The ergogenic impact of the glucocorticoid prednisolone does not translate into increased running motivation in mice
Introduction
The World Anti-Doping Agency (WADA) has established lists of substances that are considered to be sport doping agents on the basis of three criteria: performance enhancers, health risk factors and/or spirit of sport threateners (World Antidoping Agency (WADA), 2019). Owing to their ergogenic properties, glucocorticoids, such as prednisolone, are forbidden when used in-, but not out-, competition (World Antidoping Agency (WADA), 2019). These physical performance-enhancing properties are thought to be accounted for by their peripheral actions as both endogenous (cortisone and cortisol produced from the human adrenal cortex) and exogenous glucocorticoids primarily stimulate hepatic and adipose metabolic pathways as to provide muscular energy (Magomedova and Cummins, 2016). In addition, glucocorticoids are endowed with anti-inflammatory properties which ease respiration and prevent muscle and joint pains (Adcock and Mumby, 2016), all these effects facilitating physical performance and after-exercise recovery (Duclos, 2010).
Besides their peripheral origins, the doping impacts of glucocorticoids have been suggested to involve several of their central effects. Whereas evidence for centrally-mediated changes in the secretion of several hormones brings direct support to this hypothesis (Collomp et al., 2016; Duclos, 2010), it remains to establish that other central effects of glucocorticoid on e.g. mood, anxiety, hedonia (De Kloet et al., 2005; Piazza and Le Moal, 1997) are relevant to doping protocols. Corticosteroid receptors, whether of the mineralocorticoid receptor (MR) or of the glucocorticoid receptor (GR) subtype, are present in numerous brain regions, albeit in an uneven manner (De Kloet et al., 2005; McEwen et al., 1986). When bound by natural or exogenous corticoids, these receptors, located both in neurons and astrocytes, then act either at cell membranes, doing so with rapid consequences, or in cell nuclei, doing so as transcription factors, and hence with longer delays (Chaouloff and Groc, 2011; Joëls, 2018). GRs, which bear low affinity for cortisol (human) or corticosterone (rodent), as compared to MRs, are present within the mesocorticolimbic system (Ambroggi et al., 2009; Härfstrand et al., 1986), a tractus exerting a key role in motivation processes (Koob and Volkow, 2016; Nestler, 2005). Hence, glucocorticoids have been reported to impact, albeit in different directions, the motivation for natural rewards (e.g. food: Gourley et al., 2008) and drugs of abuse (e.g. cocaine and amphetamine: Goeders, 2002; Piazza and le Moal, 1997; but see Graf et al., 2013; Mantsch et al., 1998). Motivation for exercise, whether intrinsic (i.e. for the pleasure of exercise) or extrinsic (i.e. for healthy reasons or for an award, whether a medal or a record), is a prerequisite for exercise training. As such, the above mentioned results raise the hypothesis that besides its ergogenic consequences glucocorticoid administration, by targeting the mesocorticolimbic system, actually stimulates exercise motivation. To our knowledge, this hypothesis has never been tested so far. The use of wheel-running in rodents, a model of volitional exercise, has indicated that high concentrations of corticosterone (as to stimulate GRs) or administration of glucocorticoids either decrease (Menezes et al., 1985) or prove ineffective (Cobos et al., 2012; Duclos et al., 2009; Yau et al., 2011) on running performance. However, the use of running performance as an index of running motivation is misleading. Thus, overall consumption of a reward when the latter is provided freely (i.e. without any effort prerequisite for its access), as is the case for the "free" wheel-running paradigm, is accounted for by both motivation for that reward and its consumption, i.e. two reward-related dimensions with different neurobiological grounds (Salamone and Correa, 2012). An appropriate estimation of running motivation requires the measurement of the maximal quantity of efforts the animals are able to achieve ("wanting" dimension) to then exert their running activity ("consumption" of the wheel linked to the "liking" dimension) (Berridge, 2007; Salamone and Correa, 2012). The sole paradigm allowing such a measurement relies on operant conditioning procedures whereby animals first need to lever-press or to nose-poke under fixed ratio (FR) schedules of reinforcement before their motivation is tested under a progressive ratio (PR) reinforcement schedule (Hodos, 1961). Although several studies have used such a cued-reward motivated instrumental task to assess running motivation in rats (Collier and Hirsch, 1971; Iversen, 1993) or mice (Belke and Garland, 2007; Hurel et al., 2019; Muguruza et al., 2019), none of them has examined the respective impacts of glucocorticoids on the motivation and consumption dimensions of wheel-running rewarding properties.
In keeping with the data reported above, the present mouse study first investigated whether subchronic ingestion of two concentrations of the glucocorticoid prednisolone increased conditioned exercise motivation and consumption (i.e. performance) whilst proving ergogenic. For running motivation analyses, we used our recently developped model wherein mice have to nose-poke under FR, and then PR, schedules of reinforcement to unbrake a running wheel (Hurel et al., 2019; Muguruza et al., 2019). The ergogenic impact of prednisolone was assessed by the wire grid-hanging test, a paradigm that provides an index of muscular resistance (Morrison-Nozik et al., 2015). The aforementioned inability of free running paradigms to discriminate between running motivation and consumption led us to perform a second series of experiments wherein mice provided a permament free (i.e. unconditioned) access to running wheels were analyzed for the impact of ergogenic concentrations of prednisolone on running performance.
Section snippets
Animals
All protocols, which complied with the French (Décret 2013-118) and European (2010/63/EU) rules on animal experimentation, were approved by the local Ethic Committee (Comité d'Ethique 50) with agreement numbers DIR13111, 13649, 33-063-69 (F.C.) and A33-063-098 (animal facilities) provided under authority of the Préfecture de Gironde and the Ministry of Agriculture.
This study used male C57BL/6 N mice (Elevage Janvier, Le Genest Saint Isle, France), aged 8-week-old upon arrival in our animal
The ergogenic effects of prednisolone occur without increased running motivation
In a first series of experiments, operant conditioning procedures (Fig. 1A) were used to study the impact of the repeated ingestion of 5 μg/ml (n = 10) and 15 μg/ml (n = 9) prednisolone, as compared to water ingestion (n = 7), on running motivation. Post hoc analysis of daily prednisolone intakes through drinking water (Fig. 1B) and their inhibitory consequences on body weights ([F(223) = 27.95; p < 0.001]; Fig. 1C) indicated effectiveness of the administration protocol. These body weight
Discussion
Whether (Duclos, 2010) or not (Orchard, 2008) glucocorticoids should be considered doping agents has been a matter of discussion. Thus, besides their obvious potential negative impacts on health, evidence for the ergogenic impact of glucocorticoids has been reported in several, but not all, studies (Collomp et al., 2016). Although one main argument for the ergogenic effect of glucocorticoids stems from their metabolic and anti-inflammatory properties at peripheral tissues, their potential
CRediT roles
F.G., G.M., and F.C. contributed to the conception and design of the study, B.R., C.V., and F.C. participated in acquisition and analyses of the data, F.C. drafted the article, B.R., C.V., F.G., G.M., and F.C. revised the article and approved its final version.
Declaration of Competing Interest
The authors declare no conflicts of interest.
Acknowledgements
We thank all the personnel from the Animal Facility and the Genotyping platform of the NeuroCentre Magendie, Virginie Morales and the other members of Marsicano's laboratory for their daily support and for useful discussions. This work was supported by the Agence Française de Lutte contre le Dopage (AFLD, to F.C. and F.G.), Institut National de la Santé et de la Recherche Médicale (to G.M.), la Région Aquitaine (to G.M.), the University of Bordeaux (to G.M.), and l'Agence Nationale de la
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