Introduction

Methylenedioxypyrovalerone (MDPV) is a designer drug belonging to the class of synthetic cathinones, a group of psychoactive substances that emerged in the mid-2000s as alternatives to illicit psychostimulants such as amphetamine, MDMA, or cocaine [1, 2]. Currently, MDPV is a popular cathinone found in products marketed as ‘bath salts’, which are easily available online. Its use is predominant among youth populations particularly in social contexts, such as festivals and rave parties [3,4,5]. Due to their high abuse potential, the constantly growing market and the lack of any registered medicinal use, most of the common cathinones, including MDPV, have been classified as Schedule I controlled substances in the United States (Drug Enforcement Administration, 2011) and they are monitored by the European Monitoring Center for Drugs and Drugs Addiction [6]. Still, MDPV continues to be available and its use by young people is a matter of concern [6].

It has been recently reported that synthetic cathinones are β-keto analogs of amphetamine with pharmacological effects resembling those of cocaine and amphetamines [7]. In particular, the behavioral effects of MDPV have been mainly ascribed to dopaminergic and noradrenergic neurotransmission, as it acts in the brain as a potent uptake inhibitor at plasma membrane transporters for dopamine (DAT) and noradrenaline (NET) [8]. However, despite the fact that the use of synthetic cathinones among young people is a growing public health concern, their impact on brain and behavior is still a matter of debate. Users report MDPV-related positive psychoactive effects resembling those of cocaine, such as euphoria, talkativeness, sexual arousal, alertness, motor excitation, and increased concentration, sociability, productivity, motivation, and libido [1, 6]. However, several adverse effects have also been reported following acute intoxication, such as hallucinations, psychosis, delirium, depressed mood, anxiety, agitation, disorganized thoughts, and cognitive alteration, that also occur in users of amphetamine and cocaine [1, 9, 10].

Preclinical findings in rodents have shown that MDPV induces dose-dependent stereotyped behaviors, alterations in thermoregulation and that it has abuse potential [8, 11,12,13]. Psychostimulants are known to disrupt a variety of social behaviors [14,15,16,17,18] and it has recently been shown that MDPV enhances aggressive behavior in adult mice with greater potency than cocaine [19]. Surprisingly, however, despite the popular use of synthetic cathinones by young people in social contexts, it is not known whether MDPV produces psychostimulant-like disruptions of social behavior at young age.

One characteristic social behavior displayed by the young of most mammalian species, including rats and humans, is social play behavior. This form of social behavior is thought to be of great importance for the development of cognitive and social competence [20,21,22,23,24]. Abnormalities in social play are also observed in childhood psychiatric disorders such as autism, early-onset schizophrenia, and attention deficit/hyperactivity disorder [25,26,27,28,29]. Therefore, because of the scarce information about the effects of MDPV on social behavior and the importance of social play for behavioral development, we here investigated the effects of MDPV on social play in male rats, and the underlying neurobehavioral mechanisms.

Materials and methods

Animals

Male Wistar rats (Charles River Laboratories, Italy) arrived in our animal facility at 21 days of age and were housed in groups of five in Macrolon cages (43 × 26 × 20 cm) under controlled conditions (temperature 21 ± 1 °C, 60 ± 10% relative humidity and 12/12 h light cycle with lights on at 07:00 a.m). Food and water were available ad libitum. After 1 week of acclimation, animals were tested at postnatal days (PND) 28–30. One experiment (experiment 2) was performed in male Wistar rats at PND 80 to evaluate whether the effects of MDPV could be also extended to young adult animals. Overall, a total number of 392 juvenile rats (weighting around 70–100 g) and a total number of 69 young adult rats (weighting around 300–350 g) were used. Animals were experimentally naive and were used only once. Sample size (n) for each experiment is indicated in the figure legends. The experiments were approved by the Italian Ministry of Health (Rome, Italy) and performed in agreement with the ARRIVE (Animals in Research: Reporting In Vivo Experiments) [30, 31] guidelines, and the guidelines of the Italian Ministry of Health (D.L. 26/14) and the European Community Directive 2010/63/EU.

Drugs

3,4-MDPV (0.025, 0.05, 0.1, 0.25, and 0.5 mg/kg), flupenthixol-dihydrochloride (0.125–0.06 mg/kg), and RX821002-hydrochloride (0.2–0.1 mg/kg) were dissolved in saline and given intraperitoneally (i.p.). Solutions were freshly prepared on the day of the experiment and were administered in a volume of 2 ml/kg in juvenile rats and 1 ml/kg in young adult rats. MDPV was administered 30 min before testing while both RX821002 and flupenthixol were administered 15 min prior to MDPV administration. Saline was used as control treatment for all the drugs used.

Experimental design

Eight experiments were performed. To assess the effects of MDPV on social play behavior, in experiment 1 juvenile rats (PND 28–30) were treated with a broad range of doses of MDPV (0.025, 0.05, 0.1, 0.25, or 0.5 mg/kg) or saline (control group). As MDPV is widely used by young adults, in experiment 2 we tested the effects of MDPV (0.025, 0.05, 0.1, 0.25, or 0.5 mg/kg) on social behavior in young adult rats (PND 80).

Social play behavior is influenced by the level of social activity of the partner [32,33,34,35]. Therefore, in experiment 3 we investigated whether the effects of MDPV on social play in juvenile rats depended on the behavior of the test partner and/or if the social repertoire of saline-treated rats was influenced by MDPV-treated partners. To this aim, we treated none, one, or both members of a test pair with MDPV (0.5 mg/kg), and behavior of both test partners was scored separately. Next, we investigated whether tolerance (experiment 4) or sensitization (experiment 5) would occur to the effect of MDPV on social play behavior after repeated treatment. To this aim, animals were pretreated with either MDPV (0.5 mg/kg) or saline for 5 consecutive days (PND 25–29). On day 30 (i.e., 1 day after the last pretreatment injection), animals were isolated for 3.5 h. Next, half of both pretreatment groups (MDPV or saline) was treated 30 min before testing with either saline or MDPV, given at the effective dose of 0.5 mg/kg to assess the occurrence of tolerance (experiment 4), or at the sub-effective dose of 0.1 mg/kg to assess the occurrence of sensitization (experiment 5).

MDPV acts as a potent uptake inhibitor for dopamine and noradrenaline, but not serotonin [8]. Both dopaminergic and noradrenergic neurotransmission have been implicated in the modulation of social play [36, 37]. Furthermore, we have previously shown that the play-suppressant effects of methylphenidate and amphetamine are mediated through α2 noradrenergic but not dopaminergic receptors [14, 38,39,40]. Therefore, to clarify whether MDPV exerts its effects on social play through dopaminergic (experiment 6) or noradrenergic (experiment 7) neurotransmission, animals were treated with either the dopamine receptor antagonist flupenthixol (0.125 mg/kg) or the α2 adrenoceptor antagonist RX821002 (0.2 mg/kg) before MDPV (0.5 mg/kg). Last, in experiment 8 we investigated whether dopaminergic and noradrenergic neurotransmission are simultaneously involved in the effect of MDPV on social play, by treating the animals with either sub-effective doses of both RX821002 (0.1 mg/kg) and flupenthixol (0.06 mg/kg), or with saline (control group) before treatment with MDPV (0.5 mg/kg).

Social play behavior in juvenile rats

All the experiments were performed in a sound-attenuated chamber under dim light conditions. The testing arena consisted of a Plexiglas cage (40 × 40 × 60 cm) with ~2 cm of wood shavings covering the floor. Rats were tested starting at PND 28–30 and social play behavior was assessed as previously described [41]. Rats were individually habituated to the test cage for 10 min on 2 days prior to testing. On the test day, rats were socially isolated for 3.5 h before testing. This isolation period has been shown to induce a half-maximal increase in the amount of social play behavior [42]. At the appropriate time before testing, pairs of animals were treated with drugs or vehicle. In all experiments except for experiment 3, both animals of a pair received the same drug treatment. The test consisted of placing two animals into the test cage for 15 min. The animals in a test pair did not differ more than 10 g in body weight and had no previous common social experience (i.e., they were not cagemates). A pair of rats was considered as one experimental unit and the behavioral parameters were therefore scored per pair of animals, except for experiment 3, in which the behavioral parameters were scored per each individual animal of a test pair. The Observer 3.0 software (Noldus Information Technology BV, Wageningen, The Netherlands) was used to score behaviors related to play. In rats, a bout of social play behavior starts with one rat soliciting (‘pouncing’) another animal, by attempting to nose or rub the nape of its neck. The animal that is pounced upon can respond in different ways. If the animal that is pounced upon fully rotates to its dorsal surface, ‘pinning’ is the result, i.e., one animal lying with its dorsal surface on the floor with the other animal standing over it. From this position, the supine animal can initiate another play bout, by trying to gain access to the other animal’s neck. Thus, during social play, pouncing is considered an index of play solicitation, while pinning can be regarded as the terminal component of a single play bout as well as a releaser of a prolonged play bout [43]. Pinning and pouncing frequencies can be easily quantified and they are considered to be the most characteristic parameters of social play behavior in rats [36]. During the social encounter, animals may also display social behaviors not directly associated with play, such as sniffing or grooming the partner’s body.

The following parameters were scored:

Social behaviors directly related to play:

  • Frequency of pinning.

  • Frequency of pouncing.

Social behaviors unrelated to play:

  • Time spent in social exploration: the total amount of time (s) spent in nonplayful forms of social interaction (i.e., one animal sniffing or grooming any part of the partner’s body).

Locomotor activity during the social encounter:

  • Crossing: a grid, dividing the arena into equally sized squares (6 × 6 cm), was projected over the recordings, and the number of line crossings made by the animal was recorded.

Social interaction in young adult rats

The test was performed as previously described [44]. Eighty-day-old rats were individually habituated to the test cage for 5 min on each of the 2 days prior to testing. Before testing, animals were socially isolated for 24 h to enhance their social motivation and thus facilitate the expression of social behaviors during testing. The animals of each pair were similarly treated, did not differ more than 10 g in body weight and were not cagemates. The test was performed between 9 a.m. and 2 p.m. under low light conditions. Behavior was assessed per pair of animals and analyzed by a trained observer who was unaware of treatment condition using the The Observer 3.0 software (Noldus Information Technology BV, Wageningen, The Netherlands).

The total number of play-related behaviors (pouncing, pinning, and boxing) and the total time spent in social exploration (sniffing any part of the body of the test partner, social grooming, following/chasing, crawling under/over, and kicking) were scored for 10 min [45]. Moreover, locomotor activity during the social encounter was assessed by a grid dividing the arena into equally sized squares (6 × 6 cm) projected over the recordings and the number of line crossings made by the animal was counted.

Statistical analysis

Data are expressed as mean ± SEM and statistical significance was set at p < 0.05. To assess the effects of single or combined treatments on social play behavior, data were analyzed using one-way or two-way analysis of variance, respectively, followed by Student–Newman–Keuls post-hoc tests where appropriate.

Results

MDPV suppresses social play behavior

The results of experiment 1 show that systemic administration of MDPV inhibited social play behavior in juvenile rats (pinning: F5,47 = 5.70, p < 0.001; pouncing: F5,47 = 3.18, p = 0.015). MDPV, administered at the highest tested dose (0.5 mg/kg), reduced pinning (p < 0.01, Fig. 1a) and pouncing (p < 0.05, Fig. 1b), whereas lower doses (0.025–0.05–0.1–0.25 mg/kg) had no effect on play behavior. General social exploration (i.e., sniffing and grooming any part of the body of the test partner, including the anogenital area) was not affected by MDPV treatment (F5,47 = 1.72, n.s., Fig. 1c). The effects of MDPV on social play were not secondary to changes in locomotor activity, since at the dose that decreased social play behavior (0.5 mg/kg), MDPV did not affect the number of crossings (F5,47 = 1.57, n.s., Fig. 1d). Thus, MPDV selectively suppresses social play behavior in juvenile rats. To support the idea that MDPV induces selective effects on social play behavior, the results of experiment 2 show that MDPV, administered at the dose of 0.5 mg/kg, reduced the total frequency of play-related behaviors (F5,27 = 2.95, p = 0.03, Fig. 1e) in young adult rats (PND 80) without affecting general social exploration (F5,27 = 1.62, n.s., Fig. 1f) and locomotor activity (F5,27 = 0.13, n.s., data not shown), confirming that MDPV selectively suppresses play-related behaviors even in young adult rats.

Fig. 1: Acute administration of MDPV (0.5 mg/kg) suppresses social play behavior without affecting general social exploration and locomotor activity in both juvenile and young adult rats.
figure 1

MDPV (0.5 mg/kg) suppressed pinning (a) and pouncing (b) behaviors in juvenile (i.e., 28–30-day-old) rats. Social exploration (c) and locomotor activity (i.e., the number of crossings (d)) were not affected by MDPV. Similarly, MDPV reduced play-related behaviors (e) without affecting social exploration (f) in young adult (i.e., 80-day-old) rats (juveniles: n SAL = 10, n MD0.025 = 7, n MD0.05 = 7, n MD0.1 = 11, n MD0.25 = 8, n MD0.5 = 10; young adults: n SAL = 6, n MD0.025 = 6, n MD0.05 = 5, n MD0.1 = 5, n MD0.25 = 6, n MD0.5 = 5). Data represent mean values ± SEM; *p < 0.05, **p < 0.01 vs SAL group (Student–Newman–Keuls post-hoc test).

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An additional experiment (experiment 3) was performed to investigate whether the effects of MDPV on social play behavior in juvenile (e.g., PND 28–30) rats depended on the behavior exhibited by the test partner and/or if the social repertoire of saline-treated rats was influenced by MDPV-treated animals. None, one, or both members of a test pair were treated with MDPV (0.5 mg/kg) and behavior of both test partners was scored separately. The two-way ANOVA analysis gave the following results: pinning F(subject)1,54 = 9.06, p = 0.004; F(partner)1,54 = 4.14, p = 0.05; F(subject×partner)1,54 = 5.19, p = 0.03; pouncing F(subject)1,54 = 2.28, p = 0.03; F(partner)1,54 = 4.02, p = 0.05; F(subject×partner)1,54 = 1.72, n.s.; social exploration F(subject)1,54 = 0.00, p = n.s.; F(partner)1,54 = 0.83, n.s.; F(subject×partner)1,54 = 0.16, n.s. Post-hoc analyses revealed that pinning (p < 0.001, Fig. 2a) and pouncing (p < 0.05, Fig. 2b) were reduced when both members of a test pair were treated with MDPV. When only the scored animal of a test pair was treated with MDPV, there was also a decrease in pinning (p < 0.001) and pouncing (p < 0.05) events, indicating that MDPV-treated rats pin and pounce their partner less, regardless of whether the partner is treated with saline or MDPV. Interestingly, saline-treated rats interacting with MDPV-treated partners also showed reduced pinning (p < 0.01) and pouncing (p < 0.05) compared with pairs in which both animals were treated with saline. These results suggest that MDPV-treated rats displayed both reduced initiation to play and responsiveness to play solicitation, indirectly decreasing playfulness in their saline-treated partners as well.

Fig. 2: MDPV reduces social play behavior regardless of the treatment received by the test partner, affecting social play in vehicle-treated animals as well.
figure 2

When behavior of the members of a pair was scored separately, there was a reduction in pinning (a) and pouncing (b) either when one or both rats in a pair were treated with MDPV. In addition, MDPV-treated rats affected behavior of their saline-treated partners: saline-treated rats interacting with MDPV-treated partners showed indeed reduced pinning (a) and pouncing (b) frequency. These results suggest that MDPV-treated rats displayed both reduced initiation to play and responsiveness to play solicitation, indirectly decreasing playfulness in their saline-treated partners as well. Social exploration was unaffected (c). n SAL–SAL = 16, n SAL–MD = 12, n MD–SAL = 12, n MD–MD = 18. Data represent mean values ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001 vs SAL–SAL group (Student–Newman–Keuls post-hoc test). ‘Subject’ represents the treatment of the animal whose behavior was scored; ‘Partner’ represents the treatment of its test partner.

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The effects of MDPV on social play are subject to tolerance but not sensitization

Experiment 4 evaluated whether tolerance occurs to the effects of MDPV on social play behavior after repeated treatment. To this aim, juvenile rats were treated with the dose of MDPV that reduced social play in the previous experiments (0.5 mg/kg, i.p.) or saline (control group) once daily for 5 consecutive days. On the sixth day, half of both pretreatment groups were tested after acute treatment with MDPV (0.5 mg/kg, i.p.), or saline (control group). A two-way ANOVA analysis gave the following results: pinning: F(repeated)1,26 = 0.45, n.s; F(acute)1,26 = 9.20, p = 0.005; F(repeated×acute)1,26 = 1.32, n.s; pouncing: F(repeated)1,26 = 0.01, n.s; F(acute)1,26 = 9.90, p = 0.004; F(repeated×acute)1,26 = 2.17, n.s. Post-hoc analyses revealed that acute administration of MDPV (0.5 mg/kg, i.p.) decreased pinning (p < 0.01, Fig. 3a) and pouncing (p < 0.01, Fig. 3b) in animals repeatedly treated with saline but not in animals repeatedly pretreated with MDPV (0.5 mg/kg, i.p.), indicating that tolerance to the effect of MDPV on social play occurs. Social exploration was not affected by either the repeated or acute treatment (F(repeated)1,26 = 0.80, n.s.; F(acute)1,26 = 0.91, n.s.; F(repeated×acute)1,26 = 0.072, n.s., Fig. 3c).

Fig. 3: Following repeated treatment, tolerance occurs to the play-suppressant effects of MDPV.
figure 3

Acute administration of MDPV (0.5 mg/kg, i.p.) decreased the amount of pinning (a) and pouncing (b) in animals pretreated with saline (SAL–MD0.5 group) but not in animals pretreated with MDPV (MD0.5–MD0.5 group), indicating that tolerance to the effect of MDPV had occurred. Social exploration was not affected by either the repeated and acute treatment (c) (n SAL–SAL = 7, n SAL–MD0.5 = 8, n MD0.5–SAL = 7, n MD0.5–MD0.5 = 8). Data represent mean values ± SEM; **p < 0.01 vs SAL–SAL group (Student–Newman–Keuls post-hoc test).

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Next, to evaluate whether sensitization occurs to the effects of MDPV on social play behavior after repeated treatment, animals were treated with MDPV (0.5 mg/kg, i.p.) or saline once daily for 5 consecutive days. On the sixth day, half of both pretreatment groups were treated with a dose of MDPV that does not affect social play by itself (0.1 mg/kg, i.p.), or saline (experiment 5). Acute administration of a low dose of MDPV (0.1 mg/kg, i.p.) did not affect social play in rats repeatedly treated with either saline or MDPV (0.5 mg/kg, i.p), indicating that sensitization to the effect of MDPV on social play behavior had not occurred after repeated treatment (pinning: F(repeated)1,26 = 0.14, n.s.; F(acute)1,26 = 1.51, n.s.; F(repeated×acute)1,26 = 0.68, n.s., Fig. 4a; pouncing: F(repeated)1,26 = 0.01, n.s.; F(acute)1,26 = 1.16, n.s.; F(repeated×acute)1,26 = 1.47, n.s., Fig. 4b). Social exploration was not affected by either the repeated or acute treatment (F(repeated)1,26 = 0.77, n.s.; F(acute)1,26 = 1.55, n.s.; F(repeated×acute)1,26 = 1.05, n.s., Fig. 4c).

Fig. 4: Following repeated treatment, sensitization to the play-suppressant effects of MDPV does not occur.
figure 4

Acute administration of a low dose of MDPV (0.1 mg/kg, i.p.) did not affect pinning (a) and pouncing (b) in rats repeatedly pretreated with an effective dose of MDPV (0.5 mg/kg, i.p.), revealing that sensitization to the effects of MDPV on social play behavior had not occurred. Social exploration was not affected by either the repeated and acute treatment (c) (n SAL–SAL = 7, n SAL–MD0.1 = 8, n MD0.5–SAL = 7, n MD0.5–MD0.1 = 8). Data represent mean values ± SEM.

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Pharmacological mechanisms underlying the effects of MDPV on social play behavior

It has recently been shown that certain behavioral effects of MDPV are mediated by activation of dopaminergic receptors, as a result of DAT blockade by MDPV [12, 46, 47]. To investigate whether the effects of MDPV on social play in juvenile rats also depend on dopaminergic neurotransmission (experiment 6), we administered the dopamine receptor antagonist cis-(Z)-flupenthixol, at a dose (0.125 mg/kg i.p.) that does not affect social play by itself [40], before administration of MDPV (0.5 mg/kg i.p.). Systemic administration of flupenthixol blocked the effects of MDPV on social play (pinning: F(pretreatment)1,26 = 0.025, n.s.; F(treatment)1,26 = 4.57, p = 0.042; F(pretreatment×treatment)1,26 = 0.95, p = n.s., Fig. 5a; pouncing: F(pretreatment)1,26 = 0.33, n.s.; F(treatment)1,26 = 5.11, p = 0.032; F(pretreatment×treatment)1,26 = 0.62, n.s., Fig. 5b). Indeed, post-hoc analysis revealed that MDPV reduced both pinning (p < 0.05) and pouncing (p < 0.05) in animals pretreated with saline but not in animals pretreated with flupenthixol (Fig. 5a, b). No differences were found in the time spent in social exploration (F(pretreatment)1,26 = 1.03, n.s.; F(treatment)1,26 = 1.01, n.s.; F(pretreatment×treatment)1,26 = 1.15, n.s., Fig. 5c).

Fig. 5: The effects of MDPV on social play depend on both dopaminergic and noradrenergic neurotransmission.
figure 5

The effects of MDPV on pinning (a, d) and pouncing (b, e) were blocked by either the dopamine receptor antagonist flupenthixol (0.125 mg/kg i.p.; n SAL–SAL = 9, n SAL–MD = 8, n FLU–SAL = 7, n FLU–MD = 6) or the α2 adrenoceptor antagonist RX821002 (0.2 mg/kg i.p.; n SAL–SAL = 6, n SAL–MD = 7, n RX–SAL = 5, n RX–MD = 6). Moreover, combined administration of sub-effective doses of the α2 noradrenergic receptor antagonist RX821002 (0.1 mg/kg i.p.) and the dopamine receptor antagonist flupenthixol (0.06 mg/kg i.p.) antagonized the effects of MDPV on pinning (g) and pouncing (h) (n SAL–SAL = 6, n SAL–MD = 8, n FLU/RX–SAL = 8, n FLU/RX–MD = 8). Social exploration was not affected by treatments (c, f, i). Data represent mean values ± SEM; *p < 0.05, **p < 0.01 vs SAL–SAL group (Student–Newman–Keuls post-hoc test).

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We have previously shown that psychostimulants like amphetamine and methylphenidate exert their play-suppressant effects through stimulation of α2 noradrenergic receptors [14, 38, 40], and MDPV has been shown to block the NET [48]. Therefore, to investigate whether α2 adrenoceptors are also involved in the reduction of social play induced by MDPV, juvenile rats were treated with the α2 adrenoceptor antagonist RX821002 (0.2 mg/kg, i.p.) before administration of MDPV (0.5 mg/kg, i.p.) (experiment 7). Systemic administration of the α2 adrenoceptor antagonist RX821002 (0.2 mg/kg, i.p.) 15 min before MDPV administration counteracted the effects of MDPV on social play behavior (pinning: F(pretreatment)1,20 = 7.35, p = 0.013; F(treatment)1,20 = 4.25, p = n.s; F(pretreatment×treatment)1,20 = 4.98, p = 0.04; pouncing: F(pretreatment)1,20 = 13.2, p = 0.002; F(treatment)1,20 = 6.71, p = 0.02; F(pretreatment×treatment)1,20 = 4.68, p = 0.04). Post-hoc analysis revealed that MDPV reduced both pinning (p < 0.01) and pouncing (p < 0.01) in animals pretreated with saline but not in animals pretreated with RX821002 (Fig. 5d, e). No differences were found in social exploration (F(pretreatment)1,20 = 0.21, n.s.; F(treatment)1,20 = 0.56, n.s.; F(pretreatment×treatment)1,20 = 0.16, n.s., Fig. 5f).

To test whether dopaminergic and noradrenergic neurotransmission are simultaneously involved in the play-suppressant effects of MDPV (experiment 8), we treated juvenile rats with either a combination of sub-effective doses of both the α2 noradrenergic receptor antagonist RX821002 (0.1 mg/kg i.p.) and the dopamine receptor antagonist flupenthixol (0.06 mg/kg i.p.), or with saline (control group) 15 min before treatment with MDPV. The combined administration of sub-effective doses of RX821002 and flupenthixol antagonized the play-suppressant effects of MDPV (pinning: F(pretreatment)1,26 = 1.22, n.s.; F(treatment)1,26 = 0.93, n.s.; F(pretreatment×treatment)1,26 = 4.61, p = 0.04, Fig. 5g; pouncing: F(pretreatment)1,26 = 1.27, n.s.; F(treatment)1,26 = 0.64, n.s.; F(pretreatment×treatment)1,26 = 14.2, p < 0.001, Fig. 5h). Post-hoc analyses revealed that MDPV reduced both pinning (p < 0.05) and pouncing (p < 0.01) in animals pretreated with saline but not in animals pretreated with sub-effective doses of both RX821002 and flupenthixol (Fig. 5g, h). No differences were found in social exploration (F(pretreatment)1,26 = 0.63, n.s.; F(treatment)1,26 = 0.95, n.s.; F(pretreatment×treatment)1,26 = 0.016, n.s., Fig. 5i).

Discussion

Synthetic cathinones are widely abused by young people, often in a social setting [3,4,5]. However, scarce information is available about their direct effect on social behavior. Here, we provide evidence that the widely abused cathinone, MDPV, suppresses social play behavior, a characteristic and rewarding form of social interaction displayed by young mammals [22, 36, 49]. The play-suppressant effect of MDPV in juvenile rats was behaviorally specific: it was the result of reduced play initiation and diminished response to play solicitation, but it was not associated with changes in social exploratory behavior or locomotor activity during social interaction. Interestingly, MDPV reduced play-related behaviors without affecting social exploration and locomotor activity also in young adult rats, suggesting that the detrimental effects of MDPV on social play are not exclusive to juvenile animals, i.e., are not age dependent. The play-suppressant effects of MDPV were subject to tolerance but not sensitization, and were mediated by dopaminergic and noradrenergic neurotransmission. Together, these data demonstrate that MDPV has selective detrimental effects on social play behavior and shed light on the pharmacological mechanisms involved.

To dissect the effects of MDPV on either play initiation or the responsiveness to play solicitation, we performed an experiment in which MDPV was injected to none, one, or both partners of the test dyad. Pouncing, which is an index of play solicitation, and pinning, which is the most characteristic response to play initiation, were significantly reduced in all MDPV-treated rats, irrespective of the treatment of their test partners. Interestingly, the altered behavior of MDPV-treated rats also affected the behavior of saline-treated partners. Indeed, saline-treated rats interacting with MDPV-treated partners also showed reduced pinning and pouncing frequencies compared with test pairs in which both animals were treated with saline. These results support the notion that reciprocity is necessary to maintain appropriate levels of playfulness and they are consistent with findings showing that social play behavior is influenced by the level of social activity of the partner [32,33,34,35, 40].

In humans, the development of tolerance, dependence, and withdrawal has been reported after the frequent use of high doses of MDPV [50]. A recent study showed that the aggressive responses induced by MDPV in adult mice are subject to sensitization, being enhanced after repeated administration [19]. Therefore, we tested whether tolerance or sensitization, or both, would occur to the effect of MDPV on social play behavior after repeated treatment. We found that the reduction in social play induced by systemic administration of MDPV was suppressed following repeated treatment, indicating that tolerance to the effects of MDPV in social play had occurred. Conversely, social play was unaffected when animals were repeatedly pretreated with MDPV and acutely treated with a sub-effective dose of MDPV on the test day, showing that sensitization to the effects of MDPV on social play behavior had not occurred. Therefore, the play-suppressant effects induced by MDPV are different by those induced by methylphenidate, effects of which on social play are not subject to tolerance or sensitization [40].

In the brain, MDPV acts as a potent uptake inhibitor at plasma membrane transporters for both dopamine (DAT) and noradrenaline (NET), while it lacks significant activity at the serotonin transporter (SERT) and it is not considered a substrate releaser [18, 48]. In particular, in vitro studies reported that MDPV is 50-fold more potent at DAT, 10-fold more potent at NET, and 10-fold less potent at SERT than cocaine [1, 8]. On this basis, we hypothesized that changes in dopaminergic and/or noradrenergic neurotransmission underlie the play-suppressant effects of MDPV. To test this possibility, we treated rats with the α2 adrenoceptor antagonist RX821002 or the dopamine receptor antagonist flupenthixol prior to MDPV. We found that both the α2 adrenoceptor antagonists RX821002 and the dopamine receptor antagonist flupenthixol, given alone, counteracted the effects of MDPV on social play. Furthermore, combined administration of sub-effective doses of both RX821002 and flupenthixol also antagonized the play-suppressant effects of MDPV, indicating that the effects of MDPV on social play are mediated by activation of both α2 adrenoceptors and dopamine receptors. Together, these data show that MDPV and other psychostimulants suppress social play through distinct, but overlapping neurochemical mechanisms [14, 40]. That is, both amphetamine and methylphenidate exert their play-suppressant effects through stimulation of α2 noradrenergic receptors but not dopamine receptors [14, 38,39,40]. On the other hand, cocaine reduces social play by simultaneous increases in dopamine, noradrenaline, and serotonin neurotransmission [14].

Dopaminergic and noradrenergic neurotransmission have dissociable roles in social play behavior [37]. Systemic administration of drugs that either stimulate or reduce dopaminergic neurotransmission can disrupt social play, likely by altering dopaminergic neurotransmission in a variety of brain regions, leading to a dysbalance in dopamine signaling between different circuits [37]. On the other hand, it has been shown that dopamine stimulates the motivation for social play [39], acting in the nucleus accumbens [41]. Therefore, the dopaminergic mechanisms that underlie the reduction in social play induced by MDPV may either occur through the integration with other brain structures and neurotransmitters not primarily involved in the motivational aspect of social play, or may be related to dopaminergic control of more cognitive components of social behavior, such as adaptive behavior and decision making [51, 52]. In support of this possibility, it has recently been shown that the effects of MDPV on memory consolidation and associative memory are also related to activation of dopamine receptors [12]. Alongside dopamine, noradrenergic neurotransmission is also involved in the play-suppressant effects of MDPV. Increases in noradrenergic neurotransmission have been found to negatively modulate the motivation for social play behavior and its expression [39]. Furthermore, noradrenaline is involved in cognitive processes, such as learning, attention, impulse control, decision making, and behavioral flexibility [53], that all need to be optimally aligned for the proper performance of social play [37]. Thus, although the experimental setup used in the present study does not allow to separate the incentive-motivational and pleasurable properties of social play, the fact that the play-suppressant effects of MDPV are mediated through both dopaminergic and noradrenergic neurotransmission leads to hypothesize that MDPV may reduce social play by affecting cognitive processes that are necessary for the proper execution of social play. For instance, MDPV may alter the ability of the animals to interpret and adequately reciprocate social stimuli, or to adjust their social behavior to the changing circumstances in the social and physical environment.

Notably, MDPV (and bath salts in general) is often used in social contexts to facilitate social interactions/approaches [1]. However, we here found that MDPV reduced play-related behaviors in rats without altering general social exploration. These results suggest that the acute effects of MDPV on social play behavior are comparable to the effects of other psychostimulants, which are also abused in social settings, such as amphetamine, methylphenidate, or cocaine, that also exert play-suppressant effects in male rats [14, 24, 40]. Importantly, sex-dependent differences in the effects of MDPV [54,55,56] and in the structure and intensity of social play behavior [23] have been documented. Therefore, investigating the effects of MDPV on social play behavior in female rats, and the neural mechanisms involved, is an intriguing issue which deserves further investigation.

Collectively, our data provide evidence of the deleterious effects of MDPV on social play behavior in both juvenile and young adult male rats and they also indicate potential neural mechanisms for the social dysfunctions induced by this popular cathinone. Social play behavior is critical for proper neurobehavioral development, and altered or reduced patterns of social play may lead to enduring behavioral deficits [37]. Since MDPV can induce a wide range of behavioral alterations in humans [1, 6], more research into the persistent behavioral effects following both acute and chronic exposure to this synthetic cathinone at early ages is warranted.

Founding and disclosure

This work was supported by Marie Curie Career Reintegration Grant PCIG09-GA-2011-293589 (VT) and by Excellence Departments, MIUR‐Italy, Grant/Award Numbers: ARTICOLO 1, COMMI 314‐337 LEGGE 232/2016, ARTICOLO 1. The authors declare that, except for income received from their primary employers, no financial support or compensation has been received from any individual or corporate entity over the past 5 years for research or professional service and there are no personal financial holdings that could be perceived as constituting a potential conflict of interest.