Play fighting and the development of the social brain: The rat’s tale

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Neuroscience and Biobehavioral Reviews 145 (2023) 105037

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Play fighting and the development of the social brain: The rat’s tale Sergio M. Pellis *, Vivien C. Pellis, Jackson R. Ham, Rachel A. Stark Department of Neuroscience, University of Lethbridge, Lethbridge, Alberta T1K3M4, Canada

A R T I C L E I N F O

A B S T R A C T

Keywords: Rough-and-tumble play Orbital frontal cortex Medial prefrontal cortex Social coordination Dendritic arbor Socio-cognitive skills

The benefits gained by young animals engaging in play fighting have been a subject of conjecture for over a hundred years. Progress in understanding the behavioral development of play fighting and the underlying neurobiology of laboratory rats has produced a coherent model that sheds light on this matter. Depriving rats of typical peer-peer play experience during the juvenile period leads to adults with socio-cognitive deficiencies and these are correlated with physiological and anatomical changes to the neurons of the prefrontal cortex, especially the medial prefrontal cortex. Detailed analysis of juvenile peer play has shown that using the abilities needed to ensure that play fighting is reciprocal is critical for attaining these benefits. Therefore, unlike that which was posited by many earlier hypotheses, play fighting does not train specific motor actions, but rather, improves a skill set that can be applied in many different social and non-social contexts. There are still gaps in the rat model that need to be understood, but the model is well-enough developed to provide a framework for broader comparative studies of mammals from diverse lineages that engage in play fighting.

1. Introduction Play behavior occurs in a variety of animals, especially in mammals and birds (Burghardt, 2005; Kaplan, 2020). For most species that play, it is especially prevalent in the juvenile period (Fagen, 1981), the age spanning from weaning to sexual maturity (Pagel and Harvey, 1993). Why juveniles expend time and effort in what seems a frivolous activity has puzzled researchers for well over a century since Groos’ seminal book circulated widely in its English translation (1898). More than two dozen different hypotheses have been posited to explain the benefits gained by playing (Baldwin, 1986), but none have received unequivocal support (Martin and Caro, 1985; Sharpe, 2019). Groos (1898) posited that playing as juveniles trains animals in the use of the motor patterns needed in adulthood. Variations of this explanation are still commonly espoused not only in the professional literature but also in nature doc­ umentaries (Pellis and Pellis, 1998). This is especially the case for play fighting or rough-and-tumble play which is one of the most frequently reported forms of social play (Pellis and Pellis, 2009). As the name im­ plies, this form of play resembles serious fighting and from a Groosian perspective, it has been argued that, by play fighting, juveniles practice combat-related behavior patterns that will make them better fighters as adults (Smith, 1982; Symons, 1978). But as for play more generally, empirical tests of this hypothesis have yielded mixed results, with pos­ itive associations found in some species (e.g., Blumstein et al., 2013) but

not in others (e.g., Sharpe, 2005). Despite these contradictory findings, there is growing evidence across several species that, by engaging in social play, especially play fighting, juveniles can improve their survival and reproductive pros­ pects (e.g., Ahloy Dallaire and Mason, 2017; Fagen and Fagen, 2009; Nunes, 2014; Perret, 2021). Thus, playing as juveniles can be beneficial, but even in species that can play extensively, this is not universally the case for all members of the species, as local or seasonal conditions can markedly vary its occurrence (e.g., Baldwin and Baldwin, 1974; Barrett et al., 1992; Pellis, 1981; Stone, 2008). Quite simply, play is conditional, so that if resources permit, animals play and gain whatever benefits are associated with performing such behavior, but if not, animals can still mature into functional adults (Martin and Caro, 1985). The problem is that of identifying and characterizing the mechanisms by which play can exert such flexible benefits. In the present review, we explore the in­ sights gained on this issue from the study of laboratory rats (Rattus norvegicus). In rats, most play is social (Meaney and Stewart, 1981; Panksepp, 1981), involving play fighting, whereby the animals compete for an advantage (Aldis, 1975; Pellis and Pellis, 1987). While solitary play, involving locomotor-rotational movements, occurs in rats, most such movements are performed in a social context, and are used to solicit playful approaches from partners or to help facilitate gaining the advantage over their partner (Pellis and Pellis, 1983; Thor and

* Corresponding author. E-mail address: [email protected] (S.M. Pellis). https://doi.org/10.1016/j.neubiorev.2023.105037 Received 27 October 2022; Received in revised form 29 November 2022; Accepted 3 January 2023 Available online 5 January 2023 0149-7634/© 2023 Elsevier Ltd. All rights reserved.

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Neuroscience and Biobehavioral Reviews 145 (2023) 105037

Holloway, 1983). As play fighting peaks in the juvenile period (Thor and Holloway, 1984), and juveniles are highly motivated to engage peers in this behavior (Varlinskaya et al., 1999), social play interactions provide a major source of social experience at this age. Therefore, the first issue to explore are the consequences of denying juveniles the opportunity to play with peers.

much as is the case with rats that have been reared in complete isolation (e.g., Byrd and Briner, 1999; van den Berg et al., 1999). However, ju­ venile rats are hyper-social (Douglas et al., 2004; Varlinskaya and Spear, 2008; Varlinskaya et al., 1999), so failure to have direct social contact may be stressful and the ensuing deficits could be accounted for by the negative effects of stress and not the lack of social play. Indeed, rats reared across barriers show signs of hyper-defensiveness (Bell, 2014), not unlike the hyper-defensiveness present in rats reared in complete isolation (Einon and Potegal, 1991). An alternative is to rear rats together, but with partners that provide reduced or atypical play experience. When play first begins to emerge in the latter part of the third week of life, young rats preferentially target siblings, while mothers rarely, if ever, initiate play with their offspring (Cramer et al., 1990; Pellis and Pellis, 1997; Thiels et al., 1990). Therefore, one approach for social rearing with reduced/atypical play is to house a juvenile with an adult (Fig. 1B). In such a rearing environ­ ment, not only does the juvenile rat have an impoverished play expe­ rience (Pellis et al., 2017), but it also results in an adult with altered development of the prefrontal cortical neurons (Bell et al., 2010; Himmler, Pellis and Kolb, 2013) – a brain area that has been implicated with some of the behavioral deficits associated with reduced play experience (Pellis et al., 2014; Vanderschuren and Trezza, 2014). Sup­ porting this association is that, like rats, hamsters (Mesocricetus auratus) reared with an adult also exhibit atypical prefrontal neuronal develop­ ment and deficiencies in social skills (Burleson et al., 2016). However, there are limitations with this method as well. Critically, this rearing arrangement works well for juvenile female rats but not males. Juvenile females can be reared with an adult female, that will huddle with and groom the juvenile, but mostly avoid the ju­ venile’s playful overtures (Pellis et al., 2017). Juvenile males cannot be reared with an adult female because, with the onset of sexual maturity, the male’s attention to the female becomes sexual (Pellis and Pellis, 1990), thereby creating a very different experience to that of the juve­ nile females. Rearing a juvenile male with an adult male is not equiva­ lent to rearing a juvenile female with an adult female, as adult males are less tolerant of young rats, leading to periodic pummeling, as a domi­ nance relationship is enforced, which results in the experience of social defeat (e.g., Burke et al., 2010, 2013). The intolerance exhibited to playful youngsters by the adults in the adult rearing paradigm may cause some level of stress, which, in turn, may influence development, and so account for some of the neural and behavioral effects, muddying the effects of the lack of play. To avoid this adult-induced stress, another paradigm is to rear a juvenile with a same sex peer from a less playful strain of rat (Fig. 1C). Pioneering this approach, Schneider and colleagues reared Wistar rats, a highly playful strain (B.T. Himmler, Modlinska et al., 2014; S.M. Himmler, Modlinska et al., 2014) with a Fischer 344 (F344) partner, a low playing strain (Siviy et al., 1997, 2003). Female Wistar rats reared with either a single F344 partner or with multiple F344 partners, experienced less and atypical play as juveniles and exhibited neural and pain threshold changes, as well as reduced social skills (Schneider et al.,

2. The deficits associated with play deprivation Social isolation during the juvenile period results in adults with a range of physiological, emotional, cognitive and social deficits, a phe­ nomenon that has been well-documented in rats (e.g., Baarendse et al., 2013; Byrd and Briner, 1999; Cuesta et al., 2020; da Silva, Ferreira, Carobrez, and Morato, 1996; Einon and Potegal, 1991; Fone and Pork­ ess, 2008; Hall, 1998; Hermes et al., 2011; Potegal and Einon, 1989; Potrebi´c et al., 2022; van den Berg et al., 1999; von Frijtag et al., 2002). These are chronic deficits that do not arise if rats are subjected to comparable periods of isolation at older ages (e.g., Arakawa, 2002, 2003; Seffer et al., 2015), and they are not rectified by social housing after the juvenile period (Baarendse et al., 2013; Einon and Potegal, 1991; Potegal and Einon, 1989). At least some of these deficits have been attributed to the lack of experiences normally derived from social play (Pellis and Pellis, 2006). A compelling case for such a role for play was made by Einon and her colleagues. First, they found that, when observed in litters, juvenile rats play about an hour per day (Hole and Einon, 1984). Second, they determined, using spatial and cognitive tests, that rats being reared in isolation as juveniles have deficits when adult (Einon, 1980; Einon and Morgan, 1976; Einon et al., 1975). Third, they showed that if juveniles reared in isolation were given daily exposure to a same-age peer for one hour per day they did not exhibit these deficits. However, if that peer was injected with a drug that rendered them non-playful, but still mo­ bile, the experimental rats exhibited the same deficits as the isolated rats not given daily exposure to peers (Einon and Morgan, 1977; Einon et al., 1981). That is, it was the opportunity to play that made the difference between the two conditions, although recent evidence shows that while a short period of daily exposure to a peer is better than complete isolation it is not as good as continuous exposure to a playful partner (Bijlsma et al., 2022). Modern standards of animal welfare make both total isolation and the use of repeated injections of the same rats difficult to justify. To avoid injecting the same rat multiple times, new rats could be used each day, but ethical issues are raised as the number of rats used is increased dramatically. Consequently, less invasive, approaches have been developed to abolish or reduce the amount of play experienced over the juvenile period. One approach is to rear rats next to one another, but separated by a transparent, perforated barrier (Fig. 1A). This enables them to see, smell and hear one another, as well as huddle up against each other, but not play together. When tested as adults, such rats exhibit reduced cognitive flexibility (Bijlsma et al., 2022) and a reduced ability to coordinate their movements with a partner (Pellis et al., 1999),

Fig. 1. Depriving rats of social play without social isolation. A. Pairs of juveniles separated by a perforated barrier; B. juveniles reared with an adult; and C. mismatched juveniles, in which a rat from a high playing strain is housed with a peer from a low playing strain. (Reprinted from Stark, 2021). 2

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¨tz et al., 2016; Schneider, Bindila et al., 2016). 2014; Schneider, Pa Using the F344 paradigm, we reared Long Evans rats (LE), another highly playful strain (Himmler, Stryjek et al., 2013; Himmler, Modlinska et al., 2014), with F344 peers. LE rats were used because they were the strain initially used in the adult rearing paradigm (Bell et al., 2010; Himmler, Pellis and Kolb, 2013), and the F344 paradigm allowed both males and females to be tested and compared. When reared with a same sex F344 peer, both male and female LE rats experienced atypical play as juveniles (Stark et al., 2021), and as adults had the same atypical development of the prefrontal neurons (Stark et al., 2023) and deficient social skills (Stark and Pellis, 2020, 2021). These latter studies more precisely target play as having an important role in the development of brain mechanisms associated with social skills. Importantly, Wistar rats reared with Sprague Dawley rat partners, another high playing strain (B.T. Himmler, Modlinska et al., 2014; S.M. Himmler, Modlinska et al., 2014), do not exhibit the deficits associated with being reared with a F344 partner (Schneider, Bindila et al., 2016). This is the case even though Wistar and Sprague Dawley rats have some differences in how they play (B.T. Himmler, Modlinska et al., 2014; S.M. Himmler, Modlinska et al., 2014; Manduca, Campolongo et al., 2014; Manduca, Servadio et al., 2014). LE rats have a style of play that is similar to that of Wistar rats (B.T. Himmler, Modlinska et al., 2014; S.M. Himmler, Modlinska et al., 2014), and when they are reared with Sprague Dawley partners, their play is modified (S.M. Himmler, Lewis and Pellis, 2014). These findings suggest that some degree of modifi­ cation in play can occur without major developmental consequences, but that greater disruption to play experience, as when Wistar or LE rats are reared with a F344 partner, leads to deficits in the development of the brain and associated behavioral skills. The questions that arise are which skills are dependent on play, which neural mechanisms do these involve and what specific play experiences are critical?

Pellis et al., 1989). For most strains used to study play in rats, around 90% of attacks are directed to the nape (Himmler, Stryjek et al., 2013; Himmler, Modlinska et al., 2014). This makes it relatively easy to distinguish playful from serious fighting and also to detect when a play fight escalates to serious fighting (Smith et al., 1999; Stark and Pellis, 2020) — a feature that can prove useful in exploring the interface be­ tween friendly and aggressive interactions (Pellis et al., 2022). Play fighting in rats mostly involves complex, if brief (around 3–5 s), bouts of wrestling in which the nape is attacked and defended. Fig. 2 shows the sequence of events during a bout between two juvenile males. The rat on the left approaches its partner (a) and reaches toward its nape from the rear (b), but before contact can be made, the partner rotates around its longitudinal axis (c) to face its attacker (d). By moving for­ ward, the attacker pushes the defender onto its side (e). The defender then rolls over onto its back as the attacker continues to reach for its nape (f–h). Once in the supine position, the defender launches an attack on its partner’s nape (i), but fails due to its partner’s use of its hind foot (j, k). Eventually, the rat on top (l) is pushed off by the supine animal (m), which then regains its footing (n). The latter illustrates the other feature of play fighting — turn taking — as once he wriggles free, the original defender lunges towards its partner’s nape (o), restarting the interaction in which he is now the attacker. Depending on the strain, age and sex of the partner, about 80–90% of nape attacks are defended by the recipient (Himmler, Himmler, Pellis and Pellis, 2016). To do so, a rat has two main options – evade contact by running, jumping or swerving away, or turn to face the attacker, using its forepaws and face as a shield against nape contact. Evasive defense occurs around 20–30% of the time, although it can be higher in some strains, but the predominant tactics are those of facing defense (Himmler, Modlinska et al., 2014; Himmler, Stryjek et al., 2013; Rein­ hart et al., 2004; Siviy et al., 1997, 2003). Facing defense involves two major tactics – vertical rotation around the pelvis and rotation around

3. The social play of rats Play fighting, also known as rough-and-tumble play, is the most common form of play in rats (Pellis and Pellis, 2009), but because of its superficial resemblance to aggression, and since it is most frequent during the juvenile period, play fighting has been thought to be imma­ ture serious fighting (e.g., Hurst et al., 1996; Silverman, 1978; Taylor, 1980). However, play fighting, unlike serious fighting, involves a posi­ tive affective state (Siviy and Panksepp, 2011; Vanderschuren, 2010; Vanderschuren et al., 2016), in which the partners engage in some de­ gree of reciprocity, whereby they take turns as to which partner attacks and which defends (Palagi, Cordoni et al., 2016). Critically, because of the importance of reciprocity, playful interactions are a combination of competition and cooperation (Pellis and Pellis, 1998, 2017; Pellis, Pellis and Reinhart, 2010), unlike serious fighting, which tends to be purely competitive (Blanchard and Blanchard, 1994; Geist, 1978; Pellis, 1997). Another distinguishing feature of play fighting in rats is that the majority of interactions involve attempts to gain access to the nape of their partner’s neck which is nuzzled with the tip of the snout if con­ tacted (Pellis and Pellis, 1987; Siviy and Panksepp, 1987). This action again distinguishes play fighting from serious fighting, as in the latter the animals compete to deliver bites to the lower flanks and rump, and to the face (Blanchard et al., 1977; Pellis and Pellis, 1987). Such a distinction is not the case for all species that engage in play fighting. For many species, play fighting and serious fighting involve biting or strik­ ing the same body targets, in which case, the play fighting is a friendly simulation of serious fighting (Aldis, 1975), but in others, it is a simu­ lation of sexual, affinitive or predatory behavior (Pellis, 1988), and furthermore, in some species, play fighting can involve more than one type of simulation (Pellis and Iwaniuk, 2004; Pellis and Pellis, 2018). For rats, and many other mouse-like family of rodents (Muridae), play fighting involves competing for body targets that are nuzzled, licked or nibbled during courtship (Pellis, 1993), with gentle bites to the rump area comprising a small percentage of the encounters (Burke et al., 2021;

Fig. 2. A sequence of play fighting is shown for a pair of juvenile male rats. They compete for access to each other’s napes using a variety of maneuvers (see text for further details). (Reprinted from Pellis and Pellis, 1987, with permission). 3

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the longitudinal axis (Pellis et al., 2022). In the former, the defending rat responds to the attacker, typically when approaching from the rear, by pivoting vertically around its pelvis to face the oncoming partner. In the latter, the defending rat responds to the attacker, typically when approaching from the rear, by rotating around its longitudinal axis, starting with the head and progressing to the pelvis, again creating space between its nape and the partner’s snout (Pellis and Pellis, 1987; Pellis et al., 1989; Whishaw et al., 2021). The complete rotation tactic usually leads to a ‘pin configuration’ (Panksepp, 1981), in which one rat is on its back and its partner stands over it (see panels g and h in Fig. 2). However, this pin configuration can also arise from the defending rat adopting the partial rotation defense tactic and then being pushed over by the attacking rat or by one partner pushing the other over from the mutual upright position (Himmler, Himmler, Stryjek et al., 2016; Pellis and Pellis, 1987, 1997). Detailed descriptions, diagrams and videos of these various tactics can be found elsewhere (Himmler, Pellis and Pellis, 2013; Pellis et al., 2022). Developmentally, play fighting begins to appear around 17 days of age (Baenninger, 1967; Bolles and Woods, 1964; Pellis and Pellis, 1997), peaks in frequency in the juvenile period, 30–40 days of age, then gradually declines with the onset of puberty, but continues, albeit at a lower frequency, well into adulthood (Pellis and Pellis, 1990, 1991a; Thor and Holloway, 1984). There are age-related changes in which defense tactics are used most often, but facing defense remains the predominant form of defense at all ages (Pellis and Pellis, 1990, 1997). Of the facing defense tactics, complete rotation – one of the longitudinal rotation tactics – becomes predominant around 28 days of age (Pellis and Pellis, 1997) and remains predominant during the juvenile period in both sexes (Pellis and Pellis, 1990). However, there are differences be­ tween males and females with the onset of sexual maturity. For males, partial longitudinal rotation and vertical rotation become more preva­ lent after sexual maturity, whereas for females, complete longitudinal rotation remains the predominant facing defense (Pellis, 2002). Once dominance relationships form between mature males, sub­ ordinates will mostly perform complete rotation when playfully attacked by a dominant male but will use the standing defenses when attacked by another subordinate (Pellis and Pellis, 1991b, 1992; Pellis et al., 1993). Both dominant and subordinate males mostly use standing defenses when attacked by females (Pellis et al., 2006; Smith et al., 1998). As juveniles, males are frequently reported to play more than females (e.g., Meaney, 1988; Meaney and Stewart, 1981; Pellis and Pellis, 1990; Thor and Holloway, 1983), but this is not invariably the case (e.g., Himmler, Stryjek et al., 2013; S.M. Himmler, Modlinska et al., 2014; Panksepp, 1981). Males playing more than females is most likely to occur when rats are reared in multi-animal, mixed sex groups, and least likely when reared with one, same-sex partner (Himmler, Himmler, Pellis and Pellis, 2016). Other factors, such as the duration of the pre-test social isolation, the sex of the partner and the duration of the test period may also affect whether sex differences in play are revealed (Argue and McCarthy, 2015; Pellis et al., 2022; Thor and Holloway, 1984). At least for the strains that have been studied, the age-related changes in the use of defensive tactics after sexual maturity is robustly different between the sexes (Pellis, 2002).

birds (Kruska, 1988, 2005), it was long held that ‘brainier’ animals, that is, those with larger forebrains, are more playful (Fagen, 1981). Sur­ prisingly, rats and hamsters decorticated at birth grow into juveniles that play just as much as their intact siblings and are able to use all the defensive tactics available in their species-typical repertoire (Murphy et al., 1981; Panksepp et al., 1994; Pellis et al., 1992), indicating that the cortex is not necessary to motivate play nor to regulate how to play — sub-cortical mechanisms suffice in this regard (Pellis and Pellis, 2009). Particularly surprising is that the degree of reciprocation is not depen­ dent on the cortex (Himmler, Himmler, Pellis and Pellis, 2016), again suggesting that a base-line degree of reciprocation during play fighting is regulated by subcortical neural circuits (Pellis, Pellis and Reinhart, 2010). Nonetheless, it is not that the cortex is not involved in regulating play, just that it does so in subtle ways (Pellis et al., 1992; Schneider and Koch, 2005). To date, we have identified three distinct ways in which cortical circuits can modulate how rats play, and moreover, this involves a triple dissociation (Table 1). The first is the modulation of one of the devel­ opmental changes in which defense tactics are most often used. As noted above, from the early onset of play to its peak performance in the ju­ venile period, both sexes switch from mostly standing defenses to supine defenses. Rats that are decorticated at birth or have ablations restricted to the motor cortex do not undergo this switch, but rather, retain the predominant use of standing defenses from infancy to adulthood (Kamitakahara et al., 2007; Pellis et al., 1992). The second is the mod­ ulation of which defense tactics are most often used, irrespective of age. Evasive tactics involve less coordination with the movements of the partner than do facing defense tactics, and neonatal decortication or selective ablation of the medial prefrontal cortex (mPFC) produces rats that perform significantly more evasive than facing defenses (Bell et al., 2009; Pellis et al., 1992). A subsequent study using a non-playful social test paradigm confirmed that rats with damage to the mPFC have reduced ability to coordinate their movements with those of their partner (B.T. Himmler, Bell et al., 2014). The third is the modulation of which defense tactics are most often used depending on the relationship with the partner, a form of modulation most evident in post-pubescent males, when dominance relationships become more clearly delineated. Subordinate male rats with ablations restricted to the orbital frontal cortex (OFC) fail to modify their play when interacting with a dominant male, but rather, treat all partners similarly (Pellis et al., 2006). That this failure to modulate one’s behavior with the identity of one’s partner is not limited to dominance-subordinate relationships was confirmed by testing in a non-play social paradigm – rats with OFC damage do not modulate their behavior based on their partner’s identity (Pellis et al., 2006). Such failure can also be detected in juveniles by subtle changes in their play behavior with either complete cortical ablations or ones limited to the OFC (Pellis et al., 1992, 2006). Importantly, none of these lesions reduce the age-typical frequency of the play performed, and the effects of lesions to each area is restricted to a deficit in only one type of modulation (Table 1). While play may provide the opportunity to practice motor patterns used later in serious contexts by adults (e.g., Carter et al., 2019; Dawson

4. The role of the cortex

Table 1 Three distinct types of modulation during play are associated with three distinct cortical areas.

All levels of the nervous system are engaged during play, including the mid- and forebrain (Siviy, 2016, 2019; Vanderschuren et al., 2016; VanRyzin et al., 2020). Circuits in the mid-brain are crucial for moti­ vating play, and these connect to cortical circuits, especially in the prefrontal cortex, which are activated when playing (Gordon et al., 2002, 2003; van Kerkhof et al., 2013, 2014). Comparatively, as overall brain size is correlated with the increased likelihood of playing in both mammals and birds (Byers, 1999; Iwaniuk et al., 2001; Kaplan, 2020; Lewis, 2000), and since brain size increases are usually associated with a disproportionate increase in the size of the cortex or its equivalent in

Cortical area ablated/type of modulation*

Age-related modulation of defense

Action-related modulation of defense

Partner-related modulation of defense

Motor cortex Medial prefrontal cortex Orbital frontal cortex

↓

0

0

0

↓

0

0

0

↓

*

4

0 = no effect; ↓ = impaired

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with the assumption that the juvenile living with an adult would expe­ rience less play than one living with a peer (Bell et al., 2010); an assumption subsequently confirmed empirically (Pellis et al., 2017). Moreover, since rats are raised within litters (Barnett, 1975), and the presence of multiple partners can elevate the amount of play present (Pellis and McKenna, 1992), we reasoned that rats reared with multiple, same-age peers would experience more play than ones reared with a single peer, and that more play would be better than less play (Fig. 3). To assess the effects of these rearing conditions on the development of the prefrontal cortex, the rats were sacrificed at 60 days, their brains removed and histologically prepared with the Golgi-Cox stain. This stain penetrates individual neurons completely, allowing the dendritic arbor and the cell body to be viewed under the microscope (e.g., Crombag et al., 2005; Kolb et al., 2003). There were several surprises in store. The presence of a peer led to the pruning of the dendritic arbor of the neurons from the mPFC (specifically area Cg3 – Zilles, 1985), but the magnitude of this pruning was the same whether the rat was housed with one peer or three peers (Bell et al., 2010). However, the dendritic arbor of the OFC neurons (specifically area AID – Zilles, 1985) was larger in rats reared with three peers compared to rats reared with a single cage mate – either one peer or one adult. The experiment was repeated, but this time, rats were reared with either one adult or three adults. Whether reared with three peers or three adults, the dendritic arbor of the OFC neurons was enlarged compared to rats reared with one partner (Bell et al., 2010). Since complexity of many cortical neurons is experience dependent (Kolb, 1995), and since the natural rearing environment is one with multiple animals (Schweinfurth, 2020), it can be concluded that the pruning of the mPFC neurons is dependent on the experience of play with peers, whereas the enlarged complexity of the arbor of OFC neurons is dependent on interacting with multiple animals, irrespective of whether those interactions involve play or not (Pellis and Pellis, 2009). Given what we know about the effects of selective damage to the mPFC and the OFC, there may be some logic to these findings. The mPFC is involved in coordinating actions with partners (Bell et al., 2009; B.T. Himmler, Bell et al., 2014), and since effective coor­ dination is critical for sustaining play fighting in many species (e.g., Llamazares-Martín et al., 2017; Palagi et al., 2015, 2019; Pellis and Pellis, 2017), such play provides an ideal context for training the skills associated with the mPFC. In contrast, the OFC is involved in modulating one’s behavior depending on the identity of the partner (Pellis et al., 2006), and what provides the critical experience is having to interact with multiple animals, learning to adapt behavior depending on the idiosyncrasies of particular individuals. Once rats are fully weaned, around 34 days (Cramer et al., 1990), they mainly interact with peers (Barnett, 1975; Thiels et al., 1990), particularly with ones of a similar age, as older juveniles avoid playing with younger ones (Pellis et al., 2018). So, it is likely that, in naturally occurring colonies of rats, most experience of multiple partners arises in the context of peers playing together. Therefore, play experiences directly influence some neural

et al., 2022; Sharpe, 2005), it does not mean that the lack of play fighting experience as juveniles that results in impoverished reproductive suc­ cess or combat effectiveness and the ability to gain dominance (e.g., Blumstein et al., 2013; Nunes, 2014; Perret, 2021), results from the lack of practicing associated behavior patterns as posited by Groos (1898). Rather, rats that have been reared in social isolation, and so are devoid of play experience, react with heightened anxiety to fearful situations (da Silva et al., 1996), have an exaggerated stress response to such sit­ uations (von Frijtag et al., 2002), overreact to benign social contact (Einon and Potegal, 1991), fail to behave submissively when confronted by a dominant rat (van den Berg et al., 1999), have difficulty coordi­ nating movements with a partner in both sexual and nonsexual contexts (Duffy and Hendricks, 1973; Hole et al., 1986; Pellis et al., 1999) and are less competent in solving cognitive tasks (Einon et al., 1975, 1978, 1981). That is, they have all the hallmarks of impoverished executive functions. Executive functions refer to a collection of control processes neces­ sary for the organization of complex—and often goal-ori­ ented—sequences of movements, including monitoring behavior, attention, resistance to interference, behavioral inhibition, planning, decision making, short-term memory and task switching (Dalley et al., 2004; Diamond, 2013). Many of these deficits have been found to emerge in rats and hamsters reared in less draconian conditions than complete social isolation (see Fig. 1) - that is, with some social contact, but with limited or atypical play experience (Bijlsma et al., 2022; Bur­ leson et al., 2016; Pellis et al., 1999; Schneider, Bindila et al., 2016; Stark and Pellis, 2020, 2021). Moreover, there is growing evidence that play experiences in the juvenile period affect the development of the prefrontal cortex, the area of the cortex known for its role in regulating executive functions (Baarendse et al., 2013; Bell et al., 2010; Bijlsma et al., 2022; Burleson et al., 2016; Himmler et al., 2013; Stark et al., 2023). Therefore, rather than practicing particular motor patterns (Groos, 1898), play fighting is more likely to help train the brain to use executive functions more effectively, which, in turn, leads to improved performance in a variety of social and non-social contexts (Pellis and Pellis, 2009; Pellis, Pellis and Bell, 2010). 5. Play and the development of the prefrontal cortex Given that reduced play experience in the juvenile period leads to impoverished executive functions (Pellis et al., 2014; Vanderschuren and Trezza, 2014), and that selective damage to regions of the prefrontal cortex produces deficits similar to some of those reported in play deprived rats (Bell et al., 2009; B.T. Himmler, Bell et al., 2014; Pellis et al., 2006), it was reasonable to predict that play influences the development of these prefrontal cortical areas. To avoid the effects of complete social isolation, female LE rats were reared, from shortly after weaning (24 days) to just after sexual maturity (60 days), with a same-sex partner, either an adult or a same-age peer,

Fig. 3. Three rearing conditions in the original experiment designed to test whether social play influenced the development of the prefrontal cortex (Bell et al., 2010). The conditions, based on the assumption that the magnitude of play would be increased along a gradient, involved having three same age peers (A), which would give the subject more play experience than when reared with a single peer (B) and this, in turn, would provide more play experience than when reared with an adult (C). 5

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circuits (e.g., mPFC) and, given juveniles’ high motivation to play with same age peers (Varlinskaya and Spear, 2008; Varlinskaya et al., 1999), play indirectly ensures the experience of multiple partners, and so in­ fluences other neural circuits (e.g., OFC). Play in the juvenile period can thus influence the development of both these prefrontal areas (Pellis and Pellis, 2009). There is a problem with this nicely packaged story. The original study of peer versus adult rearing (Bell et al., 2010) was repeated, but the rats were sacrificed at 100 days, so that they could be tested phar­ macologically and behaviorally after 60 days of age. The neurons of the mPFC showed the same effect as previously demonstrated, but there was no difference between the OFC neurons in rats reared with a single partner or those reared with multiple partners (Himmler, Pellis and Kolb, 2013). We hypothesized that the play effects on the mPFC were permanent, whereas the multiple partner effects on the OFC were transient, eroding over time. The experiment was again repeated; however, this time, juveniles were reared either with one peer or three peers, and one sample of animals was sacrificed at 60 days and one at 100 days. For the mPFC neurons, whether reared with one or multiple peers and whether sacrificed at 60 or 100 days, the neurons showed the same degree of pruning as previously found for peer-reared rats. How­ ever, an enlarged dendritic arbor of the OFC neurons was only present in rats reared with multiple peers, and only at 60 days. By 100 days, the dendritic arbor of the OFC neurons in multiple peer-reared rats had shrunk to that of rats reared with a single peer (Himmler et al., 2018). That is, the multiple partner effects on the OFC are transient. Consid­ ering that the natural niche for rats is to live in colonies provides insight as to why this may be so. The need to coordinate movements with partners is likely to remain invariant throughout life, so it is reasonable that the play-induced changes in the mPFC neurons remain as they age. However, over the course of a lifetime, the composition of a colony changes, with new rats being born, some rats migrating to new colonies and others dying (Barnett, 1975; Calhoun, 1962; Schweinfurth, 2020). Consequently, if the enlargement of the OFC neurons reflects adaptation to the cohort of multiple partners at a particular time in their lives, then there should be re-adaptation when new partners are confronted. Starting at 90 days of age, pairs of adult female rats were housed together, then, at 48 h in­ tervals over a 40-day period, rats were removed from their home cage and placed in a novel cage for an hour, either with the same cage mate (control condition) or with a novel partner (experimental condition). The behavior of the rats in the first 10 min of each cage change was recorded and then, at the end of the experiment, the rats were sacrificed

so that the brain could be prepared for comparison of the neurons from the mPFC and the OFC. Strangers interacted more, including playful encounters, than did cage mates, and the histology showed that the neurons of the OFC of subjects that had exposure to strangers had significantly increased dendritic arbor compared to subjects reintro­ duced to their cage mates. No group showed any changes to mPFC neurons (Hamilton et al., 2020). These findings support the view that the changes induced in the mPFC by play experiences in the juvenile period are permanent and help refine skills related to coordinating movements with partners — skills that last a lifetime. In contrast, the changes induced by exposure to multiple partners during the juvenile period is not contingent on play and is transitory, with OFC neurons increasing and decreasing in complexity as they encounter new partners over the course of the rat’s lifetime (Fig. 4). The evidence strongly supports the hypothesis that play fighting in the juvenile period has a direct effect on the physiology and anatomy of neurons of the mPFC (Baarendse et al., 2013; Bell et al., 2013; Bijlsma et al., 2022; Burleson et al., 2016; Himmler, Pellis and Kolb, 2013) and that these changes are associated with improved social skills and exec­ utive functions (Baarendse et al., 2013; Bijlsma et al., 2022; Burleson et al., 2016; Schneider, Bindila et al., 2016; Stark and Pellis, 2020, 2021). There is one last issue that needs to be explored; sex differences in the role of play fighting on brain development. Overall, for most species, play fighting is more frequent in males than females (Ellis et al., 2008; Marley et al., 2022), and, as shown above, this is the case for rats, at least for those reared in more naturalistic social groupings (Thor and Hollo­ way, 1984). In most studies, only females or only males have been tested (e.g., Baarendse et al., 2013; Bell et al., 2010; Bijlsma et al., 2022; ¨tz Burleson et al., 2016; Schneider, Bindila et al., 2016; Schneider, Pa et al., 2016), but the discordant strain peer rearing paradigm (Fig. 1C; Schneider, Bindila et al., 2016) offers the opportunity to compare males and females being reared with comparable levels of impoverished play during the juvenile period. The atypical play experienced by male and female LE rats when reared with a same sex F344 peer is statistically the same (Stark et al., 2021), and although the socio-cognitive test used was more appropriate for males, both sexes exhibited impoverished social skills (Stark and Pellis, 2020, 2021). Importantly, the comparable rearing paradigm allows for a direct comparison between the sexes of the effects of play on the dendritic pruning of mPFC neurons. The brains of LE rats reared with either an LE same sex peer or an F344 same sex peer were compared in adulthood as previously done for LE rats reared in the adult paradigm (Bell et al., 2010; Himmler, Pellis

Fig. 4. This schematic diagram illustrates the areas from the prefrontal cortex that were sampled (see central coronal brain section), with the dendritic arbor of the neurons from the medial prefrontal cortex (mPFC) on the right being permanently pruned by juvenile peer play experiences, whereas the dendritic arbor of the neurons from the orbital frontal cortex (OFC) increases with exposure to novel or multiple partners at all ages. (Created with BioRender.com). 6

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experienced that has these effects?

and Kolb, 2013), but this time, both sexes were used (Stark et al., 2023). Moreover, anticipating that the sex differences would likely be smaller than the condition effects, a different approach was used to analyze the dendritic arbor of the mPFC neurons. Instead of using hand drawn pic­ tures derived from inspection with a camera lucida, which compresses the three-dimensional structure into two dimensions (Bell et al., 2010; Himmler, Pellis and Kolb, 2013; Himmler et al., 2018), the Golgi-stained slides were converted into virtual slides using an Olympus VS120 digital slide scanner with a 40x oil objective (UPlanFL N, 40x/1.30 oil, ∞/0.17/FN26.5) and Olympus VS-ASE FL software. Virtual slides con­ sisted of a z-stack of 147 images spaced 0.68 µm apart throughout the section, yielding 99.96 µm of working distance. Once digital images were created, they were uploaded into Neurolucida 360® (Micro­ BrightField, Williston, VT, USA) for reconstruction of the neurons. The traced neurons were analyzed using NeuroLucida Explorer®, which then allowed a 3-dimensional reconstruction of the dendritic arbor, allowing the size and shape of each cell to be reconstructed. Having a 3-dimen­ sional rendering of the neurons allows the ends of each dendrite belonging to a single cell to be connected to form a polygonal geometric structure – referred to as the convex hull (Fig. 5). This geometric cellular structure can then be quantitatively compared using two convex hull measures – surface area and volume (Brinkman et al., 2022; Rojo et al., 2016) – the bigger the dendritic arbor, the larger the surface area and volume, and so the less pruning. For both measures, there was a significant condition effect with the mPFC neurons of the LE rats reared with F344 peers being larger, but for neither measure was there a significant sex difference (Fig. 6). These findings indicate that both male and female brains are susceptible to the pruning effects of juvenile play experience. Of course, there may be differences in synapse number (Kolb et al., 2003) or in the physiology (Baarendse et al., 2013; Bijlsma et al., 2022) of the neurons. Still, the evidence does suggest that play is an important behavioral experience for refining executive functions and social skills in both sexes (e.g., Baarendse et al., 2013; Bijlsma et al., 2022; Burleson et al., 2016; Schneider, Bindila et al., 2016; Stark and Pellis, 2020, 2021), although given sex differences how rats play (Pellis, 2002) and the differences in the neural mechanisms that regulate how the sexes play (VanRyzin et al., 2020), it may not be that all skills are equally influenced in males and females. This remains to be determined. But from the perspective of the role of play, is it how much they play or the quality of the play

6. Reciprocity: a key to play fighting As noted above, juvenile females reared with an adult female fail to exhibit the dendritic pruning of mPFC neurons that occurs when reared with a peer (Bell et al., 2010; Himmler, Pellis and Kolb, 2013). That this difference reflects the greater amount of play experienced by juveniles reared with other juveniles was assumed to be the case because adults or even older adolescents initiate little play with young juveniles (Pellis and Pellis, 1997; Pellis et al., 2018; Thiels et al., 1990). As LE rats were used in the neuronal studies, female juvenile LE rats were housed with either a LE peer or an LE adult, and then tested in a paradigm to evaluate dyadic play (Pellis et al., 2022). Fig. 7 A shows that, in the mixed age pair, the juveniles initiate more playful attacks than the adult, but closer inspection shows that the total amount of play does not differ between mixed-age and same age pairs. That is, the total amount of play expe­ rienced does not differ – indeed, the juvenile interacting with an adult, experiences initiating more playful attacks, as it compensates for the reduced playfulness of the adult. However, there is more to play fighting than attacking, there is an interaction in which one animal attacks and the other defends (Aldis, 1975; Pellis and Pellis, 1987), and critically, for play fighting to remain playful, some degree of reciprocity is needed (Palagi, Cordoni et al., 2016; Pellis and Pellis, 2017), which can be seen when, during an interaction, the partners switch roles, with the original attacker becoming the defender (see Fig. 2). Most often, role reversals occur in rats when the defender launches a successful counterattack to its part­ ner’s nape (Pellis et al., 2022). Comparing play fights between same age and mixed age pairs reveals that there are significantly fewer role re­ versals in the mixed-age pairs (Fig. 7B). So, although juveniles may experience the same amount of play whether interacting with peers or adults, the quality of that experience differs. How the role reversals occurred further highlights the qualitative differences. When interacting with an adult, a role reversal occurs when the ju­ venile persists in attempting to gain access to the nape of the prone adult and after the adult repeatedly kicks the juvenile with its hind foot or pushes it with its hip. The adult then rotates its forequarters toward the juvenile and pushes it over. Once the adult has pushed the juvenile over, it holds it down and then, when the juvenile stops struggling, the adult

Fig. 5. Two neurons reconstructed from NeuroLucida analyses are shown. The cell body and dendritic projections are shown in green and the enclosing polygon is shown with straight red lines. A neuron from an LE rat reared with an LE partner (A) is visibly smaller than a comparable neuron from an LE rat reared with a F344 partner (B). (Adapted from Stark, 2021). 7

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Fig. 6. The convex hull surface area (A) and volume (B) of the neurons for both sexes subjected to the two rearing conditions are shown. The box plots show the mean, standard deviation and confidence intervals for each group, with outliers indicated by dots. A 2-way ANOVA revealed a significant condition effect for both measures but not a significant sex difference (asterisks denote a significant difference at the p < .05). (Adapted from Stark, 2021).

lets it go and moves away. Unlike the case between juveniles (Fig. 2), the role reversals by adults did not arise from counterattacks directed at the juvenile’s nape. Further highlighting the asymmetry in the pattern of role reversals involving juveniles and adults is that when an adult attacked a juvenile, no cases led to a role reversal (Pellis et al., 2017). That is, when interacting with an adult, the juvenile has few opportu­ nities to modulate its cooperative and competitive actions to facilitate role reversals. The exception that proves the rule: Rats were domesticated from the late 1800s (Castle, 1947) and Syrian golden hamsters from the 1930s (Murphy, 1985), so one concern about the relationships found between play fighting by juveniles and the development of the mPFC is that it may be an artifact of domestication. Indeed, the seemingly less ‘aggressive’ play of laboratory rats could be due to domestication (Hurst et al., 1996). As predicted from the general pattern seen when comparing domesticated animals with their wild counterparts (Budiansky, 1999; Coppinger and Coppinger, 2001; Dugatkin and Trut, 2017), wild rats do initiate fewer playful encounters, but they mostly attack and defend the nape and use the same repertoire of defensive tactics as do domesticated strains (Himmler, Stryjek et al., 2013; S.M. Himmler, Modlinska et al., 2014). There is no evidence that the play fighting of juvenile wild rats is more aggressive than that of juvenile laboratory rats. However, when the adult rearing paradigm (Bell et al., 2010; Himmler, Pellis and Kolb, 2013) was applied to wild rats, no play-induced pruning of the mPFC neurons was present. Indeed, the degree of dendritic pruning in both the peer-reared and the adult reared wild rats was greater than that present in the LE rats reared with a peer (Pellis et al., 2018). One possibility is that wild rats cannot afford to have their executive functions be dependent on a prolonged period of playful training, as free-living rats have to be able to make life and death de­ cisions once they are no longer cared for by their mothers (Calhoun, 1962). However, this explanation is unlikely. Play fighting is common in many wild species of mammals (Aldis, 1975; Burghardt, 2005; Fagen, 1981), and for some species, including Belding ground squirrels (Urocitellus beldingi), another rodent, it has been found that individuals which play more as juveniles have greater

social skills when mature (Blumstein et al., 2013; Marks et al., 2017; Pellegrini, 1995). Moreover, for many of these species, play fighting involves simulation of serious fighting (Pellis, 1988), so it is not the specific behavior patterns rehearsed that is critical, but the kinds of experiences that can be derived from all kinds of play fighting (Pellis and Pellis, 2017). Another possibility is that it is not domestication-induced changes in brain plasticity that is crucial, but in the possible effects of domestication on adult responses toward juveniles. With water and food freely available, humidity and temperature invariant and the absence of predation, it is possible that mothers in the laboratory setting do not care for their young as well as they would under more naturalistic conditions (Bateson and Martin, 2000). Such laxity in infant care may be exacerbated by domestication. Associated with such changes in maternal behavior, an overall reduced interest in interacting playfully with juveniles by adolescents, mothers and other adults (Pellis and Pellis, 1997; Pellis et al., 2017, 2018) may similarly follow as a byproduct of domestication. To test this, juvenile female rats from both a wild strain and the LE strain were housed with a same strain adult female. The dyadic play paradigm (Pellis et al., 2022) was used to test the play of each pair. Comparing trials by juvenile-adult pairs showed some marked differences between the wild and LE rats (Pellis et al., 2019). As expected, the wild pairs engaged in significantly less play than did the domesticated rats (LE: 87.93 + 21.92; wild: 40.50 + 18.95; t = 4.00, df = 13, p < .01), and also, as expected, if domestication dampened adult interest in playing with juveniles, wild adults, as predicted, initi­ ated significantly more play fights than their domesticated counterparts (Fig. 8 A). Moreover, play fights with wild adults were significantly more likely to involve close-quarter wrestling (Fig. 8B) and lead to role reversals (Fig. 8 C). Critically, while wild juveniles were responsible for causing nearly a third of the role reversals, LE juveniles were responsible for less than 6% (Fig. 8D). Overall, the wild adults provided more op­ portunity for juveniles to engage in play fights that resembled those of juvenile pairs, including not only the experience of role reversals, but also the opportunity to be in control of producing role reversals (Pellis et al., 2019 cf. Himmler, Stryjek et al., 2013; Pellis et al., 2017). Thus, 8

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Fig. 7. Two features of play fighting are shown comparing female pairs of juveniles and pairs with a juvenile female and an adult female. Playful nape attacks are dispropor­ tionately distributed between pair mates with an adult, as the juvenile initiates more than the adult, whereas in ju­ venile pairs, the distribution is more even (A). Role re­ versals are more than twice as likely to occur in juvenile pairs as compared to pairs with an adult (B). All measures are represented as the mean ± the SEM, (**) denotes sig­ nificant p < .01. (Adapted from tabular data in Pellis et al., 2017 with permission).

the lack of difference in the pruning of the dendritic arbor between ju­ veniles reared with peers or adults in wild rats (Pellis et al., 2018) may be because the wild adults, unlike domestic adults, provide sufficient experiences to train executive functions. To have an equitable number of role reversals, rats need to monitor ongoing behavior, attend to the forcefulness of their partner’s actions, inhibit inappropriate actions, use short-term memory to recall the relative balance of wins and losses, decide on when to hold back and allow their partner to win and switch tactics if the currently used ones fail to produce the desired effect (Pellis and Pellis, 2017; Pellis et al., 2014). That is, the rats need to use their executive function skills (Dalley et al., 2004). Social play that involves such negotiation between partners in young children has been demonstrated to improve executive func­ tions (e.g., Coelho et al., 2020; Diamond et al., 2007). Therefore, reci­ procity, as reflected by turn taking, may be one of the critical experiences derived from play fighting that contributes to the pruning of the dendritic arbor, changes in the physiology of the mPFC neurons and the enhancement of the executive functions associated with the mPFC. But experience of role reversals may not be enough. Reciprocity revisited: Juvenile LE rats playing with an adult expe­ rience only a small fraction of the role reversals that are experienced by juveniles playing with peers. Moreover, when interacting with peers, there is about a 50:50 split between partners in the number of role re­ versals they initiate (Himmler, Himmler, Pellis and Pellis, 2016), but when playing with an adult, the juveniles only initiate a fraction of the role reversals (Pellis et al., 2017, 2019). However, when LE rats are reared with F344 rats and they play together, the LE rats initiate 2–3

Fig. 8. Four features of play fighting are shown for female pairs containing an adult and a juvenile, comparing domesticated (LE) and wild rats. Adult wild rats initiate significantly more nape attacks (A), significantly more of the play fights of the wild pairs involve wrestling (B), with significantly more of these leading to role reversals (C), and significantly more of the role reversals are initiated by the juvenile in the wild pairs (D). All measures are represented as the mean ± the SEM, (**) denotes significant p < .01. (Adapted from tabular data in Pellis et al., 2019 with permission).

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play to last for prolonged durations, the interactions need to be ‘fair’ (Palagi, Cordoni et al., 2016), whereby both animals have a chance to gain the advantage at least some of the time. Originally, this was thought to require that each partner win about 50% of the encounters (Altmann, 1962; Dugatkin and Bekoff, 2003), but it has since become clear that depending on the species and the relationship between the pair mates, this ratio can deviate markedly from 50:50, but it cannot be 0:100. Rather, whatever a pair perceives as fair is what needs to be honored (Pellis and Pellis, 2017). Signaling can be important for facilitating fairness in play fights (Palagi, Burghardt et al., 2016). In rats, this is mostly achieved by vocalizations, especially those in the ultrasonic range (Burgdorf et al., 2008; Burke et al., 2018, 2020). In both social and non-social contexts, rats emit 22-kHz calls in

times more role reversals than do LE rats playing with other LE rats (Stark et al., 2021). That is, even though there is a lack of dendritic pruning in the mPFC neurons of LE rats reared with F-344 peers (Stark et al., 2023), and they have reduced social skills (Stark and Pellis, 2020, 2021), the LE rats reared with F344 peers experience more, not less, role reversals, which the LE rats mostly initiate (Stark et al., 2021). What this suggests is that it is not the experience of role reversals or even being able to initiate them that provides the key experience during play fighting, but rather, that the partners have to balance the experiences relatively evenly. Indeed, on most measures of play fighting, including role reversals, LE-F344 pairs exhibit 2-4 times greater asymmetry than do LE-LE pairs (Fig. 9). For animals to sustain repeated, playful encounters or for bouts of

Fig. 9. The proportion of asymmetry in several measures of play fighting is shown for juvenile experimental LE rats paired with a F344 rat (right) and the control LE rats paired with another LE rat (left). Data for both male pairs (dark grey) and female pairs (light grey) are presented. In no case was there a sex difference, but there were significant group differences. Nape attacks were significantly more asym­ metrical in LE-F344 pairs (A), and although not significant, there was a trend to greater asymmetry in the likelihood of defending against attacks in the mixed strain pairs (B). Both facing defense (C) and the use of the complete facing tactic (D) were significantly more asymmetrical in the mixed strain pairs. The chance that a facing defense ended in a pin configuration was significantly asymmetrical in the mixed strain pairs (E) as was the likelihood of facing de­ fense leading to a successful counterattack or role reversal (F). All measures are represented as the mean ± the SEM, (*) denotes significant p < .05. (Reprinted from Stark et al., 2021 with permission).

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aversive situations and a variety of 50-kHz calls in positively affective situations (Knutson et al., 2002; Wright et al., 2010). During play fighting, rats emit 50-kHz calls, especially ones that are frequency modulated, when most intensely engaged in play, but may also emit 22-kHz calls when satiated and avoiding further playful contact (Burg­ dorf et al., 2008). In this way, the rate of emission of frequency modu­ lated 50-kHz can be an index of the playful mood of the animals. In addition, though, different types of 50-kHz calls are emitted during different playful actions (Burke et al., 2018) and these can influence the behavior of the partner (Burke et al., 2020). Indeed, in the absence of producing or hearing such vocalizations, playful attacks, wrestling and role reversals are reduced (Kisko, Himmler et al., 2015; Siviy and Pan­ kespp, 1987). However, even if only one partner in a pair can vocalize, play in pairs of juveniles is the same as that between pairs in which both can vocalize (Kisko, Euston and Pellis, 2015) — although rougher play between unfamiliar adult males is more likely to escalate to serious fighting if one partner cannot vocalize (C.J. Burke, Kisko et al., 2017; Kisko, Euston and Pellis, 2015). But given that even without vocalizing, rats can still engage in play fighting, and do so repeatedly with the same partner (Kisko, Himmler et al., 2015), it would suggest that it is by following the rules that play fighting is made playful (Pellis and Pellis, 2017). Signals may be particularly important when there is a chance of misinterpretation of a partner’s actions (Aldis, 1975; Bekoff, 1995; C. J. Burke, Kisko et al., 2017), but signals would not be heeded after a while if after emitting them the partner consistently broke the rules (Pellis and Pellis, 1996). As seen in Fig. 9, excessive asymmetry in most aspects of play sug­ gests that the pair mates are unable to follow the rules of fairness, and it is the lack of training on how to be fair that likely causes the develop­ mental anomalies seen in LE adults that have been reared with F344 peers. This conclusion takes us a long way away from Groos (1898) hypothesis that play trains animals to use specific behavior patterns, and leads us to play, at least some forms of social play, having a role in ˇ psychological training (Spinka et al., 2001; Pellis, Pellis and Bell, 2010). As noted above, improved executive functions can result in better per­ formance in many tasks. But to achieve this does not require perfect symmetry, and again, as seen in Fig. 9, the interactions between same-strain, same-sex peers deviate to some degree from parity, but whatever the degree of asymmetry in a pair, the animals need to use their executive functions to ensure that they do not deviate too far from that level. In species in which juveniles are limited to only playing with their mothers, such as is the case for most giant panda cubs (Ailuropoda melanoleuca), the mother has to initiate many of the play fights and engage in restrained actions to enable the cub occasionally to gain the upper hand (Kleiman, 1983; Snyder et al., 2003). Our hypothesis is that, in species in which juveniles are dependent on adults for the majority of their play fighting experiences, the adults bend the 50:50 rule in the juvenile’s favor, but not completely, as the juvenile has to train in how to achieve fairness, as seems to be the case with wild adult rats playing with juveniles (Pellis et al., 2019). This hypothesis remains to be tested, but for the argument central to the present review, there is a missing component to the rat’s tale that still needs to be added.

However, play in mice is mostly locomotor-rotational, and the little social play present involves approach and withdrawal, not wrestling (Pellis and Pasztor, 1999), unlike the social play of ground squirrels which involves complex wrestling (Nunes et al., 1999; Pasztor et al., 2001), much like that present in rats and hamsters (Pellis and Iwaniuk, 2004; Pellis and Pellis, 2009). These contrasting findings support the possibility that it is the experiences derived from social play that are particularly important for training executive functions (Pellis and Pellis, 2017). Moreover, the findings from Marks et al. (2017) show that, within a group of juveniles living together, not all may gain the same level of play experience, as is true for some other group living mammals (Ham et al., 2022; Weller et al., 2020), and this has implications for the development of executive functions and associated social skills. Rats not only give birth to litters (usually 6–12), but they also live in colonies with multiple adult females that tend to synchronize estrous cycles and so births (McClintock, 1978; Barnett, 1975; Calhoun, 1962; Schweinfurth, 2020). In this milieu, once fully weaned from the mother, young rats have siblings and other same age peers from other litters with which to play. This variety of partners not only provides the experiences needed for training and retraining the OFC, but also the opportunity to choose partners most suitable to gain the experiences needed to train the mPFC. Play partner selection has been an understudied feature of social development in rats, which is unfortunate as there are likely real choices faced in a group setting, choices that could influence social development. Not all juvenile rats are equally playful, with some consistently playing more than others (Lampe et al., 2019; Lesscher et al., 2021; Melotti et al., 2014; Pellis and McKenna, 1992). A period of social deprivation, from as little as 2–3 h to as much as several days, can in­ crease an individual rat’s motivation to engage in social play (Hole, 1991; Niesink and van Ree, 1982; Panksepp and Beatty, 1980; Pellis and Pellis, 1990). Interestingly, such isolation does not simply increase so­ cial interest, as social investigation is not increased, but selectively in­ creases social play (Panksepp, 1981). So, when following a period of isolation rats are tested in dyads, the rats will eagerly play together, potentially dampening individual differences (Pellis et al., 2022). However, high playing rats in such dyadic tests also initiate more play in their home cages (Lampe et al., 2017; Melotti et al., 2014), suggesting that some individuals are intrinsically motivated to play more than others. Moreover, the play fighting of high playing pairs involves more wrestling and role reversals than that of low playing pairs (Pellis et al., 2022), suggesting that high and low players not only differ in their preferred level of play, but also in their style of play. If so, this would suggest that, in a home environment with multiple peers present, an individual could modify its level of play, and the types of experiences gained therefrom, by selecting the partners it finds most suitable. Thus, even though consistent individual differences can be detected using the dyadic test paradigm (Lesscher et al., 2021), testing rats in a group setting offers them an opportunity to show us what kind of play experiences they seek in a partner. After a period of social isolation, when given the choice of multiple play partners in a test chamber, some rats play together more than they do with other members of the group. A simple basis for selecting play partners could be to play with the rat that is closest. This is not the case. Rats will actively move from one corner of the cage where they are near to some group members to another corner to initiate play with a particular rat (Pellis et al., 2022). That is, specific play partners are selected in preference to others. What constitutes a ‘suitable’ partner remains to be determined, but some interesting pre­ liminary data provide a clue. Groups of six male juveniles housed together were tested after a short period of isolation (2.5 h) over several, consecutive days. We found that there were play partner preferences on every day of testing, but as illustrated in Fig. 10, the preferred partners on one day were not necessarily the same partners on the next (e.g., Rat 1 played more with Rat 4 on the first day, but more with Rat 6 on the next day). Moreover, some rats were rarely chosen as play partners on any day. It is possible

7. A playful life within the group Most mammals either give birth to a litter or, if they give birth to singletons, they are often reared within a social group with the young of other females present (Eisenberg, 1981); consequently, juveniles typi­ cally have many options for playing with peers (Ham et al., 2022; Lilley et al., 2020; Turner et al., 2020). Belding’s ground squirrels are reared within the context of a litter, and it was found that those members that play more as juveniles have better social skills later in life. Importantly, it was those engaging in more social play, not more locomotor-rotational play, that gained this benefit (Marks et al., 2017). In contrast, in a study of domestic mice, it was found that those playing more performed more poorly in a task in which they were stressed (Richter et al., 2016). 11

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Fig. 10. The pattern of play between pairs in a group of six juveniles, familiar with each other, when tested in the group setting. The directed social networks show that some members engage play more than others (size of the circles) and that some partners play together more often than others (thickness of lines connecting the circles). The pattern of play among the group members is shown for two consecutive days. The preferred partners can change from one day (A) to the next (B), but it should also be noted that some members remain infrequently chosen as a play part­ ner as highlighted by ‘Rat 1.’ On the first day (A), Rat 1 plays the most with Rat 4, playing infrequently with Rat 6. On day two (B), Rat 1 now plays the most with Rat 2, but again, plays infrequently with Rat 6.

et al., 2013; Bijlsma et al., 2022), suggesting that the sensitive period for training executive functions is indeed the juvenile period and that the training is achieved via engagement in play. How the cellular changes in the mPFC produced by play result in improved executive functions remains to be determined, but a recent paper provides an interesting clue (Bijlsma et al., 2022). The balance of the flood of neurotransmitters released during play (Vanderschuren et al., 2016) may be altered by play that is deficient (e.g., Achterberg et al., 2014, 2015; Schneider et al., 2014; Schneider, P¨ atz et al., 2016), and these changes may, in turn, alter the development of inhibitory neurons in the mPFC (Bijlsma et al., 2022), which then leads to reduced impulse control (Baarendse et al., 2013), and so overreaction to other­ wise benevolent contexts, such as being investigated by another rat (e.g., Byrd and Briner, 1999; Einon and Potegal, 1991; van den Berg et al., 1999). The details of the causal pathway by which play experience in the juvenile period influences the developmental trajectory of these systems remains a major focus for further research, but the outlines of what that causal pathway may look like is starting to become clearer (Bijlsma et al., 2022). Although much is left to be understood, what the rat’s tale tells us is that, unlike what was proposed by Groos (1898), the function of play is not the practice of specific behavior patterns, but to influence the development of executive functions, which, in turn, can influence skills important for the effective deployment of many behavior patterns in a variety of contexts. Thus, play fighting does not simply produce better fighters (Smith, 1982), but more socially skilled animals in general (Pellis and Pellis, 2009; Pellis, Pellis and Bell, 2010). Given that different lineages of animals follow different rules to achieve reciprocity, and so fairness, a major unresolved comparative question is whether some styles of play are better suited for training these general skills than are others (Pellis and Pellis, 2017). A more specific experimental issue that remains to be resolved is the influence of the lack of play on the animal’s ability to cope with stressful situations. Rats that have been reared in isolation exhibit an exaggerated hormonal stress response, taking longer to bring stress hormones back to baseline following a stress-inducing event (von Frijtag et al., 2002), and the ability to attenuate the hormonal stress response in marmosets (Callithrix geoffroyi) is correlated with whether they had prior play experience (Mustoe et al., 2014). These findings are consistent with the finding that being reared in complete social isolation results in rats that are more anxious and fearful in both social and non-social situations (e. g., da Silva et al., 1996; van den Berg et al., 1999). Similarly, rats reared in proximity with other rats, but are not able to play with them (Fig. 1A), end up exhibiting poor impulse control and are hyper-defensive (Bell, 2014; Bijlsma et al., 2022), as are rats reared in total social isolation

that some individuals simply play in a manner that makes them unat­ tractive play partners and so are ostracized. Indeed, this has been shown to be the case in both human children and in juvenile rhesus macaques (Macaca mulatta) (Suomi, 2005; Wilmer, 1991). Similarly, in some species, such as rats and squirrel monkeys (Saimiri sciureus), with the onset of adolescence, the play fighting of males becomes rougher and females increasingly avoid playing with them (Biben, 1986; Meaney and Stewart, 1981). Some potential partners may be ‘good enough’ and so interchange­ able as suitable play partners, but this still leaves some members of the group lagging in their overall play experiences (Fig. 10). If corroborated, these findings in groups of rats indicate that not all individuals within the group may have the same experience with play and this could have developmental consequences (Ham, work in progress). Given the find­ ings on ground squirrels (Marks et al., 2017), it would seem reasonable that rats ostracized from gaining access to the most suitable partners over the juvenile period should show less pruning of the mPFC and be less socially skilled than group members with greater ability to select partners that can provide the optimal play-derived experiences. 8. Conclusion Social interactions during the juvenile period are important for the maturation of social behavior (A.R. Burke, McCormick et al., 2017; Potrebi´c et al., 2022) and for many mammals, such as rats, playful in­ teractions are an important part of that early experience (Pellis et al., 2014; Vanderschuren and Trezza, 2014). Based on multiple ways of preventing social play from occurring during the juvenile period, the evidence converges on play having a role in pruning both the number of neurons in the mPFC (Markham et al., 2007) and the dendritic arbor of the remaining cells (Bell et al., 2010; Burleson et al., 2016; Himmler et al., 2013; Stark et al., 2023), along with inducing physiological and biochemical changes in those cells (Baarendse et al., 2013; Hermes et al., 2011; Van Kerkhof et al., 2013). It should also be noted that while different studies with rats have housed the rats in a play deficient context for varying lengths of ages, spanning from early post-weaning (21–25 days post birth) up to young adulthood (60–100 days post birth) (e.g., Arakawa, 2007; Baarendse et al., 2013; Bell et al., 2010; Himmler, Pellis and Kolb, 2013; Schneider, Bindila et al., 2016; Stark and Pellis, 2020), in all cases, that period of deficiency has included the juvenile period in which play peaks in occurrence (30–40 days post birth). Moreover, studies that have limited that period of deficiency to the post weaning period and the ages of peak play of juveniles (e.g., 21–42 days post birth) have found changes in the development of the mPFC and reduced effectiveness of executive functions (e.g., Baarendse 12

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(Baarendse et al., 2013; Byrd and Briner, 1999; Einon and Potegal, 1991). Hamsters reared in the adult paradigm (Fig. 1B) exhibit an exaggerated response when tested for defeat stress (Burleson et al., 2016). All these findings are consistent with the hypothesis that emotional regulation is an important part of the training offered by play experience ˇ as juveniles (Pellis and Pellis, 2009; Spinka et al., 2001). However, for rats reared in the peer paradigm (Fig. 1C), even though they exhibit social deficiencies later in life (Schneider, Bindila et al., 2016; Stark and Pellis, 2020, 2021), they are not more anxious (Schneider, Bindila et al., 2016). This suggests that the stresses young animals exhibit in total isolation, partial isolation or being confined with an adult, are what influences the development of impoverished emotional regulation, not the absence of play. As reduced executive functions are correlated with altered development of the mPFC cells (Baarendse et al., 2013; Bijlsma et al., 2022; Burleson et al., 2016; Stark et al., 2023), it may not be a coincidence that adult rats with ablation of the mPFC exhibit impov­ erished inter-animal coordination skills but do not exhibit greater stress or anxiety (S.M. Himmler, Bell et al., 2014; B.T. Himmler, Bell et al., 2014). That is, impoverished play experience may diminish cognitive aspects of executive functions, but may not be directly responsible for diminished emotional regulation (Pellis and Pellis, 2016). Indeed, while the LE rats reared with F344 peers, like LE rats reared with adults, have enlarged apical dendrites, unlike the LE rats reared with adults, the basilar dendrites are as pruned as those of LE rats reared with juvenile peers (Stark et al., 2023). This difference suggests that some of the neuronal changes in some play deprivation studies are due to the stresses to which the animals are exposed and not to the lack of play. Nonetheless, the amygdala, a sub-cortical brain area instrumental for emotional regulation (Aggleton, 1993), is activated during play (Alu­ gubelly et al., 2019; Gordon et al., 2003) and may be critically involved in ensuring reciprocity (Pellis, Pellis and Reinhart, 2010), so an emotional component to the benefits gained from play cannot be ruled out. Moreover, given the connections between sub-cortical structures like the amygdala and frontal cortical circuits (Banks et al., 2007), it should not be surprising that changing the neuronal architecture of one part of the network (e.g., amygdala) results in correlated changes in another (e.g., mPFC) (Reinhart et al., 2021). While in the present review we focus on the changes that play experience has on prefrontal cortical systems, studying the influence of play on the development of subcor­ tical systems should not be ignored (Siviy, 2019). Further refinements in how play experience is manipulated during the juvenile period are needed to identify exactly which skills are influenced by play and how this is achieved. Similarly, new methods and technologies that can correlate neural activity of areas such as the mPFC, with ongoing behavior (e.g., Bagi et al., 2022; Reinhold et al., 2019) may provide further insights on the developmental effects of juvenile play experience on complex neural circuits that span both cortical and sub-cortical networks. We would be remiss in ending this review without briefly consid­ ering the implications of the rat’s tale on the role of social play in humans. Young boys that engage in more play fighting have been shown to be more socially skilled (Pellegrini, 1995). The findings on rats would suggest that this results from the collaborative negotiation needed to ensure that the play fighting remains playful. Indeed, boys that try to dominate all play fights are ostracized by their potential play partners (Wilmer, 1991). Programs that enhance the opportunity for social play, even if that play does not involve play fighting, but still requires the participants to negotiate the rules and then enforce them, improves executive functions (Diamond et al., 2007). It is unethical to deliberately deprive children of play, but a natural experiment tracked the executive functions of children having enforced deprivation of interactions with peers due to long-term hospitalization and found that compared to a non-hospitalized cohort, their executive function development was delayed (Nijhoff et al., 2018). Interestingly, those that played with peers at a distance via computer games were better off than those that did not

engage in such play. This is not as good as in-person play, but it is better than no play at all, and highlights that, as demonstrated for rats, it is the lack of play that contributes to this developmental deficit. However, not all studies show that children benefit from social play, especially, play fighting – some do (e.g., Freeman et al., 2022), some do not (Veiga et al., 2022). Just as has been the case for rats, how play and its consequences are measured can be an important factor in determining the divergent outcomes of studies (Pellis et al., 2022). Another factor is that context, especially social context, may be important in what value play may have for a particular population of people (Ciani et al., 2012). As a general conclusion, in some situations, social play has a positive influence on the development of executive functions and associated social skills (e.g., Diamond et al., 2007; Nijhoff et al., 2018; Lillard, 2017). Further research is needed to determine how much play is needed and the contexts in which its beneficial effects are realized. The rat model can be a useful tool to help answer these questions. Acknowledgments We thank our many collaborators and colleagues who helped develop the model articulated in this review. The majority of the research conducted in our laboratory was supported by grants from the Natural Science and Engineering Research Council of Canada (NSERC) to SMP (current grant: 2018-03706) and a NSERC PGS D award to JRH. References Achterberg, E.J.M., Trezza, V., Vanderschuren, L.J., 2014. Glucocorticoid receptor antagonism disrupts the reconsolidation of social reward-related memories in rats. Behav. Pharm. 25, 216–225. Achterberg, E.J.M., van Kerkhof, L.W.M., Damsteegt, R., Trezza, V., Vanderschuren, L.J. M.J., 2015. Methylphenidate and atomoxetine inhibit social play behavior through prefrontal and subcortical limbic mechanisms in rats. J. Neurosci. 35, 161–169. Aggleton, J.P., 1993. The contribution of the amygdala to normal and abnormal emotional states. Trends Neurosci. 16, 328–333. Ahloy Dallaire, J., Mason, G.J., 2017. Juvenile rough-and-tumble play predicts adult sexual behaviour in American mink. Anim. Behav. 123, 81–89. Aldis, O., 1975. Play Fighting. Academic Press, New York, NY. Altmann, S.A., 1962. Social behavior of anthropoid primates: analysis of recent concepts. In: Bliss, E.L. (Ed.), Roots of Behavior. Harper, New York, NY, pp. 277–285. Alugubelly, N., Mohammad, A.N., Edelmann, M.J., Nanduri, B., Sayed, M., Park, J.W., Carr, R.L., 2019. Proteomic and transcriptional profiling of rat amygdala following social play. Behav. Brain Res 376, 112210. Arakawa, H., 2002. The effects of age and isolation period on two phases of behavioral response to foot-shock in isolation-reared rats. Dev. Psychobiol. 41, 15–24. Arakawa, H., 2003. The effects of isolation rearing on open-field in male rats depends on developmental stages. Dev. Psychobiol. 43, 11–19. Argue, K.J., McCarthy, M.M., 2015. Utilization of same- vs. mixed-sex dyads impacts the observation of sex differences in juvenile social play behavior. Curr. Neurobiol. 6, 17–23. Baarendse, P.J.J., Counotte, D.S., O’Donnell, P., Vanderschuren, L.J.M.J., 2013. Early social experience is critical for the development of cognitive control and dopamine modulation of prefrontal cortex function. Neuropsychopharmacol 38, 1485–1494. Baenninger, L.P., 1967. Comparison of behavioural development in socially isolated and grouped rats. Anim. Behav. 15, 312–323. Bagi, B., Brecht, M., Sanguinetti-Scheck, J.I., 2022. Unsupervised discovery of behaviorally relevant brain states in rats playing hide-and-seek. Curr. Biol. 32, 1–14. Baldwin, J.D., 1986. Behavior in infancy: Exploration and Play. In: Mitchell, C., Erwin, J. (Eds.), Comparative Primate Biology. Vol. 2A. Behavior, Conservation, and Ecology. Alan R. Liss, New York, NY, pp. 295–326. Baldwin, J.D., Baldwin, J.I., 1974. Exploration and play in squirrel monkeys (Saimiri). Am. Zool. 14, 303–315. Banks, S.J., Eddy, K.T., Angstadt, M., Nathan, P.J., Phan, K.L., 2007. Amygdala-frontal connectivity during emotion regulation. Soc. Cogn. Affect. Neurosci. 2, 303–312. Barnett, S.A., 1975. The Rat: A Study in Behavior. The University of Chicago Press, Chicago, IL. Barrett, L., Dunbar, R.I.M., Dunbar, P., 1992. Environmental influences on play behaviour in immature gelada baboons. Anim. Behav. 44, 111–115. Bateson, P.P.G., Martin, P., 2000. Design for a Life. How Behavior and Personality Develop. Simon & Schuster, New York, NY. Bekoff, M., 1995. Play signals as punctuation: the structure of social play in canids. Behaviour 132, 419–429. Bell, H.C., 2014. Behavioral variability in the service of constancy. Int. J. Comp. Psychol. 27, 338–360. Bell, H.C., McCaffrey, D., Forgie, M.L., Kolb, B., Pellis, S.M., 2009. The role of the medial prefrontal cortex in the play fighting in rats. Behav. Neurosci. 123, 1158–1168.

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