Literature Thesis Brain and Cognitive Sciences: 3Title: The effect of voluntary wheel running onhedonic feeding behavior-induced neuroadaptations to the dopamine and endogenous opioid neurotransmitter systemsin the VTA-

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MSc. Brain and Cognitive Sciences: Behavioral Neuroscience

The effect of voluntary wheel running on hedonic feeding

behavior-induced neuroadaptations to the dopamine and endogenous opioid

neurotransmitter systems in the VTA-NAc circuit

Despoina Kortesidou

ID: 11122064

Submission date: 24-3-2020

Supervisor:

Dr. Joram D. Mul

Assesor NIN:

Prof. Dr. Susanne E. la

Fleur

Examiner FNWI:

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To Dimitra, Vaso and Stratos,

“And so, along with the seven we descent. With the rain, with the weather that rules us. Your eyes sustain a sea, i remember... The latter lulls me with a flute. Deaf Salah is sweeping the deck. -With a scraper, take the ship's paint off of me. But there is something deeper that stains me. -Son, where are you going? Mother, I'm off to sail.” – The seven dwarves of s/s Cyrenia, Nikos Kavvadias

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Title:

The effect of voluntary wheel running on hedonic feeding behavior-induced

neuroadaptations to the dopamine and endogenous opioid neurotransmitter

systems in the VTA-NAc circuit

Abstract

Voluntary wheel running (VWR) is a commonly employed rodent exercise model that is equivalent to intentional human physical activity. It robustly enhances the expression and function of the mesolimbic dopamine and endogenous opioid neurotransmitter systems in the VTA-NAc circuit that ultimately upregulate dendritic growth and cell survival. Conversely, prolonged overconsumption of palatable, energy-dense diets evokes adverse neuroanatomical adaptations in the mesolimbic dopamine and endogenous opioid neurotransmitter systems, and specifically, a drop in the expression of μ-opioid and Dopamine 1 and 2 receptors. These persistent impairments increase the risk for various metabolic syndromes and contribute to low engagement for VWR and, ultimately, physical inactivity. This literature thesis explores VWR’s impact on palatability-driven feeding behavior on a behavioral level, as well as the interaction’s influence on the shared VTA-NAc circuit dopamine and opioid systems. I examined the interaction of VWR with (i) acute and (ii) prolonged palatability-driven eating, under the prism of three co-variants: (a) rewards’ sequence of presentation, (b) rodents’ sex, and (c) different rates of VWR. Nonetheless, short-term exposure to palatability diets is not optimal for observing persistent alterations in the studied neurotransmitter systems. Therefore, since studies on VWR-chronic palatability-driven eating interaction are limited, the exact role of VWR on potentially ameliorating the obesogenic phenotypes is still unknown in its most part. To sum up, although VWR has positive effects on naïve lean rodents, its potential role as a palatable diet reward substitution or a reward intervention merits additional future research.

Keywords: Voluntary wheel running; Palatable diets; Palatability-driven eating; Hedonic eating; Reward; VTA-NAc circuit; Dopamine; Endogenous opioids; Rodents

1. Introduction

The current conceptualizations of feeding regulation propose the energy homeostasis and the hedonic reward systems as two interconnected, in both function and structure, brain circuitries, which are of paramount importance in influencing food intake (Anderson and Adolphs, 2014; Berridge and Kringelbach, 2015; Rossi and Stuber, 2018). On a meal-to-meal basis, the intersection of the homeostatic and hedonic systems mediates the integration of information related to the perception of satiety and food reward, respectively, that ultimately promote the termination of a meal – for more details on the homeostatic feeding regulation, see 1_{. Food-related reward information is carried by mesolimbic dopaminergic neurons} that project from the ventral tegmental area (VTA) to the nucleus accumbens (NAc, forming the VTA–NAc neurocircuit), wherein it is processed (Castro and Berridge, 2014; Kelley et al., 2005).

Contrary to early research, consumption of energy-rich, highly palatable foods in quantities beyond metabolic needs (termed as hedonic or palatability-driven feeding behavior; Fields and Margolis, 2015) is not merely fueled by the absence of hunger, but in fact, it is integrated with energy homeostasis (for a comprehensive review see; Morton et al., 2014). Nonetheless, hedonic eating results in persistent neuroanatomical adaptations (Cone et al., 2013; Matikainen-Ankney and Kravitz, 2018; Meyers et al., 2017) that contribute majorly to increased risks of incident diet-induced obesity, depression, cardiovascular diseases, type 2 diabetes, cancer and osteoporosis (Aydin et al., 2014; Melanson, 2017; Novak et al., 2012; Tulloch et al., 2015). The comorbidity of prolonged access to obesogenic diets and sedentary lifestyle promotes impairments in the mesolimbic dopamine and endogenous opioid signaling, which are suggested to be involved in the induction of insufficient physical activity and eventually the reinforcement of diet-induced obesity (Kravitz et al., 2016; Melanson, 2017; Ruegsegger and Booth, 2017).

Compellingly, self-motivating, structured, and repetitive voluntary physical activity, in comparison with spontaneous or forced exercise models, facilitates neuroprotective and neurogenerative processes through alterations in various neurotransmitter systems’ function+, including dopamine and endogenous opioids (Garland et al., 2011; Knab et al., 2009). Hence voluntary physical activity is commonly employed in the prevention and treatment of a plethora of neurological disorders, including dementia, Alzheimer’s and Parkinson’s disease and stress-related mental disorders (Dishman et al., 2006; Mul, 2018; Novak et al., 2012), as well as metabolic diseases with symptoms such as glucose intolerance, insulin sensitivity, and high cardiovascular risk (Haskell-Luevano et al., 2009; Manore et al., 2017; Mul et al., 2015). For instance, a fifteen-week exercise training decreased the preference for western diets and snacking in young adults, whereas higher intensity exercise increased their preference for a prudent pattern of eating

1_{On a meal-to-meal basis, the available energy stores produce satiety humoral and peptidal mediators, such as the anorexigenic,}

adipocytic leptin (Considine et al., 1996) and the orexigenic pancreatic insulin (Bruning, 2000) hormones, that enter the brain in proportion to the plasma levels (Schwartz et al., 1996). In line with homeostatic feeding behavior, these mediators evoke a cascade of signals that result in increased food consumption until deficient energy stores are replenished. The homeostatic brain circuitries in the forebrain and hindbrain (e.g., the hypothalamic arcuate nucleus and the nucleus of the solitary tract, respectively) process circulating signals regarding the bodily energy balance. These signals are transmitted onto downstream second-order neurons, including those in the paraventricular nucleus (PVN; Fulton et al., 2006) and lateral hypothalamic area (LHA; Kelley et al., 2005) and contribute to maintaining stable body fat stores over time (Begg and Woods, 2013; Grill et al., 2002). The homeostatic and hedonic systems intersect in LHA, where neurons integrate information related to the perception of food reward from nucleus accumbens (NAc) of the ventral striatum (Leinninger et al., 2009). Ultimately they project to the nucleus of the solitary tract, a terminal nucleus for vagal afferents conveying satiety signals, to promote the termination of the meal.

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(Joo et al., 2019). Regardless of the numerous effects of voluntary physical activity on physiological health maintenance, the multifactorial and complex motivation for voluntary exercise, as well as the underlying brain circuits that mediate that motivation, remain poorly understood (Garland et al., 2011). In that, employing rodent models to decipher human physical activity offers a unique opportunity to dissolve practical and ethical difficulties of studying the human brain’s structure and function. The acquisition of operant conditioning to a running wheel, a powerful reward reinforcer for rodents, establishes voluntary wheel running (VWR) as a prevalent intervention in homeostatic feeding regulation studies. For instance, long-term VWR has beneficial effects on the rodents’ metabolism (Dishman et al., 2006; Sahay et al., 2011; Sherwin, 1998), and can prevent the development of diet-induced (Patterson et al., 2007; Scarpace et al., 2010; Yang and Liang, 2018), as well as genetically-induced (Beeler et al., 2016; Ruegsegger et al., 2017) obesity models in both rats and mice.

Recent findings of rodents engaging on both hedonic eating and different rates of wheel running advanced our understanding of VWR’s potential influence on food’s value perception and the motivation to work for it, for this line of research has received less attention. Specifically, the VTA-NAc mesolimbic dopaminergic system mediates the calculation of the cost-benefit reward ratio and the motivation to work for both VWR (Volkow et al., 2017; Zheng and Berthoud, 2008) and palatable-induced eating (Lammel et al., 2014; Tulloch et al., 2015) in both humans and rodents. Additionally, within the VTA-NAc circuit, an established body of evidence indicates a role for the endogenous opioid system in modulating the euphoric properties of pleasurable experiences, including energy-dense diets and physical activity (Gosnell and Levine, 2009; Lett et al., 2001; Niikura et al., 2010; Zheng and Berthoud, 2008). The involvement of mesolimbic dopamine and opioid neurotransmitter systems in the reward processing of both VWR and palatability-driven feeding behavior raises the question of the role that VWR may have in mitigating the persistent diet-induced neuroanatomical cerebral adaptations.

In focusing on the impact that both wheel running and hedonic eating have on the reward neurocircuits of rodents, the purpose of the present literature thesis is twofold. The first one is to determine whether VWR as an intervention holds potential in alleviating established hedonic eating-induced dopamine and opioid neuroadaptation “switches” and analyze experimental circumstances under which VWR could reverse the impaired reward networks. For doing so, the second one is to identify the contribution of the shared dopamine and endogenous opioid neurotransmitter systems in regulating the rewarding effects of both VWR and hedonic eating, and suggest a potential mechanism of DA–opioid interaction to trigger the “switches” overturn. For that purpose, initially, it is pending to describe the exact role that dopamine and endogenous opioids play in the facilitation of reward signaling in the VTA-NAc circuit. Following, I will examine the behavioral effect of VWR on short-term palatability-driven food intake, and whether this interaction is dependant on three variables frequently utilized by the literature: (a) the two rewards’ sequence of presentation, (b) rodents’ sex, or (c) different rates of VWR. Following, I will define the separate impact of each pleasurable activity on the reward networks – by reviewing the influence of prolonged obesogenic diets’ consumption on sedentary rodents, as well as the impact of selectively bred high rates of VWR on homeostatic eating rodents, both behaviorally and molecularly. Finally, by examing the chronic palatability-driven eating on wheel running rodents, and given the limitations of the thesis, I will attempt to suggest whether VWR can be employed as an effective reward-intervention strategy.

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2. Methodology of Literature Research

2.1 Appropriateness of research and experimental designs

In assessing the interaction of VWR and hedonic eating, I split the results into two sections and reviewed VWR’s effect on the (i) acute and (ii) prolonged consumption of different types of palatable diets in mice and rats. In each section, I included studies’ results addressing how that interaction was affected by three co-variants: (a) the sequence of the two rewards’ presentation, (b) rodents’ sex, or (c) different rates of VWR, in selective breeding lines for enhanced wheel running. In the process of studying the available literature, I reviewed a handful of conflicting results, most likely due to a lack of uniformity in the utilized methodological approaches across the experimental designs, which often limit the comparison of results among studies. Namely, feeding and physical activity paradigms, as well as tested species often widely vary across studies. For instance, the most frequently discussed co-variables in the present literature thesis is the type of physical activity (i.e., voluntary, spontaneous or forced exercise paradigms), its duration (e.g., short- vs. long-term exercise), the composition of palatable diets (i.e., high in fat, high in sucrose, or both), the type and duration of food access (e.g., intermittent vs. continuous, and acute vs. prolonged access, respectively), palatable diets’ and VWR’s sequence of presentation (e.g., palatable diet presented before, after or simultaneously with VWR), but also the tested rodents’ species (i.e., mice or rats), as well as the rodents’ sex. I enclosed an analytical overview of the experiments’ methodological set-ups that I included in the results’ discussion (section 4) in Supplementary Tables 1 and 2 of the Appendix.

In the vast body of literature, associations have been found between the VWR-hedonic eating interaction and an abundance of additional factors. Such include the gut’s microbiota (Lee et al., 2017), circadian rhythms (Blancas Velazquez, 2018; Mifune et al., 2015; Okauchi et al., 2018), early-life stress (Eller-Smith et al., 2019), conditioned taste aversion (Moody et al., 2015; Satvat and Eikelboom, 2006; Yang et al., 2017), conditioned place preference (Greenwood, 2018; Lett et al., 2001), low receptiveness to obesity (Levin and Dunn-Meynell, 2004; Yang et al., 2017), and baseline diet preference (Glass et al., 1996; Gosnell and Levine, 2009; Liang et al., 2015; Naleid et al., 2007) among others. Nonetheless, the in-depth analysis of such interactions is out of the scope of this thesis.

2.2 Paper research methodology

The goal of the present literature thesis was in no way to construct an exhaustive, quantitative meta-analysis on the influence of VWR on rodents’ hedonic eating both on a behavioral and molecular level, but rather to aggregate reporting of experimental data and facilitate data comparison across studies. I limited the citation of historical papers, and instead, focused on recently published original articles (2014-present), to avoid overlap with similarly focused reviews from previous years. For topics outside the main scope of the thesis, I cited pertinent reviews for the interested readers. To search literature, I used a combination of search terms to filter papers addressing, in the Pubmed database (https://www.pubmed.com), and Google Scholar’s advanced search tool (https://scholar.google.com). The custom set of keywords that I applied was:

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("voluntary exercise" OR "wheel running" OR "voluntary wheel running") ("palatable *" OR "high fat" OR "high sugar) ("reward *" OR "dopamine *" OR "opioid *" OR VTA OR NAc) (rodents OR rats OR mice) NOT treadmill NOT swim (for an explanation of the different Boolean search

elements, see 2₎

2.3 Comparison of different exercise models

Apart from voluntary physical activity models, spontaneous (e.g., home-cage activity) and forced (e.g., forced treadmill running, forced swim test) physical activity models are, also, prevalent rodent exercise paradigms in metabolic syndrome studies. Spontaneous physical activity better represents non-exercise activity thermogenesis and lacks the exercise physiology characteristics of voluntary and forced paradigms (Garland et al., 2011). Thus, it is not suitable for measuring the influence of physical activity on reward and reinforcement of food. To date, forced exercise models, also, do not constitute a suitable methodological approach for such a measurement, because they act as chronic stressors by increasing the signaling of both adrenergic and corticosteroid systems in rodents. For example, corticosterone promotes a decrease in the neuroprotective brain neurotrophic factor’s expression (BDNF; Huang et al., 2006) and an elevation in the NLRP3 inflammasome’s expression (Brown et al., 2007; Ke et al., 2011). In turn, NLRP3 induces the secretion of pro-inflammatory IL-1b cytokines, while simultaneously decreasing the anti-inflammation cytokine levels, fluctuations associated with neuronal damage (Svensson et al., 2016).

Contrary to forced physical activity models, VWR promotes stress resilience in mice (Mul et al., 2018) and rats (Greenwood et al., 2011) by upregulating the expression of various proteins involved in dendritic growth and cell survival (Greenwood, 2018; Greenwood et al., 2011; Mul et al., 2018; Thompson et al., 2015). Taking into consideration that excessive consumption of palatable diets also induces the activation of inflammatory stress pathways, along with evoking neuronal cell death in the hypothalamus (Morton et al., 2014; Sousa-Ferreira et al., 2014), forced exercise paradigms were excluded from the results to minimize the stressor stimuli.

2.4 Comparison of different palatable diet models

Regarding the type of presented diets, I mainly considered the two-choice diet feeding paradigms, in which rodents have simultaneous, free-choice (fc-) access to both a palatable, energy-dense and a control standard chow diet (SC). Such palatable diets are: (a) high in fat (HF), (b) rich in carbohydrates [either in sucrose (HS) or cornstarch (HC)], or (c) high in fat and in sugar (HFHS). Fc-HFHS diets reflect some

2_{We used multiple Boolean operators (like OR, NOT, *) to limit the searched publications for the particular topics of this literature}

thesis. Specifically, parentheses contain similar query terms that could be used in rotation between and within papers and all cover our field of research (e.g., the first parenthesis contains frequently rotating terms in literature for VWR). Within them, double quotations serve the purpose of marking the searching of multiple words in the exact written order. The OR operator between the searched keywords in the parentheses ensures the search of all alternative terms that are used in rotation in the published literature. The asterisk (*) is used within the search as a placeholder for any unknown or wildcard terms (e.g., in the second parenthesis, the asterisk allows the search of multiple key sentences, such as palatable food or palatable diet). Finally, the NOT operator forces the definite withdrawal of forced exercise models from the results, such as the forced treadmill running and forced swimming tests.

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significant similarities with the dietary environment of humans; namely the vast choice between multiple food components varying in texture, taste and caloric content and the free access to them (for an elaborate review on the implementation of free-choice diets in researching metabolic syndromes, see Mul and La Fleur, 2016; Slomp et al., 2019). Another commonly employed feeding paradigm in literature is the concurrent access to two or more palatable diets (e.g., the presentation of HF vs. HS diets), which addresses the ratio of diet preference. However, the introduction of confounding variables limits the interpretation of results when it comes to the effect of palatability-driven eating on the signaling of the reward neurotransmitter systems and the interaction of VWR and palatability-driven eating. For any feeding paradigm that is radically different from the ones explained above, I will enclose an explicit explanation of the methodology’s set-up in the results’ analysis. I enclosed a full list of HF, HS, and HFHS diets’ nutritional components of all considered palatable diets in Supplementary Table 3 of the Appendix.

3. Reward Signaling Pathways in the VTA-NAc Circuit

Before proceeding to the VWR and palatability-induced feeding behavior interaction, it is imperative that I offer some insight into the reward processing by two major neurotransmitter signaling systems in the VTA-NAc circuit. The mesolimbic dopamine and endogenous opioid systems encode different aspects of positive/rewarding stimuli both for VWR and hedonic feeding behavior.

3.1 The mesolimbic dopaminergic system mediates several aspects of reward reinforcement

The mesolimbic dopaminergic (DA) system is a central component of the brain’s reward and aversion circuit, and it comprises of midbrain VTA DA neurons that predominantly project to NAc of the ventral striatum, forming the VTA-NAc circuit, see 3_{(Hicks et al., 2016; and for a recent review, see Volkow et al.,} 2017). Mesolimbic DA cells’ firing controls the cost-benefit reward ratio calculation, through consistently encoding reward prediction errors (Hamid et al., 2015; for more on reward prediction patterns, see 4_{). As} microdialysis testings over slow timescales (of tens of minutes) have demonstrated, mesolimbic DA release is correlated with motivational, behavioral activity (Horvitz, 2000; Narayanaswami et al., 2013). Extensive evidence also indicates that reward prediction triggers the motivational process for obtaining a

3_{The VTA-NAc circuit is heterogeneous in its afferent and efferent connectivity with several limbic and sensory regions. For}

instance, VTA is largely composed of DA neurons (~60%), a small population of GABAergic interneurons (30%), including ones that express calretinin, parvalbumin, somatostatin, or calbindin, and even fewer glutamate neurons (~3%; Dobi et al, 2010). Furthermore, NAc innervates neocortical brain areas, such as the medial prefrontal cortex, and several allocortical brain regions, such as the central and basolateral amygdala and hippocampus (Berridge and Kringelbach, 2015; Luo and Huang, 2016).

4_{The reward prediction firing pattern suggests that if the probability for the rewarding stimuli’s delivery is high, then the DAergic}

neurons’ magnitude of firing is stronger ahead of its presentation. Conversely, when the probability is low, then the DA neurons’ activity is stronger during the reward receipt (Romo and Schultz, 1990). Reward prediction errors’ role is to update the estimated values of prediction outcomes (i.e., an error exists when the reward outcome differs from its prediction, and no-error exists when the reward outcome matches the prediction). These updated values affect future decisions under similar situations (Hamid et al., 2015). Interestingly, a reward stimulus that develops into a full reward-value elicits no DA response (and an absence of a prediction error). In contrast, the reward’s omission generates a negative prediction error that induces a DA depression. However, and by contrast, an unfamiliar stimulus that is succeeded by a surprising reward elicits a positive prediction error and dopamine activation, whereas no reward fails to generate a prediction error and dopamine response (for a comprehensive review see; Schultz, 2016).

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reinforcer, a core psychological component of working for a reward, termed as “wanting” (Berridge and Kringelbach, 2008). Ultimately, the gratifying effects produced by “wanting” the pleasurable reinforcer, serve as a motivational cue that holds the potential to increase the likelihood for future engagement, e.g., optogenetic DA manipulations in the NAc indicated that DA both enhances motivation and positively reinforces choices to guide future behavior (Hamid et al., 2015). That reinforcement is evoked by a Pavlovian establishment of neuronal representations between the context, sensory cue, and behavioral outcome, that ultimately triggers changes in the synaptic strength of the reward system, another core psychological component of the reward circuit, termed as “learning” (Berridge and Kringelbach, 2008; Lowe and Butryn, 2007; Russo and Nestler, 2013). The “learning” mechanism establishes DA as one of the most potent factors in mediating attention, motor movement, reward, reinforcement, goal-directed, and habitual learning.

Regarding feeding behavior, upon processing peripheral satiety signals, the DA system modifies the effort-related decision-making to obtain palatable food (Volkow et al., 2017; Zheng and Berthoud, 2008). Cues that predict food consumption increases the NAc levels of DA, especially in food-deprived rodents. Although the DA system does not necessarily alter food’s hedonic properties, it modifies the incentive salience for food intake across time (Corwin et al., 2011; Kelley et al., 2005; Lammel et al., 2014; Tulloch et al., 2015; Volkow et al., 2017). Intriguingly, DA receptor antagonist, flupenthixol, and DA system lesions attenuate the animals’ exerted effort to obtain food but do not abolish feeding or impair the orofacial liking response to HS diets (for a review, see Baldo et al., 2013). Next, a wealth of research on both animals and humans has described that the DA system, also, plays a central role in mediating the motivation to engage in voluntary physical activity, and processing the VWR-successive reward (Clark et al., 2014; Volkow et al., 2017). For instance, Foley and Fleshner (2008) suggest that habitually physically active animals, compared to their sedentary counterparts, may present increased dopamine 2 receptor (DRD2)-mediated inhibition of the indirect pathway of the basal ganglia, which attenuates the stimulation of the motor cortex (for more on the direct and indirect pathways of movement, see 5_).

VWR, also, generates an automatic reinforcement that alters the control exerted by extrinsic reinforcement on exercise and evokes a hyperdopaminergic state characterized by sensitization of DA responses to non-exercise stimuli, including aversive stressors (see Figure 1; Belke et al., 2017; Lammel et al., 2014; Wise, 1996). Although three weeks of VWR access did not alter baseline firing rates of VTA DA neurons, compared to sedentary housing (Dremencov et al., 2017), six weeks of VWR altered the neuroplasticity of the mesolimbic DA pathway by increasing the VTA tyrosine hydroxylase (Th) mRNA levels and decreasing the NAc core Drd2 mRNA levels (Greenwood et al., 2011). To illustrate how distinct

5_{DA neurons of the nigrostriatal pathway, another DA circuit, project from the substantia nigra to the dorsal striatum, and release}

DA in response to reward-related stimuli and occasionally to aversion-related stimuli as well (Foley and Fleshner, 2008; Russo and Nestler, 2013; Tulloch et al., 2015). The nigrostriatal pathway influences movement through two pathways, the direct and the indirect pathway of movement, which are involved in the facilitation and suppression of movement, respectively. The direct pathway consists of D1 MSNs striatal projections onto tonically active GABAergic cells in the substantia nigra pars reticulata and in the internal segment of the globus pallidus, which in turn disinhibit key neurons in the thalamus. Finally, the thalamus sends stimulatory signals to the motor cortex, which translate to muscle movement. On the other hand, the indirect pathway's D2 MSN projections in the caudate nucleus and putamen synapse onto tonically active GABAergic cells in the external segment of the globus pallidus, which in turn project to the substantia nigra pars reticulata via the excitatory subthalamic nucleus. The activation of the indirect pathway creates an overall net inhibitory effect on the thalamus and in turn, on the motor cortex (for reviews by Horvitz, 2000; Wise, 2009).

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DA receptors operate in the presence/absence of rewarding stimuli, a computational model demonstrated that VTA tonic, low-frequency DA release (of 1-5Hz) results in a baseline DAergic “pacemaker tone,” to which high-affinity D2-like receptors (i.e., DRD2, DRD3, and DRD4) are sensitive to (Dreyer et al., 2010). In the presence of a rewarding stimulus, the neurons burst a high-frequency, phasic firing (⩾20 Hz; Grace, 1991), increasing the synaptic DA sufficiently to post-synaptically occupy low-affinity D1-like receptors (i.e., DRD1 and DRD5; Dreyer et al., 2010). On the contrary, DA burst pauses translate into low occupancy of both D1- and D2-like receptors (for more on temporal signaling patterns, see 6_).

Figure 1. Exercise adaptations in mesolimbic and nigrostriatal DA circuits that contribute to a hyperdopaminergic state. In the

midbrain, physical activity

increases BDNF, pCREB, and TH in VTA DA neurons. Downstream second-order neurons convey the signal to the dorsal and ventral striatum, where exercise elevates the release of DA by D2 MSNs, the release of BDNF, mTOR, and ΔFosB by D1 MSNs, and the expression of inhibitory OPRD (or δR) receptors by GABArergic neurons. At the same time, exercise attenuates the expression of excitatory 5HT2C receptors. Also, the A1R and A2AR

levels decrease in D1 and D2 MSNs, and, therefore, they interfere significantly less with DA binding to D1- and D2-like receptors in striatal MSNs. Together, these neuroadaptations lead to sensitization of local DA release in the striatum during aversive stimuli. They also lead to a shift towards the activation of D1 MSNs, which contribute to the chemical cascade induces positive emotional valence and stress resilience. From Greenwood, 2018. Abbreviations: BDNF: neuroprotective brain neurotrophic factor; TH: Tyrosine hydroxylase; pCREB: phosphorylated cAMP response element-binding protein; 5-HT-2C: a subtype of serotonin 5-HT receptor; D1 and D2 MSN: D1- and D2-like receptor-expressing medium spiny neurons, respectively; GABA Ach: GABAergic acetylcholine neuron; dδR: δ-opioid receptor-expressing neurons; mTOR: mammalian target of rapamycin; A1R and A2AR:

adenosine 1 and 2A receptors; ΔFosB: a member of the Fos family of transcription factors.

To further examine D1- and D2-like receptors’ association with VWR, using cfos mRNA radioactive in

situ hybridization, Clark et al. (2014) demonstrated that in 344-Fischer rats engaged in VWR for six weeks, Drd2 mRNA levels modestly elevated in both the NAc shell and NAc core, as well as in the dorsal striatum.

On the other hand, Drd1 levels were left unaffected. The same study also demonstrated that prolonged running frees DA receptor signaling from antagonistic adenosine 1 and 2A receptor interactions (A1R and A2AR, respectively). As shown, A1R and A2AR levels attenuated in the dorsal striatum, and both NAc shell and NAc core of running rats; a decrease that was associated with a potentiation in cfos mRNA levels in

6_{The precise control of DA’s temporal signaling patterns – afforded by optogenetics– demonstrates that the antagonism of D1-}

but not D2-like receptors in the NAc core, a hedonic coldspot, decreases active avoidance of the footshock (Wenzel et al., 2018). Another optogenetics study shows that brief but not prolonged pauses in the firing of midbrain DA dopamine neurons are sufficient to produce conditioned signaling inhibition in response to a warning –preceding a footshock– signal (Chang et al., 2018). Although an in-depth analysis of phasic and tonic firing modes of DA neurons is not the main interest of this literature thesis, insights on these mechanisms could illuminate how the transition between different rewards impacts the signaling of reward circuits.

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dynorphin opioid peptide expressing “direct pathway” neurons, and an attenuation of cfos mRNA levels in encephalin opioid peptide expressing “indirect pathway” neurons (Clark et al., 2014). In accordance, when Ghasem (2014) investigated the effect of six weeks VWR on Drd1 and Drd2 mRNA levels, they observed increased Drd2 mRNA levels and attenuated A1R and A2AR levels in the dorsal striatum, but no significant change in the Drd1 mRNA levels, compared to the non-running controls (Ghasem, 2014). By using designer receptor exclusively activated by designer drugs (DREADDs) to control the neural activity of D1- and D2-Cre dependant mouse lines, Zhu et al. (2016) demonstrated that the activation of DRD1 containing neurons in NAc decreased VWR engagement, while activation and blockage of DRD2 containing neurons enhanced or decreased, respectively, running behavior.

Summarizing these results and in conjunction with a revised perspective on how DA neural circuit mechanisms are involved in movement and reward (Greenwood, 2018; epitomized in Figure 1), while D2-like receptors are involved in the mediation of locomotor activity, D1-D2-like receptors are thought to be involved in the mediation of the motivational process for obtaining a rewarding reinforcer. The absence of abundant results or the discrepancies in the documented neuroadaptations in the expression of D1-like receptors does not necessarily imply that they are not involved in the mechanisms of exercise habituation. Contrariwise, the variance of experimental methodology variables (e.g., different exercise durations, studied brain regions, and used strains of mice and rats) may perplex the interpretation of results.

3.2 The opioid system co-modulates DA transmission and the euphoric properties of reward

While research suggests that DA signaling controls several aspects of reward reinforcement, such as motivation, incentive salience, and prediction, the rewarding stimuli that evoke pleasurable experiences do not appear to be strictly DA-dependent (Fields and Margolis, 2015). A significant body of evidence indicates that through the activation of the mesolimbic DA system, the endogenous opioid and endocannabinoid (eCB) neurotransmitter systems mediate and reinforce the euphoric properties of pleasurable experiences, i.e., the third psychological component of reward-seeking, termed as “liking” (Berridge and Kringelbach, 2008; Gosnell and Levine, 2009; Lett et al., 2001; Niikura et al., 2010; Wenzel and Cheer, 2018; Zheng and Berthoud, 2008).

Τhe endogenous opioid and endocannabinoid neurotransmitter systems are uniquely positioned to serve the orchestration of DA response in the midbrain, in response to the dense VTA glutamatergic (e.g., from the medial prefrontal cortex, the lateral dorsal tegmentum, and the extended amygdala, after reward-related signals) and GABAergic (e.g., from the extended amygdala and the rostromedial tegmentum, after aversion-related signals) innervation (Morton et al., 2014; Russo and Nestler, 2013). Unlike midbrain DA neurons, the projecting to NAc glutaminergic and GABAergic neurons post-synaptically express opioid and cannabinoid type-1 (CB1) receptors (Fallis, 2013; Le Merrer et al., 2009). Upon binding to the specific receptors, opioids and eCBs excite indirectly VTA DA cells (for a schematic illustration of this interaction, see Figure 2). Accordingly, opioid- or eCB-mediated activation of NAc D1-like receptors increases cAMP levels and activates the phosphorylation of the cAMP response element-binding protein (CREB). Then the phosphorylated transcription factor (pCREB) induces the mRNA expression of transcripts linked directly to addiction- and stress-related behaviors, such as the

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CREB-13

dependent expression of ΔFosB transcription factor in the NAc. Evidence shows that the overexpression of ΔFosB promotes the upregulation of various proteins (e.g., Cdk5, pTrkB, BDNF, PSD-95, Cadm4, and other actin-related proteins) involved in dendritic growth and cell survival, with positive effects on stress susceptibility, addictive compounds intake, but also relapse from such addictive compounds (Greenwood, 2018; Greenwood et al., 2011; Mul et al., 2018; Thompson et al., 2015).

Figure 2. Schematic of proposed eCB and opioid interaction with the mesolimbic dopamine system in (A) the ventral tegmental area and (B) the nucleus accumbens. In VTA, the glutamatergic activation of DA neurons promotes the synthesis of eCBs. eCBs are released “on-demand” to the synaptic shaft and bind to post-synaptic CB1s on GABA projecting neurons to further disinhibit the release of DA. Likewise, the upregulation of μ-opioid signaling, through palatable food or physical activity, disinhibits the VTA DA neurons through the inhibition of GABA cells, which synapse directly on DA neurons or glutamate projections. In turn, these VTA GABAergic neurons project to the NAc where they synapse with cholinergic interneurons and inhibit the excitatory input onto DA terminals.The VTA GABAergic neurons projecting to the NAc, synapse predominantly onto cholinergic interneurons expressing D1- and D2-like receptors, rather than onto D1- and D2-like medium spiny neurons (MSNs). Ultimately, the iMSNs CB1 or OPRM-mediated inhibition disinhibits acetylcholine’s release, resulting in DA terminal stimulation. From Wenzel and Cheer, 2018. Abbreviations: ACh, acetylcholine; CB1, endocannabinoid receptor 1; DA, dopamine; MOPR, μ-opioid receptor.

However, not all “liking” processes are carried by DA-dependant reward circuits. Although pharmacological DA suppression reduces the incentive salience for sweetness (measured with a sucrose solution lickometer) and evokes a dissociation that alters “wanting,” ultimately, it does not affect the “liking” of the food incentive (Galistu and D’Aquila, 2012; Pecina, 2005). For example, NAc μ-opioid receptor (OPRM) stimulation in male SD rats elevates fatty tastant intake through an increase of orosensory cue-dependant palatability mechanism and the suppression of satiety signals (Katsuura et al., 2011; Tulloch et al., 2015). In further support of DA non-dependent circuits of reward, the expression of OPRMs and CB1s on cholinergic interneurons indicates the existence of a direct opioid or eCB inhibition pathway that may attenuate DA’s concentration in NAc (Fields and Margolis, 2015). As an illustration of that mechanism, using cyclic voltammetry in mouse brain slices, opioid agonists bind on NAc cholinergic interneuron OPRMs, thereby reducing terminal DA release on the neighbor VTA DA cells (see Figure 2; Yorgason et al., 2017). However, whether this mechanism occurs, also, in vivo remains unknown. Our knowledge of DA non-dependent circuits for OPRM reward is limited; nonetheless, we refer the interested

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reader to a recent review that synthesizes a comprehensive view of the current conceptualization of the DA–eCBs–opioids interaction’ molecular mechanisms (see, Wenzel and Cheer, 2018).

Nevertheless, for the sake of brevity, this literature thesis focuses only on the opioid system’s role in the “liking” processes of hedonic eating and VWR, regardless of the importance of the eCBs system in these processes. In brief, the neuromodulatory opioidergic system consists of four major classes of G-protein (Gi/o) coupled opioid receptors – the mu (μ-opioid receptor; OPRM), delta (δ-opioid receptor; OPRD), kappa (κ- opioid receptor; OPRK) and the newly identified nociceptin (NOPR). Opioid receptors are widely expressed throughout the brain, including in the VTA-NAc circuit. Specifically, the medial NAc shell brain structure acts as a cognitive “hedonic hotspot and amplifier,” and the pharmacological stimulation of OPRMs in it enhances the orofacial liking response to sucrose (Castro and Berridge, 2014). The endogenous opioid receptors’ binding ligands belong in three groups of peptides that activate the receptors in a semi-specific manner; endorphins have high-affinity for OPRMs, enkephalins preferentially bind to DOPRs and dynorphins typically bind to OPRKs (Le Merrer et al., 2009; Nogueiras et al., 2012). For hedonic feeding behavior, the most consistent and robust opioid-mediated reward requires functional OPRMs (Baldo et al., 2013), whereas functional OPRDs are associated with reduced VWR (Sisti and Lewis, 2001). OPRK and NOPR are primarily involved in counter-reward mechanisms; OPRK agonism blocks the rewarding effects of OPRMs and induces aversion, and while NOPR agonism is not inherently aversive, its stimulation opposes the opioids’ reward-inducing action (Chefer et al., 2013).

Given the OPRMs’ primary role in reward-related processes, their functional and structural interaction with the other neurotransmitters in the VTA-NAc circuit has been studied intensely over the recent years. Transient DA signaling via OPRM (or CB1)-mediated disinhibition of GABAergic inputs on NAc DA release, promulgates singular reward signals, whereas prolonged DA signaling through long-term exercise or palatability-induced feeding behavior, alters NAc cellular morphology and reinforces the associated reward responses (Hamid et al., 2015; Wenzel and Cheer, 2018). Selective OPRM agonists, like the natural opioid peptide morphine or [D-Ala2, N-MePhe4, Gly-ol]–enkephalin (DAMGO; a synthetic opioid peptide) increase the firing rate of VTA DA neurons, by disinhibiting GABAergic inputs on them (Acquas and Di Chiara, 1994; Melis et al., 2000), and subsequently promoting the consumption of HFHS diets (Liang et al., 2015; Ottaviani and Riley, 1984; Taha, 2010). In contrast, selective OPRM antagonism attenuates hedonic eating (Bodnar, 2015; Taha, 2010; Yeomans and Gray, 1996), and the administration of the non-selective opioid receptor antagonist naloxone only decreases food consumption in sated but not hungry rats (Barbano et al., 2009). As for the OPRMs’ role in mediating VWR, in a selective breeding line for low and high rates of VWR, the intrinsic NAc expression of opioid transcript Oprm1 was lower in low running rats (Ruegsegger et al., 2016; Ruegsegger et al., 2015b). Interestingly, also, both NAc administration of DAMGO and naltrexone, an OPRM’s specific antagonist, decreased running in high, but not low wheel-running rats, independently of hedonic food intake (Ruegsegger et al., 2015b), and additionally, naltrexone reduced DA-related mRNAs, and free feeding in only HR rats (Ruegsegger et al., 2016).

The extensive body of data showcasing the implication of the partially overlapping DA and opioid signaling profiles, in response to VWR engagement and palatability-induced feeding behavior, raises the question of whether VWR could intervene with the craving response for palatable food, by modifying the latter’s “liking,” “wanting” and eventually “learning” psychological responses. That question will be

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explored thoroughly in the next section. Nonetheless, the three psychological responses to reward will be addressed with caution for the rest of the thesis’ analysis, as in the absence of known molecular underlying mechanisms, behavioral tests alone prove to be inadequate in deconvolving the different aspects of palatability-induced eating and VWR.

4. VWR’s Influence on Palatability-Driven Feeding Behavior

Following, I presented the efficacy of VWR in influencing palatability-driven feeding behavior and the extent to which this interaction is modulated by three different variables frequently addressed in the literature. These three variables are: (a) the presentation sequence of VWR-palatable diets, (b) rodents’ sex, and (c) the microevolutionary control of wheel running rates through the development of selective mouse and rat breeding lines for enhanced, high versus low rates of VWR. Across the following subsections, I summarized the research papers’ experimental designs and significant results in Tables 1-4, as well as the extended experimental design set-up for each paper in the Supplementary Table 1 and Table 2 of the Appendix.

4.1 VWR’s role in short-term hedonic food intake: the presentation’s sequence effect

Across literature’s experimental designs, the VWR and palatable diets presentation sequence can be broken down into three distinct conditions: (a) presenting the palatable diet(s) precedently of VWR, (b) presenting the palatable diet(s) afterward VWR, or (c) presenting both activities simultaneously. By way of example, sequential presentation of one week of VWR and a 60% fc-HF diet evoked a strong avoidance for the HF diet to male F344xBrown Norway (F344xBN) rats (Scarpace et al., 2012). However, presenting an fc-HF diet two days prior to the VWR caused no preference for HF and SC diets among the running rats (Scarpace et al., 2010); in other words, the preference for the SC and HF diets was equivalent. Simultaneous access to VWR and a 24% fc-sucrose solution caused an elevation in the preference for the, previously avoided, HS diet in male Sprague-Dawley (SD) rats to a no-preference level (Satvat and Eikelboom, 2006). In mice, regardless of the VWR-palatable diets presentation sequence, one and two weeks of acute VWR exposure were associated with a high preference in a 45% fc-HF diet both in the running and sedentary C57BL6 males (Yang et al., 2017).

Interestingly, when Scarpace et al. (2012) simultaneously presented short-term VWR and an HF diet to male F344xBN rats, the rats resulted in avoiding the HF diet altogether and demonstrating a subsequent anorexic behavior. Then when the running wheels were removed, and SC was provided, the rats enhanced its consumption compared to the pre-experimental HF intake. It is unclear whether the complete HF diet avoidance is gender- or species-dependent. Another interpretation of the results could argue that when VWR (conditioned stimulus) is simultaneously paired together with a novel palatable diet (unconditioned stimulus), it produces a place preference in rats that is displayed as a taste aversion (Lett et al., 2001; Yang et al., 2017). To my knowledge, no other study observed complete avoidance of a palatable diet paired with VWR in mice.

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To summarize, the extent to which the sequence of presentation affects the interaction of VWR and short-termed hedonic eating, independently of other factors, is not concrete. Therefore the sequence of presentation co-variable will be acknowledged following in the results’ discussion, but the discussion’s focus will shift to the other variables under review; rodents’ sex and the different rates of VWR.

4.2 VWR’s role in short-term hedonic food intake: the rodents’ sex effect

Considering previous (and lack of thereof) findings that demonstrate the VWR’s potential sex-dependent impact on diet preference, this section explores the different motivation levels for working for VWR and hedonic eating between males and females rodents. Patients of eating disorders are predominantly female (Hudson et al., 2007), yet the lack of studies on sex’s direct effect on the formation of reward-induced neuroadaptations created a misleading stream of research that attributed VWR engagement and diet preference to sex differences based on the metabolism of only male subjects. Apart from balancing the predominance of male rodent results in the literature, the rationale for including female rodents is because they typically run higher distances than males (Looy and Eikelboom, 1989; Scarpace et al., 2012; Shapiro et al., 2011) and display a body mass plateau when engaging in hedonic eating in comparison with the continued body mass growth of males, uncomplicating the measurement of engagement to hedonic food intake.

4.2.1 Behavioral observations of VWR’s influence on hedonic food intake in male and female rodents

For these reasons, several experiments investigating VWR’s influence on hedonic eating incorporated intact animals from both sexes, as well as animals that went into bilateral orchiectomy (GDX) and ovariectomy (OVX). Simultaneous access to acute VWR and a 60% fc-HF diet for six days, evoked a significantly higher preference in SC compared to HF diet, in both male and female SD rats (Moody et al., 2015). Repeating Satvat and Eikelboom’s (2006) sucrose preference experiment on both sexes, revealed that the simultaneous presentation of VWR and a 50% fc-HS diet, caused a significant decrease of HS preference in VWR males and females, which evolved to no preference for either SC or HS diets in the females (Moody et al., 2015). However, when rats had intermittent access to an fc-HF diet, concurrently to more prolonged exposure to VWR (three weeks), the HF diet intake in both male and OVX female rats attenuated, whereas it increased in intact females, evolving to a no SC/HF diet preference. The avoidance of fat consumption by OVX females disappeared when VWR was presented before the palatable diet, and similarly, with intact females, then the OVX rats showed no diet preference (Moody et al., 2015).

In another study with similar conditions (i.e., simultaneous introduction of VWR and a 45% fc-HF diet, for two and a half weeks), apart from testing OVX and intact female, as well as GDX and intact male rats, the OVX females were treated with β-estradiol 3-benzoate (E), progesterone (P) or with both of them for hormone replacement (E+P; Yang and Liang, 2018). Consistently with previous results, all sedentary rats consumed significantly more of the HF diet, whereas the hormone replacement created a VWR-induced palatable diet intake in running rats. That is, while intact and GDX males persistently avoided the HF diet, the intact and OVX E+P females reversed their diet avoidance by the end of the experimental procedure. OVX E and OVX P rats expressed an HF diet preference pattern that lies between the ones by GDX males and intact females. Contrary to the female hormone replacement, the administration of androgens did not reverse the diet avoidance of GDX male rats (Yang and Liang, 2018). Taken together, these results

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suggest that in females, hormonal replacement may play a role in running-induced changes in short-term palatable diet intake.

In line with these observations, more work addressed the topic of diet preference under the influence of VWR, by utilizing the concept of diet novelty and familiarity in concurrent access with VWR. Initially, SD rats of both sexes acclimatized to a 45% HF diet, and then they simultaneously accessed VWR and a new diet rich in carbohydrates (70% HC). The results of this experimental set-up (lasting three and a half weeks) demonstrated that both male and female running rats avoided the novel HC diet, but neither group exhibited a reduction in the intake of the familiar HF diet. The second part of the experiment questioned the the impact of the novel-familiar diets’ reversal. Namely, the sedentary and running groups were reversed for another three and a half weeks, and the new groups were re-habituated to the HC diet until intake stabilized, while both being sedentary. When a novel 60% HF diet was presented concurrently with the unlocking of the running wheels, the HF diet preference and intake significantly decreased for both male and female running, previously sedentary, animals (Yang et al., 2017). Corroborating these results with previous research, when Wistar rats of both sexes were presented initially to one week of VWR and subsequently to a novel ad libitum three-choice 60% HF vs. 70% HC vs. 70% HS diet paradigm for four additional weeks, VWR animals demonstrated robust disfavor for the HF diet. Accordingly, VWR males consumed less HF diet compared with their sedentary counterparts, while elevating the consumption of both carbohydrate diets, and achieving an overall caloric intake equivalent to the sedentary rats. Intriguingly, this pattern of feeding behavior was not observed in females; VWR females consumed more calories in total compared to their sedentary littermates, by enhancing the HF diet intake, while keeping the HC and HS intake levels low (Lee et al., 2017). Similarly to the study by Yang and Liang (2018), Lee et al. (2017) observed no correlation between running distance and diet preference.

4.2.2 Molecular observations of VWR’s influence on hedonic food intake in male and female rodents

Aside from the diet novelty and familiarity experiments, Lee et al. (2017), also, explored the opioid signaling patterns of VWR rats engaging in hedonic feeding behavior for four weeks (see Table 1). According to their results, the mRNA levels of both μ-opioid receptor 1 (Oprm1) and preproenkephalin (Penk) transcripts in the ventral striatum were elevated in VWR females compared to sedentary ones. On the other hand, VWR males showed no significant changes in the mRNA levels of Oprm1 and Penk. However, the absence of mRNA expression fluctuations in VWR males may be expected due to the robust drop in their HF diet intake, compared to their sedentary male counterparts (0.47 fold of decrease), and both female group (0.55 and 0.5 fold of decrease, respectively; Lee et al., 2017). In a like manner, male SD rats exposed to a 60% fc-HF diet followed by a two-week VWR access, exhibited no changes in neither VTA nor NAc Oprm1 mRNA levels (Liang et al., 2015). Granted these observations, Liang et al. (2015) did not test female rats. In an additional study, Lee et al., (2019b) demonstrated that VWR female Wistar rats that concurrently accessed a 56% HC and 75% HF diet for one week, displayed significantly higher sensitivity to DAMGO-induced HC diet consumption, compared to sedentary littermates. Yet in males, DAMGO-driven food consumption raised baseline preference of both diets regardless of the exercise condition.

A handful of studies also explored the effects of concurrent access to VWR and palatability-driven feeding behavior in the DA system receptors’ expression profiles (see Table 1). For example, in sedentary

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male rats receiving on average the same amount of an fc-HF diet that was consumed by the VWR group, NAc Drd1 and Drd2 mRNA levels were significantly lower, compared to the wheel running and control sedentary rats. Besides, no difference was observed in the VTA DA transporter (DAT) levels between VWR and the sedentary control rodents (Liang et al., 2015). On the other hand, access to acute VWR and a three-choice 60% HF vs. 70% HC vs. 70% HS diet model, caused no significant change in the NAc Drd1 and

Drd2 mRNA levels in both male and female rats (Lee et al., 2017), whereas the levels of neither DA nor

DAT levels were recorded. When Correa et al. (2016) administrated haloperidol, a DRD2 antagonist, to A2AR knockout (A2AKO) mice that had access to running wheels and a 50% HS diet, they observed no haloperidol-induced shift from wheel-running activity to sucrose intake. Oppositely, haloperidol reduced the time wild-type (WT) mice spent running and increased the time consuming the HS diet. Besides the behavioral measurements, haloperidol elevated the c-Fos immunoreactivity in the anterior cingulate cortex (ACg) and the NAc core of WT, but not A2AKO mice. Interestingly, prefeeding attenuated the HS diet intake in the T-maze for both WT and A2AKO strains, indicating that hedonic eating may be sensitive to the motivational devaluation emerging from the DRD2 antagonism. Therefore the strain-specific DRD2 levels in NAc core and ACg seem to be involved in the regulation of the intrinsic motivation reinforcing physical activity, but not in the regulation of the primary reinforcing motivation for reward (Correa et al., 2016).

Table 1. Overview of alterations in mesolimbic DA and opioid pathways in female and male rodents in response to the interaction of regular rates of VWR and palatable diets. From left to right, the studied transcripts for each experiment (Agent); measurements from the ventral tegmental area (VTA); measurements form the nucleus accumbens (NAc); intraperitoneal injection of an agonist or antagonist to the studied agents’ action (Agent injection); behavioral effect after the injection (Behavioral effect). Abbreviations: DA: Dopamine; DAT: Dopamine transporter; Drd1: Dopamine 1 receptor mRNA; Drd2: Dopamine 2 receptor mRNA; Oprm1: μ-Opioid receptor mRNA; Penk: Proenkephalin mRNA; ↑: increase in the agents’ levels; ↓: decrease in the agents’ levels; ↔: no difference in the agents’ levels; +: exogenous-induced agonism; –: exogenous-induced antagonism; R: Rats; M: Mice; (X)HF, (X)HS, (X)HF,HS,HC, or (X) HF,HC: number of weeks exposed to an fc-high fat, fc-high sugar, fc-high fat, sugar, and carbohydrates, or fc-high fat, and carbohydrates diet, respectively; c-Fos: expression of the c-Fos gene; ⇒: induction of downstream action.

Females Males

Agents VTA NAc Agent

injection

Behavior al effect

VTA NAc Agent

injection Behavior al effect DA DAT ↔ (R, 4HF; Liang et al., 2015) Drd1 ↔ (R, 4HF,HS,HC; Lee et al., 2017) ↓ (R, 4HF; Liang et al., 2015) ↔ (R, 4HF,HS,HC; Lee et al., 2017) Drd2 ↔ (R, 4HF,HS,HC; Lee et al., 2017) ↓ (R, 4HF; Liang et al., 2015) ↔ (R, 4HF, HS, HC; Lee et al., 2017) – (M, 1HS; Correa et al., 2016) ; ⇒ ↑ c-Fos Oprm1 ↑ (R, 4HF,HS,HC; Lee et al.,

2017) ↔ (R, 3HF,HC; Lee et al., 2019b) ↔ (R, 4HF; Liang et al., 2015) ↔ (R, 4HF; Liang et al., 2015) ↔ (R, 4HF,HS,HC; Lee et al., 2017) ↓ (R, 3HF,HC; Lee et al., 2019b) + (R, 4HF; Liang et al., 2015) ; ⇒ ↓ HF

Penk ↑ (R, 4HF,HS,HC; Lee et al., 2017) ↔ (R, 3HF,HC; Lee et al., 2019b) ↔ (R, 4HF; Liang et al., 2015) ↓ (R, 4HF; Liang et al., 2015) ↔ (R, 4HF, HS, HC; Lee et al., 2017) ↔ (R, 4HF, HC; Lee et al., 2019b)

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A prevalent researched principle in studies exploring VWR’s effect not on hedonic feeding behavior, but drug self-administration is the reward substitution principle, see 7_{(Berridge and Kringelbach, 2008).} In such studies, there has been observed a sex-dependent VWR substitution of the drug (ab)use (Peterson et al., 2014). Namely, while running males display a dose-dependent decrease in cocaine self-administration, females are more resistant to that effect (Bardo et al., 2015; Peterson et al., 2014; Wise, 1996). The degree to which reward substitution occurs has been shown to be sensitive to (a) the intensity of each rewarding stimulus in the context of an individual’s reward ceiling capacity, and (b) the natural level of maximum engaging behavior expressed for each reward, or to both of them. Nonetheless, the presented data on VWR and palatability-driven feeding behavior (as summarised in Table 1) neither explicitly contradict nor support the reward substitution hypothesis of VWR substituting palatability-driven feeding behavior in a sex-dependent manner. However, I can conclude that on a behavioral level, at least, both the prevention and permission of (short-term accessed) wheel running lead to a profound sex-dependent diet preference. Nonetheless, the necessity for further exploring rodents’ response to physical activity while having access to palatable diets merits for both sexes, as the underlying molecular mechanisms of this interaction have not been fully deciphered yet. Similarly with the discussion around the presentation sequence co-variable, the rodents’ sex will be acknowledged in the next sections of the results’ presentation; however, the analysis focus will shift to the final variable under review; the different rates of VWR.

4.3 VWR’s role in short-term hedonic food intake: the different VWR rates’ effect

Next, an arising discussion topic is whether animals could evolve to choose diets that positively affect

their physical performance abilities. In other words, do rodents with enhanced motivation for VWR exhibit a differentiated sensitivity for palatable food, another natural rewarding trait? Studies in selective mouse and rat breeding lines for enhanced, high versus low rates of running (HR and LR, respectively) have explored the interaction of VWR and diet preference for palatable food.

4.3.1 Behavioral observations of VWR’s influence on hedonic food intake in HR vs. LR rodents

Measuring the physiological markers of HR mice engaging in VWR bouts (e.g., daily energy expenditure, resting metabolic rates, locomotor costs and running behavior), showed that they run roughly 50-90% more compared to their LR control counterparts. Also, HR running rodents display sex-dependent energetic cost variations due to the evolution of their running behavior; HR males ran ~40% more time than their LR counterparts but completed shorter running bouts than HR females. On the contrary, HR females run longer distances without increasing their running time (Girard et al., 2001; Rezende et al., 2009). Also, monitoring the running speed and oxygen consumption of HR mice for 24-48h, as well as controlling for body mass effects, displayed that the daily energy expenditure raised 23% in males and 6% in females, with the total VWR activity cost, see 8_{, to raise as much as 29% in males and 5% in females,} compared with their LR littermates (Rezende et al., 2009). These results suggest that the enhanced breeding lines’ effect on the running energy budgets can differ dramatically between the sexes and that

7_{According to it, aside from the researched reinforcer’s introduction, an additional competitive reward reinforcer alleviates the}

“wanting,” and ultimately the “learning” for that former, initial rewarding stimulus (Berridge and Kringelbach, 2008).

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the HR males’ energetic constraints might partly explain their apparent limitation for VWR (Rezende et al., 2009). Hence, in this line of research, a plethora of studies use female rodents, as a way to record more significant responses to experimental treatments (Girard et al., 2001; Rezende et al., 2009).

Recent evidence demonstrates that both controls and Hsd:ICR outbred mice selective for HR rates display a strong preference for a 40% fc-HFHS diet, regardless of sex. However, after two weeks of VWR acclimation, HR mice preferred the HFHS diet significantly more than the control ones (Acosta et al., 2017). Eventually, by the completion of the running period, all groups except control LR males have run significantly more (Acosta et al., 2017). Moreover, when HR running female mice were presented with several HS diets (i.e., solutions with artificial sweeteners, 3% and 10% w/v sucrose), the solutions’ consumption elevated the running levels of LR mice, whereas it left unaffected the HR mice. In accordance, when HR mice had access to running wheels, they consumed significantly less artificial sweeteners, compared to the control, LR mice (Thompson et al., 2018). The writers’ interpretation implies that HR mice have evolved a reduced incentive salience towards non-nutritive sweetener blends, due to alterations in the reward system that “may be attributed to a greater incentive salience to VWR, and is explained by the phenomenon of cross-sensitization rather than reward substitution.” Their parsimonious explanation could be justified by the existence of an inherent trade-off system between distinct rewarding pathways that “tunes” different rewarding stimuli potentially based on their intensities and duration. In other words, there is a limited capacity for the processing of reward stimuli by the reward brain network (Belke and Garland, 2007; Thompson et al., 2018). Based on this hypothesis, if sucrose, apart from being a substrate of catabolic metabolic reactions, also taxes HR mice’ reward for maximal aerobic capacity, may explain why sucrose-consuming LR, but not HR, mice enhance their VWR rates.

Moving to rats, Lee and colleagues (2019a) assessed the diet preference for a three-choice feeding paradigm (as described by Lee et al. (2017); ad libitum 60% HF vs. 70% HC vs. 70% HS diets) on Wistar rats selectively bred for HR running. Interestingly, they found a dissimilarity in the total intake between HR and LR rats only in males, whereas female HR and LR rats consumed equal amounts of calories. Nonetheless, the respective dissimilarity in calory intake between males and females may be associated with the distinct energy expenditures between the two linetypes across animals of different sex (Rezende et al., 2009). Specifically, as it has been noted, HR rats showed a preference for the HF diet, and females consumed more HF diet than males across both lines, respectively. Furthermore, there was no influence by the physical activity status alone on the diet preference within any rat group. Moving to the preference for the HS and HC diets, HR rats consumed less of them compared to their LR counterparts, and among both HR and LR lines, males had higher HS and HC preference (Lee et al., 2019a). The researchers attribute the line- and sex-dependent variation in HF diet intake to the different ceiling of animals for reward properties (Lee et al., 2017), as already proposed by the bibliography (Belke and Garland, 2007; Thompson et al., 2018). In accordance, juvenile, female HR rats consumed more of the fc-HFHS diet during an eight-week timeframe, than their LR counterparts, independently of wheel running or diet preference (Ruegsegger et al., 2015a).

4.3.2 Molecular observations of VWR’s influence on hedonic food intake in HR vs. LR rodents

The data mentioned above lead to the following hypothesis: could high rates of VWR offset the deleterious effects of palatable diets despite the higher energy intake, through alterations of the DA and

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opioid signaling in the VTA-NAc circuit? Although not an abundance of studies analyzed the molecular cascades that uniquely mediate the phenotypic behavioral differences between HR and LR rodents, monitoring these genetic factors would provide ample insight into the genetic regulation of the VWR engagement. Indeed in a follow-up study, Lee and colleagues (2019a) examined the expression of Oprm1 and Penk genes among HR and LR rats and found a trend towards elevated Penk mRNA expression among the male running rats of both linetypes. As for Oprm1 expression profiles, HR male rats demonstrated higher levels of Oprm1 mRNA compared to the LR ones, regardless of their physical activity status. In contrast, females showed no significant change in the line- or physical activity status-dependant expression of Oprm1 gene (Lee et al., 2019a). Even though the energy homeostasis neurocircuitry is beyond the literature thesis’ focus, data shows that body fat percentage, but also the expression of the

Cart and Lepr transcripts in the mediobasal hypothalamus, do not differ between HR and LR rats

(Ruegsegger et al., 2015a). Collectively, this section’s results propose that throughout selective breeding, the HR rodents develop a more robust rewarding response to physical activity, partially mediated, according to my hypothesis, by an adaptation in the VTA-NAc circuit’s opioid signaling.

Altogether, on the base of the summarized observations regarding VWR’s role in short-term palatability-driven food intake and the impact on the three analyzed co-variables on that interaction, I can not draw a definite conclusion on the underlying cross-sensitization or reward substitution of palatability-driven food intake by VWR. Nonetheless, these theories could be fully corroborated if the evidence swings towards the existence of an inherent trade-off system between distinct rewarding pathways.

4.4 The separate impact of VWR and hedonic food intake on the VTA-NAc circuit

After reviewing the current literature on VWR and hedonic feeding behavior, it is clear that acute access to palatable diets does not provide the optimal methodological framework for determining VWR’s role in relation to palatability-induced eating behavior for two main reasons. Firstly, in the absence of an abundance of research on the molecular basis of the HR VWR and palatability-induced feeding behavior interaction, it is ambiguous whether HR running rodents are micro-evolutionary more fit to ameliorate palatability-induced neuroadaptations. Secondly, given that prolonged exposure to palatable diets induces morphological, biomolecular, and plasticity impairments in the midbrain and striatal neuronal circuits, acute hedonic food intake does not establish the prerequisite dysfunctional neurocircuitry for studying a subsequent VWR intervasion. Hence, I gathered a selection of behavioral and molecular observations, respectively, on (a) HR and LR running rodents with access only to standard chow diet (section 4.4.1, and as summarized in Table 2), and (b) sedentary rodents with chronic exposure to palatable, energy-dense diets (section 4.4.2, and as summarized in Table 3) to observe the impact of each pleasurable activity on the reward neurocircuits distinctively of the other.

4.4.1 Interaction of acute HR vs. LR VWR activity status and homeostatic food intake

This section’s interest lies in recognizing whether the manipulation of different VWR running rates in non-hedonic eating control rodents, uniquely shapes the mesolimbic DA and opioid systems’ signaling in the VTA-NAc circuit. As evidenced in a range of studies, SC-fed HR mice presented alterations in the hippocampal DA signaling pathway (Bronikowski et al., 2004), the serotonergic system (Claghorn et al., 2016) and the eCB system of the VTA-NAc circuit (Keeney et al., 2008; Thompson et al., 2017). Given the