex differences are that male rats have higher basal levels of CB1 receptors than females.
associated with the ECS (or factors related to downstream or upstream Likewise, Miller et al. (2004) showed that CP 55,940, a full agonist at ECS activity). Several studies suggest that sex-based differences in CB1 both the CB1 and CB2 receptors, increased intake of a highly palatable signaling are common. For example, Fattore et al. (2007) found that food reward to a greater degree in male than female rats. In line with ovarian hormones play a crucial role in the behavioral response of rats these results, Diaz et al. (2009) found that administration of WIN to cannabinoids. Similarly, it has been shown in rats that estrogen can produced a greater degree of hyperphagia in male than female guinea affect cannabinoid receptor density (Rodriguez de Fonseca et al., pigs. It is not yet clear if these dimorphisms represent a clear sex-bias 1994), transcription (González et al., 2000), and signal transduction in ECS-influenced behaviors (see Fattore and Fratta, 2010 for review).
(Mize and Alper, 2000). Likewise, Hill et al. (2007) reported that the Given these known sexual dimorphisms in ECS physiology, we antidepressant effect of estrogen in a rat model of anxiety was prevented suggest that perhaps the psychotropic effects of ECS activity may Author's personal copy
B.K. Keeney et al. / Pharmacology, Biochemistry and Behavior 101 (2012) 528–537 play a role in our observations following CB1 receptor agonism and to have been involved in the development of the HR phenotype.
antagonism in male and female HR mice. As previously mentioned, Dopamine and endocannabinoids interact (Laviolette and Grace, it has long been hypothesized that the ECS may contribute to a 2006; Lupica and Riegel, 2005; Maldonado et al., 2006; Pillolla et al., pleasurable "runner's high" sensation associated with prolonged 2007), and in some cases, both the ECS and the dopaminergic system endurance-type exercise (Dietrich and McDaniel, 2004). It has been have been shown to influence the performance of locomotor behaviors shown that CB1 signaling can mimic the action of drugs of abuse, (Beltramo et al., 2000; Giuffrida et al., 1999; Gorriti et al., 2005). Equally thus producing a rewarding sensation that is capable of conditioning interesting, but perhaps less expected, is the fact that ECS activity via behaviors (De Vries and Schoffelmeer, 2005; Maldonado et al., 2006).
both activation and suppression of CB1 transmission results in unique It is possible that this neurobiological "reward" may motivate, or be sex differences in running behavior in HR mice. A logical next step stimulated by, high-intensity (high-speed) running. If it were true that following both selective CB1 agonism and antagonism would be to CB1 activity (at least in areas of the brain relevant to the performance administer mice of both sexes and linetypes an indirect CB1 agonist of wheel running) is intensity-dependent, then we can predict that (such as an uptake blocker or FAAH inhibitor). On a more general relatively high-speed running would be conditioned by the neural level, it is possible that over the course of selective breeding male and "pay off" of CB1 activation. Given that HR females tend to run at higher female HR mice have evolved large-scale differences in how the brain speeds than males, we would expect females to decrease their wheel motivates and/or rewards relatively high-speed wheel running.
running to a greater degree when CB1 transmission is blocked, and to Likewise, although pharmacology can be a useful tool in detecting a lesser degree (perhaps influenced by the injection itself) when trans- behavioral correspondences to neural activity, it does not allow us to mission is stimulated (as this could approach normal CB1 activity during make quantitative statements about how male and female HR mice running). This is indeed what we observed (see Table 1), although might differ with respect to CB1 distribution, regulation or activation without actual quantification of CB1 dynamics, behavioral observations in various behavioral contexts. Our two pharmacological tools (receptor alone are not sufficient evidence of any particular mechanism.
antagonism and agonism) do not fully illuminate the role of the CB1 Of course, it is also possible that the expression or function of the receptor during HR running. Therefore, additional studies aim to better CB1 receptor itself is not directly related to promoting voluntary characterize the involvement of the ECS in voluntary wheel running.
wheel running in HR mice. It has long been understood that receptor The genetic basis of sex differences in running by HR mice is now agonist dynamics can affect either a compensatory downregulation or under study (Hannon et al., 2011; Kelly et al., 2010), and future studies upregulation of a target receptor protein (Meyer and Quenzer, 2005).
may aim to characterize the extent and nature of how both sexes of HR Male or female HR mice could have any number of alterations upstream mice organize and utilize mechanisms of neural reward, with a special of the CB1 receptors that affect its functionality, including those that emphasis on an understanding of the dynamics of ECS activity in vivo.
interrupt the synthesis, release or degradation of endocannabinoids. Ifthe ECS is important to network-level neural mechanisms, such as those that may control overall "motivation" to run, even mutations inindirectly-related genes (e.g. those affecting COX-2, which would in We thank G. L. Gerdeman for comments on an earlier version of turn act on 2-arachidonoylglycerol, a common endocannabinoid) the manuscript and H. Schutz for her help during experimentation.
could have an influence on such a complex phenotype.
Alternately, HR females could be more sensitive to potential negative Appendix A. Supplementary data effects of Rimonabant administration (Pacher et al., 2006), while malesare more sensitive to the catalepsy-inducing effects of cannabinoid Supplementary data to this article can be found online at doi:10.
agonists such as WIN. It is also possible that some or all of these observed sex differences in the effects of cannabinoids could be due topatterns of drug deposition, as related to differences in body fat. WIN,THC, and other cannabinoids are highly lipophilic, and can be readily absorbed by fat cells (Nahas et al., 1981). Cortright et al. (1997) found Belke TW, Garland Jr T. A brief opportunity to run does not function as a reinforcer for that male rats have a higher percentage of body fat than females, mice selected for high daily wheel-running rates. J Exp Anal Behav 2007;88: which led Tseng et al. (2004) to hypothesize that perhaps the behavioral effects of cannabinoids are less apparent in male rodents due to their Beltramo M, de Fonseca FR, Navarro M, Calignano A, Gorriti MA, Grammatikopoulos G, et al. Reversal of dopamine D(2) receptor responses by an anandamide transport body fat levels. Contrary to this idea, however, male and female HR inhibitor. J Neurosci 2000;20:3401–7.
mice do not significantly differ in their percentage body fat (Swallow Bronikowski AM, Rhodes JS, Garland Jr T, Prolla TA, Awad T, Gammie SC. The evolution et al., 2001), which is very low compared to other common laboratory of gene expression in the hippocampus in response to selective breeding for in-creased locomotor activity. Evolution 2004;5:2079–86.
strains of mice (Nehrenberg et al., 2009), suggesting that sex differences Chaouloff F, Dubreucq S, Bellocchio L, Marsicano G. Endocannabinoids and motor be- in the behavioral response to CB1 manipulation are resultant of more havior: CB1 receptors also control running activity. Physiology 2011;26(2):76–7.
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Online Supplemental Table A. Least squares means (all traits transformed by raising to
the 0.6 power) and standard errors from repeated-measures analyses (Table 1) of wheel
running 10-70 and 70-120 minutes following injections, for females and males.
Females 10-70 mins
Females 70-120 mins
Revolutions 0.6
Average Speed 0.6
Maximum Speed 0.6
Males 10-70 mins
Males 70-120 mins
Revolutions 0.6
Average Speed 0.6
Maximum Speed 0.6
All values are means per 10-minute intervals. Time denotes number of 1-minute intervals with at least one revolution; Average Speed is revolutions/time; Maximum Speed is revolutions in the single highest 1-minute interval.
Online Supplemental Table B. Proportional response (dose/vehicle) to WIN 55,212-2 in
the first 10-70 minutes post-injection in females (log10 transformed) and males (log10
transformed). Values are least squares means from SAS Procedure Mixed. Covariates
are wheel freeness and age at time of injection (results not shown).
Females 10-70
Control Standard High
Standard
mins after
Linetype
injection
Revolutions
Average Speed
Maximum Speed
Males 10-70
mins after
injection

Revolutions
Average Speed
Maximum Speed

Online Supplemental Table C. Proportional response (dose/vehicle) to WIN 55,212-2 in
the latter 70-120 minutes post-injection in females (log10 transformed) and males (log10
transformed). Values are least squares means from SAS Procedure Mixed. Covariates
are wheel freeness and age at time of injection (results not shown).
Females 70-120
Standard
Standard
mins after
Linetype
injection
Revolutions
-0.031 0.235 -0.690 0.222 4.110 0.089 0.400 0.248 -0.111 0.239 2.200 0.188 0.446 0.202 0.222 0.191 0.640 0.453 -0.102 0.101 -0.360 0.095 3.460 0.112 0.073 0.097 -0.098 0.093 1.600 0.253 0.166 0.088 0.004 0.084 1.770 0.231 Average Speed
-0.016 0.148 -0.486 0.142 5.210 0.063 0.167 0.136 -0.144 0.131 2.690 0.152 0.207 0.111 0.079 0.105 0.700 0.434 Maximum Speed
-0.010 0.174 -0.484 0.167 3.850 0.097 0.219 0.156 -0.116 0.150 2.370 0.175 0.250 0.129 0.101 0.123 0.700 0.436 Males 70-120
mins after

injection
Revolutions
0.214 0.195 -0.408 0.205 4.770 0.072 0.338 0.226 0.068 0.240 0.660 0.447 0.535 0.243 0.486 0.253 0.020 0.894 -0.002 0.082 -0.241 0.086 4.000 0.093 0.055 0.091 -0.067 0.097 0.840 0.396 0.142 0.096 0.114 0.100 0.040 0.847 Average Speed
0.084 0.115 -0.358 0.120 7.000 0.038 0.115 0.140 -0.100 0.147 1.110 0.333 0.219 0.113 0.197 0.120 0.020 0.897 Maximum Speed
0.118 0.133 -0.356 0.140 5.930 0.051 0.191 0.166 -0.077 0.175 1.220 0.312 0.275 0.133 0.241 0.140 0.030 0.870



Online Supplemental Figure A. Daily pattern of wheel running (revolutions in 20-min
bins) for mice from selectively bred High Runner (HR) and Control lines during 3 days
before the start of WIN 55,212-2 injections (22 Oct- 24 Oct 2008). Note that females run
more than males in both linetypes. Grey bars indicate lights off.

 
 
 

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Advances in Astronomy and Space Physics, 4, 20-24 (2014) Abundances in the atmosphere of the metal-rich planet-host star HD 77338 I. O. Kushniruk1∗, Ya. V. Pavlenko2,3, J. S. Jenkins4, H. R. A. Jones3 1Taras Shevchenko National University of Kyiv, Glushkova ave., 2, 03127 Kyiv, Ukraine 2Main Astronomical Observatory of the NAS of Ukraine, Akademika Zabolotnoho str., 27, 03680 Kyiv, Ukraine

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