Assessment of aversive effects of methylone in male and female Sprague-Dawley rats: Conditioned taste avoidance, body temperature and activity/stereotypies

Abstract

Methylone’s rewarding effects have been well characterized; however, little is known about its aversive effects and how such effects may be impacted by sex. In this context, the present study investigated the aversive effects of methylone (vehicle, 5.6, 10 or 18 mg/kg, IP) in 35 male and 31 female Sprague-Dawley rats assessed by conditioned taste avoidance and changes in body temperature and activity/stereotypies. Methylone induced significant taste avoidance, changes in temperature and increased activity and stereotypies in both males and females. Similar to work with other synthetic cathinones, methylone has aversive effects as indexed by significant taste avoidance and changes in temperature and activity (two characteristics of methylone overdose in humans). The only endpoint for which there were significant sex differences was in general activity with males displaying a faster onset and females displaying a longer duration. Although sex was not a factor with taste avoidance and temperature, separate analyses for males and females revealed different patterns, e.g., males displayed a more rapid acquisition of taste avoidance and females displayed changes in temperature at lower doses. Males displayed a faster onset and females displayed a longer duration of activity (consistent with the analyses considering sex as a factor), while time- and dose-dependent stereotypies did not show consistent pattern differences. Although sex differences were relatively limited when sex was specifically assessed as a factor (or only evident when sex comparisons were made in the patterns of effects), sex as a biological variable in the study of drugs should be made to determine if differences exist and, if evident, the basis for these differences.

1. Introduction

Synthetic cathinones, commonly referred to as “bath salts”, are a class of new psychoactive substances that have recently captured interest for their use and abuse potential (Baumann et al., 2013, 2014; Capriola, 2013; Zawilska and Wojciezak, 2013; Banks et al., 2014; Riley et al., 2020). Synthetic cathinones are beta-ketone analogues of amphetamine and are derived from the psychoactive compound cathinone that is found in the khat plant (Catha edulis) which is native to East Africa and the Arabian Peninsula (DeRuiter et al., 1994; Coppola and Mondola, 2012; Capriola, 2013). Bath salts initially emerged as legal alternatives to drugs such as cocaine, methamphetamine and MDMA, and before their eventual classification as Schedule 1 drugs (Bonson et al., 2019), they were available for purchase online and at convenience stores and truck stops. As a group, these compounds are reported to produce effects similar to traditional psychostimulants (Vardakou et al., 2011; Winstock et al., 2011; Coppola and Mondola, 2012) and continue to be abused as more are synthesized and released (European Monitoring Centre for Drugs and Drug Addiction, 2019). This is unsurprising since the synthetic cathinones increase levels of dopamine (DA), serotonin (5-HT) and norepinephrine (NE) via their transporters (e.g., DAT, NET, and SERT, respectively) by functioning as either an amphetamine-like transporter substrate (e.g., methylone) or as a cocaine-like reuptake inhibitor [e.g., methylenedioxypyrovalerone (MDPV)] (Rothman and Baumann, 2003; Baumann et al., 2013; Banks et al., 2014; Simmler et al., 2013).

One of the first-generation bath salts was methylone. Although originally synthesized with the intent to be developed and distributed as an antidepressant, it subsequently emerged as a drug of abuse which was marketed under the trade name “explosion” in 2004 (Bossong et al., 2005). Human data indicate that methylone not only has profound psychostimulant effects, but also has been tied to fatal intoxications indexed by elevated body temperature and sympathomimetic toxicity (Pearson et al., 2012; Warrick et al., 2012). Numerous studies have demonstrated methylone’s action on monoaminergic systems, where it triggers the non-exocytic release of 5-HT, and to a lesser degree, DA and NE (Hadlock et al., 2011; Sogawa et al., 2011; Lopez-Arnau et al., 2012). By virtue of acting as a substrate at monoamine transporters, methylone also acts as a transporter blocker, thereby preventing the reuptake of DA, NE and 5-HT (Hadlock et al., 2011; Lopez-Arnau et al., 2012; Baumann et al., 2013; Banks et al., 2014). Behaviorally, methylone supports intravenous self-administration (IVSA) in rats (Watterson et al., 2012; Aarde et al., 2015; Creehan et al., 2015; Vandewater et al., 2015; Schindler et al., 2016; Nguyen et al., 2017; Dolan et al., 2018; Gannon et al., 2018, 2019; Javadi-Paydar et al., 2018), although the robustness of such self-administration is mixed. It also lowers the threshold for intracranial self-stimulation (ICSS) in rats (Bonano et al., 2014), conditions a place preference in mice (Karlsson et al., 2014) and substitutes for the discriminative stimulus effects of cocaine and methamphetamine in rats (Gatch et al., 2013). Preclinical data demonstrate that methylone has greater liability for compulsive use than 3,4-methylenedioxymethamphetamine (MDMA), a drug with which it shares a structural and pharmacological profile (Watterson et al., 2012; Vandewater et al., 2015; Nguyen et al., 2017).

Conventionally, studies analyzing the use and abuse of drugs have focused on their rewarding properties; however, such drugs also produce a variety of aversive effects (for a discussion of the potential interaction of these affective properties of drugs of abuse, (see Cunningham, 1979; Stolerman and D’Mello, 1981; Turenne et al., 1996; Riley, 2011; Verendeev and Riley, 2011, 2012; Lin et al., 2016). In relation to these aversive effects, a wide variety of drugs of abuse including amphetamine/mescaline (Cappell and LeBlanc, 1971), methamphetamine (Martin and Ellinwood, 1973), cocaine (Goudie et al., 1978), ethanol (Eckardt, 1976), THC (Edwin, 1975) and morphine (Sherman et al., 1980) induce significant taste avoidance (Freeman and Riley, 2008; for a review of conditioned taste avoidance with drugs of abuse, see Verendeev and Riley, 2012). Recently, several synthetic cathinones, e.g., MDPV (King et al., 2014, 2015; Merluzzi et al., 2014) and α-pyrrolidinopentiophenone [α-PVP] (Nelson et al., 2017; 2019a, 2019b), have been reported to induce conditioned taste avoidance (CTA) as well. Although methylone’s rewarding effects are well characterized, its ability to induce a CTA has not been examined. To address this issue, the present experiment examined methylone-induced taste avoidance learning (see Riley and Tuck, 1985). In addition to this behavioral index of the aversive effects of drugs, methylone-induced changes in temperature and activity were examined in the same subjects. The basis for these latter assessments stems from the fact that under a variety of conditions a number of traditional psychostimulants (Mash et al., 2009) as well as the synthetic cathinones (Spiller et al., 2011; Borek and Holstege, 2012; Murray et al., 2012; Penders et al., 2012) have been reported to induce psychomotor agitation and to elevate body temperature in humans, characteristics that often lead to multiorgan failure and death (for a review see Mash et al., 2009; see also Penders et al., 2012; Bonson et al., 2019). Although psychomotor agitation and elevated body temperature have both been examined with methylone (López-Arnau et al., 2012; Kiyatkin et al., 2015; Piao et al., 2015; Goldsmith et al., 2019), studies examining the two effects in the same subject (which is a characteristic of human case reports) is limited (though see Stefkova et al., 2017; Javadi-Paydar et al., 2018).

In addition to examining methylone for its ability to induce taste avoidance and changes in body temperature and activity/stereotypies, the present study assessed if the effects of methylone on these endpoints were sex dependent (Becker and Koob, 2016; Becker et al., 2017; Riley et al., 2018). Sex differences in taste avoidance learning have been reported with both traditional psychostimulants and synthetic cathinones (for a review of sex as a biological variable, see Riley et al., 2018), with the direction of the differences drug/preparation dependent (Haaren and Hughes, 1990; Russo et al., 2003; Busse et al., 2005; Zakharova et al., 2009; King et al., 2015; Hambuchen et al., 2017; Nelson et al., 2019a, 2019b). Although assessments of sex differences with methylone are limited, some studies have shown that the effects of methylone are sex dependent as well. For example, while methylone induces significant increases in temperature in both male and female rats, female rats become tolerant to this effect faster than male rats, and, in fact, display decreases in temperature with repeated exposure (Goldsmith et al., 2019). Further, male rats display greater anxiety following methylone exposure compared to that in females (Daniel and Hughes, 2016). To extend these analyses of potential sex differences in the effects of synthetic cathinones in general and more specifically in the adverse effects of methylone, the present study examined its ability to induce taste avoidance as well as changes in temperature and activity/stereotypies in both males and females.

2. General methods

2.1. Subjects

The subjects were 66 experimentally naïve male (n = 35) and female (n = 31) Sprague-Dawley rats. Animals were bred within the American University animal research facility and were allowed to mature undisturbed until the start of testing. For a minimum of 7 days prior to the experiment, males and females were weighed daily to index health and to reduce handling stress during subsequent experimental procedures. At the start of the experimental procedures, male subjects weighed between 314 and 489 g (mean = 357; SEM = 4.79) and females weighed between 210 and 290 g (mean = 235; SEM = 3.20). All procedures adhered to the Guidelines for the Care and Use of Laboratory Animals (National Research Council, 2011) and the National Research Council (2003) and were approved by the Institutional Animal Care and Use Committee at American University.

2.2. Drugs and solutions

Racemic methylone hydrochloride (synthesized and provided by the Drug Design and Synthesis section, MTMDB, NIDA and NIAAA) was dissolved in isotonic saline (0.9%) and injected intraperitoneally (IP) at 5.6, 10 or 18 mg/kg. Controls were administered equivolume saline (vehicle). Drug and vehicle solutions were prepared daily and passed through a 0.2-um filter prior to injection to remove any potential particulates. Saccharin (sodium saccharin, Acros Organics) was prepared as a 1 g/l (0.1%) solution in tap water.

2.3. Apparatus

Subjects of the same sex were socially housed (2–3 per cage) in OptiRat Plus cages (38.9 × 56.9 × 26.2 cm; 1181 cm²). The room in which the cages were located was maintained on a 12-h light/dark cycle (0800–2000 h) at 20–24 °C (humidity level was between 30 and 70%). All procedures took place during the lights-on phase of the cycle. Unless stated otherwise, food and water were available ad libitum. During conditioned taste avoidance training, animals were transferred to a separate testing room and placed in individual hanging, stainless-steel wire-mesh test cages (24.3 × 19 × 18 cm) on the front of which graduated Nalgene tubes were placed for fluid presentation. For activity assessments, animals were placed in one of eight identical testing apparatuses (68.5 × 34.5 × 21 cm; San Diego Instruments Place Preference System, San Diego, CA) that contained a 16 × 4 photo beam array near the floor to record ambulation (activity) and fine motor movements (stereotypies). Each apparatus was assembled in a manner such that the walls were clear Plexiglas with a grey plastic box placed around the outside to give the effect of opaque grey walls, and the floor was covered by a 68.5 × 21 cm sheet of haircell textured grey Kydex plastic. Each chamber was equipped with three LED lights set to maximum brightness within the otherwise unlit room. A white noise generator was also used to mask background noise (see Hutchison et al., 2010).

2.4. Procedure

2.4.1. Conditioned taste avoidance

At approximately post-natal day (PND) 94, subjects were deprived of water for 24 h and were then given 20-min access to tap water in the hanging test cages. Following access to water, animals were returned to their home cages. This limited access procedure was repeated for 10 days to allow water consumption to stabilize (all subjects approaching the drinking tube within 2 s with the average volume of water consumed not increasing or decreasing by more than 2 ml for 3 consecutive days). On the day following stable water consumption, all subjects were placed in the hanging test cages and given 20-min access to a novel saccharin solution. Based on saccharin consumption, male and female subjects were assigned to one of four groups and injected with either the saline vehicle or 5.6, 10 or 18 mg/kg of racemic methylone (n = 7–9 per group). This yielded a total of eight groups, i.e., MV, M5.6, M10, M18, FV, F5.6, F10 and F18, where the first letter indicates the sex of the subject and the number or second letter represents vehicle or the dose of methylone received. For the next 3 days (Day 2–4), subjects were given 20-min access to water in the test cages followed by a saline injection (IP). This 4-day cycle was repeated for a total of four times. Saccharin and water consumption were evaluated by the difference between pre- and post-consumption values.

The doses used in the present experiment were based on previous work reporting methylone’s ability to induce conditioned place preferences in mice at 5, 10 and 20 mg/kg (Miyazawa et al., 2011; Karlsson et al., 2014) and to facilitate low rates of ICSS in rats at 10 mg/kg (Bonano et al., 2014, see also Watterson et al., 2012). Other work has supported methylone’s capacity to induce hyperlocomotion at 5 and 10 mg/kg (Javadi-Paydar et al., 2018) and at 25 mg/kg (Miyazawa et al., 2011; López-Arnau et al., 2012). Methylone (10 and 20 mg/kg has also been shown to increase temperature significantly in rats (Stefkova et al., 2017, Javadi-Paydar et al., 2018).

On the day following the last conditioning cycle (Day 17), animals were placed in the test cages and given 20-min access to both saccharin and tap water in a two-bottle avoidance test with no injections occurring after fluid access. During this test, one bottle was offered (saccharin or water) on either the left or the right front of the test cage. Immediately following the sampling of the first bottle, it was removed, and a second bottle was presented on the opposite side. Once the second bottle was sampled, it was removed and both bottles were simultaneously placed on their respective sides on the front of the cage. The order of presentation and side placement were counterbalanced across animals, and consumption of both saccharin and water was recorded as differences in pre- and post-consumption values. Animals were then returned to their home cages with ad libitum water access.

2.4.2. Temperature

Following a 21- to 30-day wash out period, temperature probes (Bio Medic Data Systems, Seaford, DE; Model #IPTT-300) were subcutaneously implanted between the shoulder blades of each animal. To perform this procedure, the animals were put under isoflurane anesthesia and the surgical site was sterilized with 70% ethanol before the probe was implanted. Subjects were allowed to recover undisturbed for 1 day before being weighed for 3 consecutive days to check for health status and to test that the temperature probes were functional. Next, all subjects were scanned for temperature, weighed and then injected IP with saline for 2 additional days. The initial scans taken on these 2 days were not used in any statistical assessments; they were only for habituating the animals to the procedure and confirming proper functioning of the equipment. To acquire baseline measures, animals were weighed, scanned and injected with 0.5 ml saline IP and then additional temperature scans were taken 30 min, 1 h and again every hour up until 8-h post injection. The procedure on the next day was similar to that of the day before with the exception that subjects were randomly placed into groups and injected with either vehicle or 5.6, 10 or 18 mg/kg of racemic methylone (n = 8–9). Each animal was scanned three times, and the three measurements were averaged. All temperature assessments took place in the animal colony during the lights-on phase. Animals remained in their home cages, with the exception of when they were weighed, injected and scanned.

2.4.3. Activity and stereotypies

After an additional 18- to 21-day wash-out period, animals were weighed and handled for 3 consecutive days. Following the handling period, they were weighed and injected with 0.5 ml saline IP. Immediately following the injections, animals were taken to an adjacent testing room and placed into the activity testing apparatuses. Counts of gross activity (consecutive beam breaks) and stereotypies (repeated breaks of the same beam) were recorded during 1-h sessions that were divided into 12 5-min bins. The next day of testing was similar with the exception that subjects were randomly injected with either vehicle or 5.6, 10 or 18 mg/kg (n = 8–9). Eight activity chambers were used, and each animal was placed in the same chamber on both days. Chambers were cleaned using a solution (Sani-Plex 128 M, one-step disinfectant germicidal detergent) between animals.

2.5. Statistical analysis

The data from each assessment were analyzed using a mixed model ANOVA with the between-subjects factors of Sex (male and female) and Dose (vehicle, 5.6, 10 and 18 mg/kg) and the within-subjects factor varying depending on the assessment, i.e., Trial (1–4 for conditioned taste avoidance); TimePoint (pre-injection, 30 min, 1–8-h post-injection for temperature assessments); and TimePoint (5-min intervals over the span of an hour for activity and stereotypies). In the event of a significant three-way interaction, the effects of Sex and Dose at each within-subjects factor was examined (multivariate analysis) followed by Bonferroni-adjusted multiple comparisons.

Statistical significance was set to p ≤ 0.05.

3. Results

3.1. Conditioned taste avoidance

The 2 × 4 × 4 mixed model ANOVA on saccharin consumption in male and female rats revealed a significant main effect of Trial [F(1, 59) = 111.4, p < 0.001] and Dose [F(3, 59) = 82.403, p < 0.001] and a significant interaction of Trial x Dose [F(3, 59) = 67.888, p < 0.001]. There was no significant main effect of Sex [F(1, 59) = 2.987, p = 0.089] or significant interaction of Sex x Dose [F(3, 59) = 0.146, p = 0.932], Trial x Sex [F(1, 59) = 1.515, p = 0.223] or Trial x Dose x Sex [F(3, 59) = 0.508, p = 0.679]. Although there was no significant Trial x Dose x Sex interaction, separate analyses were run for males and for females to determine significant effects for each sex. The significance patterns for each sex were then compared to explore if such patterns were similar or different for males and females (for a description of the use of similar procedures for comparing males and females, see Miller et al., 2017; Rich-Edwards et al., 2018). For males, a 4 × 4 mixed model ANOVA on saccharin consumption revealed a significant main effect of Trial [F(3, 193) = 38.517, p < 0.001] and Dose [F(3,31) = 66.714, p < 0.001] and a significant Trial × Dose interaction [F(9, 93) = 29.488, p < 0.001]. For females, a 4 × 4 mixed model ANOVA on saccharin consumption showed a significant main effect of Trial [F(3, 84) = 35.123, p < 0.001] and Dose [F (3,28) = 32.551, p < 0.001] as well as a significant interaction of Trial × Dose [F(9, 84) = 17.070, p < 0.001].

Both males and females injected with methylone displayed significant dose-dependent conditioned taste avoidance (see Fig. 1), although the patterns in the acquisition of this avoidance differed between males and females. For example, males injected with methylone acquired the taste avoidance by Trial 2, whereas females took until Trial 3 (indexed by significantly less saccharin consumption compared to vehicle controls). On Trial 3, male and female animals displayed similar patterns with the 10 and 18 mg/kg dose groups drinking significantly less saccharin than the 5.6 mg/kg group. For males, this same pattern was observed on Trial 4, but females in the 5.6 mg/kg group only differed from animals injected with 10 mg/kg. On the final two-bottle test, males and females showed similar patterns of avoidance whereby all doses produced near complete suppression of saccharin consumption (data not shown).

Fig. 1.

Mean (+/− SEM) saccharin consumption (ml) on Trials 1–4 for male and female subjects injected with vehicle or 5.6, 10 or 18 mg/kg methylone. *significantly different from 5.6, 10 and 18 mg/kg; ^@only significantly different from 10 and 18 mg/kg; ^#only significantly different from 10 mg/kg; ⁺only significantly different from 18 mg/kg.

3.2. Temperature

The 2 × 4 × 10 mixed model ANOVA on methylone-induced temperature changes in male and female rats revealed a significant main effect of TimePoint [F(9, 522) = 2.559, p = 0.007], Dose [F(3, 58) = 3.763, p = 0.015] and Sex [F(1, 58) = 19.753, p < 0.001] as well as a significant interaction of TimePoint x Sex [F(9, 522) = 2.434, p = 0.010]. There was no significant interaction of TimePoint x Dose [F(27, 522) = 1.293, p = 0.150], Sex x Dose [F(3, 58) = 1.848, p = 0.148] or Trial x Dose x Sex [F(27, 522) = 1.173, p = 0.252]. As above, although a significant Trial x Dose x Sex interaction was not found, males and females were analyzed separately for assessments of differences in the pattern of temperature changes. For males, a 4 × 10 mixed model ANOVA on temperature demonstrated a significant main effect of Time [F(9, 279) = 35.648, p < 0.000] and Dose [F(3, 31) = 6.943, p < 0.001] as well as a significant interaction of Time x Dose [F(27, 279) = 4.216, p < 0.001]. For females, a 4 × 10 mixed model ANOVA revealed a significant main effect of Time [F(9, 234) = 12.454, p < 0.001] and a significant interaction of Time x Dose [F(9, 234) = 8.591, p < 0.001], but no main effect of Dose [F(3, 26) = 1.155, p = 0.346].

Both males and females injected with methylone displayed dose- and time-dependent changes in body temperature (see Fig. 2). Within 30 min post-injection, male subjects injected with 5.6 and 10 mg/kg displayed significantly lower body temperatures relative to controls and those injected with 18 mg/kg of methylone. At 30 min post-injection, female subjects injected with 10 mg/kg of methylone had a significantly lower temperature compared to controls, and at 60-min post-injection, those injected with 18 mg/kg of methylone had significantly lower body temperatures than subjects given vehicle or 5.6 mg/kg. Within 3 h post-injection, males injected with 10 mg/kg of methylone displayed higher body temperature than saline-treated animals, although for the remainder of testing they did not differ from controls. Furthermore, males given 18 mg/kg methylone displayed a significantly higher body temperature than those injected with saline or 5.6 and 10 mg/kg that was maintained for 4 additional hours. By 3 and 4 h post injection, females injected with 10 mg/kg displayed significantly higher body temperature compared to vehicle animals (subjects injected with 18 mg/kg had elevated temperatures within 4 h). Within 5 h post injection, females injected with 10 mg/kg of methylone had significantly higher body temperatures compared to those injected with 5.6 mg/kg, while animals given 18 mg/kg showed significantly higher body temperature at 6 and 7 h post injection compared to those given 5.6 mg/kg. By the 8th hour time period, there were no significant differences among any groups for males or females.

Fig. 2.

Mean (+/−SEM) temperature in °C for male and female subjects injected with vehicle or 5.6, 10 or 18 mg/kg methylone at pre-injection and 30 min, 1 h and every hour up until 8 h post-injection. ^{^}only significantly different from 5.6 and 10 mg/kg; ^&only significantly different from vehicle and 5.6 mg/kg; ^Xonly significantly different from vehicle; ^$only significantly different from vehicle, 5.6 and 10 mg/kg; ^#only significantly different from 10 mg/kg; ^@only significantly different from 10 and 18 mg/kg; ⁺only significantly different from 18 mg/kg.

3.3. Activity and stereotypies

3.3.1. Activity

The 2 × 4 × 12 mixed model ANOVA on methylone-induced activity in male and female rats revealed a significant main effect of TimePoint [F(11, 616) = 68.042, p < 0.001] and Dose [F(3, 56) = 72.231, p <0.001] as well as a significant interaction of TimePoint x Dose [F(33, 616) = 8.303, p < 0.001] and Trial x Dose x Sex [F(33, 616) = 1.518, p = 0.033]. There was no significant main effect of Sex [F(1, 56) = 2.148, p = 0.148]. Further, there was no significant TimePoint x Sex [F(11, 616) = 1.653, p = 0.080] or Sex x Dose [F(3, 56) = 0.311, p = 0.817] interactions. As above, males and females were analyzed separately for patterns in methylone-induced activity (Miller et al., 2017; Rich-Edwards et al., 2018). For males, a 4 × 12 mixed model ANOVA on methylone-induced activity revealed a significant main effect of Time [F (11, 319) = 40.560, p < 0.001] and Dose [F(3,29) = 41.300, p < 0.001] as well as a significant interaction of Time x Dose [F(33, 319) = 5.311, p < 0.001]. For females, a 4 × 12 mixed model ANOVA on methylone-induced activity revealed a significant main effect of Time [F(11, 297) = 29.495, p < 0.001] and Dose [F(3,27) = 31.807, p < 0.001] as well as a significant interaction of Time x Dose [F(33, 297) = 4.595, p <0.001].

Both male and female subjects displayed significant methylone-induced activity at all three doses compared to animals injected with vehicle with males having a faster onset and females having a longer duration, which is consistent with the results from the mixed model ANOVA when Sex was considered as a factor (Fig. 3). At the 5-min post-injection time point, male subjects injected with 10 and 18 mg/kg methylone displayed significantly greater activity than those treated with saline, while female subjects displayed no significant differences among groups. At the 10-min time point and for the remainder of the 1-h testing period, male and female animals injected with all three doses of methylone displayed greater activity than the saline-injected controls. Beginning at 45-min post-injection, male subjects injected with 10 and 18 mg/kg of methylone displayed significantly greater activity than those injected with 5.6 mg/kg. This significant difference was maintained for the remainder of the 1-h testing period. At the 60 min time point, females injected with 10 mg/kg had significantly greater activity compared to those injected with 5.6 mg/kg of methylone.

Fig. 3.

Mean (+/−SEM) consecutive beam breaks for male and female subjects injected with vehicle or 5.6, 10 or 18 mg/kg methylone in 5-min intervals for 1 h. ^@only significantly different from 10 and 18 mg/kg; *significantly different from 5.6, 10 and 18 mg/kg; ^{^}only significantly different from 5.6 and 10 mg/kg; ^#only significantly different from 10 mg/kg.

3.3.2. Stereotypies

The 2 × 4 × 12 mixed model ANOVA on methylone-induced stereotypies in male and female rats revealed a significant main effect of TimePoint [F(11, 616) = 8.987, p < 0.001] and Dose [F(3, 56) = 62.279, p < 0.001], as well as a significant interaction of TimePoint x Dose [F (33, 616) = 9.395, p < 0.001]. There was no significant main effect of Sex [F(1, 56) = 1.167, p = 0.285]. Further, there were no significant interactions of TimePoint x Sex [F(11, 616) = 1.327, p = 0.205], Sex x Dose [F(3, 56) = 0.849, p = 0.473] or Trial x Dose x Sex [F(33, 616) = 1.209, p = 0.198]. As above, males and females were analyzed separately for patterns of methylone-induced stereotypies. For males, a 4 × 12 mixed model ANOVA on methylone-induced stereotypies revealed a significant main effect of Time [F(11, 319) = 6.150, p < 0.001] and Dose [F(3, 29) = 43.249, p < 0.001] as well as a significant interaction of Time x Dose [F(33, 319) = 5.454, p < 0.001]. For females, a 4 × 12 mixed model ANOVA on methylone-induced stereotypies revealed a significant main effect of Time [F(11, 297) = 4.192, p < 0.001] and Dose [F(3, 27) = 21.944, p < 0.001] as well as a significant interaction of Time x Dose [F(33, 297) = 5.172, p < 0.001].

Both males and females injected with methylone displayed significant stereotypies at all three doses compared to animals injected with vehicle with males and females showing no consistent differences in their patterns (Fig. 4). At the 15-min time point and for the remainder of testing, both males and females injected with 18 mg/kg methylone displayed significantly more stereotypies than controls. From the 20-min sampling period and for the remainder of testing (with the exception of the 25-min time point), male subjects injected with 5.6 and 10 mg/kg methylone also displayed significantly more stereotypies than subjects injected with saline. This significant difference began at the 35-min time point for females. Male subjects injected with 18 mg/kg methylone displayed significantly more stereotypies compared to those injected with 5.6 mg/kg from 15 to 40 min post-injection and those injected with 10 mg/kg at the 20-, 30-, 35- and 40-min time points. Females injected with 18 mg/kg of methylone showed significantly more stereotypies compared to those injected with 10 mg/kg from 20 to 35 min post-injection and those injected with 5.6 mg/kg at the 15-, 20-, 25- and 35-min time points.

Fig. 4.

Mean (+/− SEM) repeated beam breaks for male and female subjects injected with vehicle or 5,6, 10 or 18 mg/kg methylone in 5-min intervals for 1 h. ^&only significantly different from vehicle and 5.6 mg/kg; ^{^}only significantly different from 5.6 and 10 mg/kg; *significantly different from 5.6, 10 and 18 mg/kg; ^$significantly different from vehicle, 5.6 and 10 mg/kg; ^%only significantly different from vehicle and 10 mg/kg; ^#only significantly different from 10 mg/kg.

4. Discussion

The present study attempted to characterize several aversive effects of methylone, and if any of these effects was dependent upon sex. As described above, methylone induced dose-dependent taste avoidance and dose- and time-dependent changes in temperature and activity/stereotypies (characteristics of methylone overdose in humans). The only effect for which sex was a significant factor was general activity in where males had a faster onset and females had a longer duration.

In relation to taste avoidance conditioning, both males and females displayed significant avoidance of the methylone-associated taste that was dose and trial dependent. There was no significant effect of sex. Interestingly, the patterns in the avoidance did vary. Although work assessing taste avoidance in males and females have reported differences, the direction of these differences is drug dependent (for a review, see Riley et al., 2018). Interestingly, when males and females have been directly compared with MDPV and α-PVP, the effect is also generally weaker in females than males. For example, King et al. (2015) found that 1 and 1.8 mg/kg MDPV induced weaker taste avoidance (as assessed in a combined taste avoidance/place preference procedure) compared to males, although this was only seen on specific trials. Similarly, Nelson et al. (2019b) reported that female rats injected with 3 mg/kg of α-PVP displayed weaker conditioned taste avoidance compared to males, again only on specific trials. Consistent with the data reported by King et al. (2015) and Nelson et al. (2019a, 2019b), differences in methylone-induced taste avoidance between males and females were only evident on a single conditioning trial and only with a single dose (5.6 mg/kg). These similarities are interesting given that methylone’s mechanism of action is quite different from that of MDPV and α-PVP (release facilitator and reuptake inhibitors, respectively), although all three affect brain amines that have been implicated in the aversive effects of other psychostimulants (for a review of cocaine’s aversive effects, see Serafine and Riley, 2013).

Although the underlying mechanisms for sex differences in taste avoidance in general are not known, there has been some work investigating their basis (see Chambers et al., 1997; Dalla and Shors, 2009 for reviews on sexual dimorphism in conditioned taste avoidance). For example, Chambers et al. (1981) reported that gonadectomy differentially affected threshold sensitivity to LiCl whereby it increased the proportion of females, but decreased the proportion of males, that acquired aversions. Similarly, treating gonadectomized males and females with exogenous testosterone increased the proportion of males and females that acquired aversions to that of intact animals, although the effect of ovarian hormones was not examined in this context (Chambers et al., 1981). Moreover, it has been reported that gonadectomy accelerates extinction of LiCl-induced aversions in males but has no effect in females, and treatment with exogenous testosterone increases the duration of extinction in males and females, suggesting that testosterone has an activational role in extinction (Chambers, 1976; Chambers et al., 1981). Other work has demonstrated that ovariectomized females treated with estradiol show faster extinction of the taste avoidance (Chambers, 1976; Yuan and Chambers, 1999a, 1999b). While these studies provide evidence for sexual dimorphism in conditioned taste avoidance, their application to the present results is limited due to the general absence of significant sex differences with methylone. Moreover, no such assessments have been made as to the mediation of avoidance induced by synthetic cathinones and the direction of the reported sex differences in taste avoidance learning in general are drug dependent (for a recent review, see Riley et al., 2018).

In relation to temperature, methylone induced dose-dependent changes in both males and females. Specifically, temperature was significantly lower following methylone (relative to vehicle) at the outset of testing, whereas over the testing period temperature following methylone was significantly greater than in controls. Although sex was not a significant factor in these effects, the assessment of the patterns of changes in temperature varied between males and females. For example, at the outset males injected with 5.6 and 10 mg/kg had significantly lower body temperatures than saline-treated animals, while in females those injected with 10 and 18 mg/kg of methylone had a significantly lower temperature compared to controls in the first hour of testing. As testing continued, males injected with 10 (3 h post injection) and 18 mg/kg (from 4 to 7 h post-injection) methylone displayed a significantly higher body temperature than controls, while female subjects displayed a significant increase above controls only at 10 and 18 mg/kg at a single time point (at 4 h post-injection).

At the outset of the temperature assessment (30 min following the injection), control subjects displayed a higher temperature than several of the methylone-injected groups. This initial temperature reading is likely due to stress associated with handling and injection procedures. Stress-induced increases in body temperature have been shown to occur as a result of various manipulations such as being introduced to a novel environment (Long et al., 1990), experiencing social defeat (Tornatzky and Miczek, 1993; Meerlo et al., 1996), handling (including routine husbandry procedures), body weight collection, being restrained for injections and other lab procedures (see Balcombe et al., 2004 for a review of laboratory procedures that result in stress responses). Although initially displaying elevated temperature, over the remaining sampling periods temperature for the control subjects decreased suggesting that although control animals were still handled and scanned, the subjects were adapting or habituating to these procedures. It is interesting in this context that while controls decreased temperature over the sampling period, for the high dose groups (10 and 18 mg/kg methylone) temperature was higher than that of controls and/or the subjects injected with 5.6 mg/kg indicating a prolonged effect on temperature that was not simply an effect of handling and injection stress alone.

Although assessments of methylone on temperature in both males and females are limited, Javadi-Paydar et al. (2018) reported significant methylone-induced hyperthermia in male Wistar rats but no impact of methylone on temperature in females (for hyperthermic effects assessed only in males, see Kiyatkin et al., 2015; Stefkova et al., 2017; see Piao et al., 2015 for hyperthermia in male mice). Interestingly, Goldsmith et al. (2019) reported that methylone initially produced hyperthermia in both sexes with female rats becoming tolerant to methylone-induced hyperthermia faster with repeated injections than male rats and, in fact, displaying hypothermia after chronic injections. The bases for these differences among these assessments and those of the present results are not known, but they may be a function of a range of parametric differences across studies, including doses examined (e.g., smaller or larger doses than those used in the current study), routes of administration (subcutaneous vs intraperitoneal), duration of assessment (e.g., 1 h testing period vs an 8 h period used here), method of temperature measurement (e.g., radiotelemetry, brain and/or core temperature, subcutaneous temperature probe) and differential drug histories (e.g., prior exposure to one or more drugs prior to temperature assessments).

Similar to the limited work on the effects of sex on methylone-induced thermoregulatory effects, work assessing sex differences in temperature induced by other synthetic cathinones is limited. In one recent assessment, Nelson et al. (2019a, 2019b) reported that α-PVP-increases in temperature were present in both sexes with males showing a faster onset and delayed offset of temperature increases. In the present study, significantly lower body temperatures occurred initially in both sexes with males showing sustained increases in temperature at the 18 mg/kg dose, while females showed significant increases in temperature relative to controls at 10 and 18 mg/kg at only a single time point. The differences in the patterns for males and females between those reported here and those seen with α-PVP under very comparable conditions argues that sex-dependent effects may be drug dependent as well (even within the same class of synthetic cathinones).

The present study assessed methylone’s ability to induce temperature changes and the role sex plays in these effects, but only did so at room temperature (20–24°C). A critical factor in drug-induced temperature changes is ambient temperature, as the impact of ambient temperature has been well demonstrated with other drugs of abuse such as methamphetamine (Brown et al., 2003; Kiyatkin and Sharma, 2009; Kiyatkin, 2010) and MDMA (Fantegrossi et al., 2003; Kiyatkin et al., 2015; Kiyatkin and Ren, 2017). For both methamphetamine and MDMA, warmer ambient temperature potentiates their induced increases in temperature (Fantegrossi et al., 2003; Kiyatkin et al., 2015; Kiyatkin and Ren, 2017). Fantegrossi et al. (2003) also noted that cooler ambient temperature attenuated the temperature effects induced by MDMA. Interestingly, the synthetic cathinone MDPV, whose mechanism is different than that of methamphetamine and MDMA in that it acts as a reuptake inhibitor, has been reported to produce enhanced increases in temperature under warm ambient temperature conditions (Fantegrossi et al., 2012; 2013; Kiyatkin et al., 2015; Kiyatkin and Ren, 2017). Warm ambient temperature has been reported to have an effect on methylone-induced temperature changes as well, although the degree to which this occurs varies (Kiyatkin et al., 2015; Kiyatkin and Ren, 2017). In these assessments, injections were given subcutaneously (a different route of administration) and at a lower dose range than used in the present work. Future studies assessing the differential impact of both cool and warm ambient temperatures on methylone-induced thermoregulation should examine these factors under different routes of administration and doses in order to understand the effects of such factors on methylone’s impact on body temperature.

Similar to conditioned taste avoidance, research elucidating mechanisms for sex differences in drug-induced temperature changes is limited. The few studies that have investigated sex-dependent effects of drug-induced increases in body temperature have attributed these effects to sex differences in vasomotor tone that is linked to the ability to dissipate heat as well as to differences in heat generation through augmented uncoupling protein activity (Mills et al., 2004; Wyeth et al., 2009), two factors important in sympathomimetic-induced hyperthermia (Pederson and Blessing, 2001). These factors appear to be impacted by sex in that: 1) vasomotor tone in human and animal subjects is diminished in females compared to males which is mediated through actions of estrogen on endothelium resulting in greater nitric oxide production and availability; and 2) stress-induced circulating catecholamines are less in females thereby affecting α₁ adrenergic receptor stimulation which maintains vasomotor tone (Wyeth et al., 2009). Wyeth et al. investigated these effects with MDMA, a drug with which methylone shares a similar pharmacological profile and demonstrated that: 1) female subjects have reduced sympathetic activation; 2) female vasculature is less sensitive to α₁-adrenergic stimulation and has increased sensitivity to nitric oxide; and 3) female expression of uncoupling protein activity (UCP3) in skeletal muscle is less than the expression seen in males. Although sex was not a significant factor with methylone’s effects on temperature, the fact that sex differences have been reported with other related compounds suggests that sex may be important variable under specific conditions and that the basis for these effects are important in understanding these differential effects.

All doses of methylone induced significant activity and stereotypies that in the case of activity were sex dependent. To be consistent across all assessments, patterns in methylone-induced activity were assessed separately in males and females as well. Comparing the patterns of activity in male and female subjects revealed differences between the sexes in methylone-induced activity. For example, within 5-min post-injection males injected with 10 and 18 mg/kg methylone displayed significantly greater activity than those treated with saline, while female subjects displayed no significant differences among groups at the outset. Although methylone at all doses elevated activity relative to controls in both males and females, this effect seemed greater in females. For example, by 45-min post injection, males injected with 5.6 mg/kg of methylone displayed decreased activity relative to males in the 10 and 18 mg/kg dose groups. This was not evident until 60-min post injection for females. In the current assessment, the patterns of stereotypies in males and females were comparable.

As with temperature, there are only few assessments of sex differences in activity induced by methylone or other synthetic cathinones. For example, Javadi-Paydar et al. (2018) reported comparable increases in activity in males and females following methylone administration (see also Daniel and Hughes, 2016). In relation to other synthetic cathinones, Javadi-Paydar et al. (2018) reported no differences following injections of pentylone or pentedrone (see also Daniel and Hughes, 2016; see Alsufyani and Docherty, 2017 for a similar report with cathinone), while Nelson et al. (2019a, 2019b) reported that females are more sensitive to α-PVP’s excitatory effects on activity. The basis for these differences in reactivity to the synthetic cathinones between males and females is not known, although as with temperature assessments, a range of parametric differences exists among these studies.

While specific sexual dimorphisms in activity have not been extensively investigated, several underlying mechanisms have been proposed. For instance, female rats show greater psychostimulant-induced loco-motor stimulation and robust sensitization following repeated exposure compared to males (Robinson, 1984; Camp and Robinson, 1988; Glick and Hinds, 1988; Caihol and Mormède, 1999), an effect only seen in adult animals which suggests that hormones secreted in adulthood may be responsible for locomotor responsivity to psychostimulants (Ujike et al., 1995). Further, gonadectomy in males has a limited effect on acute sensitivity to psychostimulants, whereas ovariectomy of females attenuates amphetamine- (Savageau and Beatty, 1981; Camp et al., 1986) and cocaine-induced locomotor activity (Haney et al., 1994). Estradiol treatment restores behavioral reactivity (Castner et al., 1993) and enhances behavioral sensitization to cocaine (Perris et al., 1991). Again, this work has not been explored with the synthetic cathinones, but given the role of gonadectomy, exogenous hormone treatment and repeated exposure in psychostimulant-induced activity, it is important to investigate these mechanisms with the synthetic cathinones, including methylone.

In the present study, we examined methylone’s aversive effects using conditioned taste avoidance, a well-documented tool for assessing the aversive effects of many toxins and drugs of abuse (Stolerman and D’Mello, 1981; Verendeev and Riley, 2011, 2012), as well as assessed changes in body temperature and activity, effects observed in humans following exposure to traditional psychostimulants and the synthetic cathinones (for a review, see Mash et al., 2009; see also Penders et al., 2012; Bonson et al., 2019). One issue with these indices is whether they are related, e.g., is increased temperature and taste avoidance a function of excess activity. Although it is difficult to examine such relationships directly given that the temporal assessments of these effects differed, all doses of methylone induced conditioned taste avoidance and increased activity and stereotypies in both males and females, suggesting a possible relationship between these indices (although the effects in these assessments displayed different dose-response functions). Interestingly, only 10 and 18 mg/kg of methylone induced increases in temperature. Further, while increased activity was evident immediately following injections, elevations in temperature were not observed until 2 h post-injection (with decreased temperature evident at the outset of the injection). At this point, it is not clear if these measures are causally related or are independent.

Another issue to consider is the potential impact of prior methylone exposure on the temperature and activity assessments conducted in the present study. As noted above, Goldsmith et al. (2019) have reported that methylone-induced temperature changes are altered by repeated exposure. Its relation to the present results, however, is not clear given the different parameters under which the two studies were done (see above). To limit any potential impact of methylone history in the present study, an 18- to 30-day washout period was selected as well as the randomization of the subjects into new groups for each assay. Methylone’s peak concentration is reached 30 min to 1 h after oral or intravenous injections of methylone (López-Arnau et al., 2013) and after 15 to 45 min following subcutaneous injections (Elmore et al., 2017). The elimination half-life of methylone has been reported to be 60 to 90 min (López-Arnau et al., 2013; Elmore et al., 2017). Given these temporal characteristics of methylone’s pharmacokinetics, it is unlikely that our results were affected by residual methylone, although the effect of its prior exposure to the present results is not known.

5. Conclusions

The present study examined the aversive effects of methylone and the role of sex in these effects. Methylone-induced significant dose and time (or trial) effects with taste avoidance, temperature, activity and stereotypies. The only effect for which sex was a significant factor was activity. Although sex was not a significant factor for conditioned taste avoidance and changes in temperature, subsequent comparisons of the patterns of these two endpoints (but not of stereotypies) revealed that males and females differed. While sex as a factor was the focus of the present assessment, sex is just one factor to consider in examinations of the characteristics of drug effects. Other factors, e.g., strain, age, drug history, can also impact the aversive effects of drugs and understanding these factors may be important in predicting the vulnerability to and consequences of their use.