Contribution of serotonin and dopamine to changes in core body temperature and locomotor activity in rats following repeated administration of mephedrone

The psychoactive effects of mephedrone are commonly compared with those of 3,4‐methylenedioxymethamphetamine, but because of a shorter duration of action, users often employ repeated administration to maintain its psychoactive effects. This study examined the effects of repeated mephedrone administration on locomotor activity, body temperature and striatal dopamine and 5‐hydroxytryptamine (5‐HT) levels and the role of dopaminergic and serotonergic neurons in these responses. Adult male Lister hooded rats received three injections of vehicle (1 ml/kg, i.p.) or mephedrone HCl (10 mg/kg) at 2 h intervals for radiotelemetry (temperature and activity) or microdialysis (dopamine and 5‐HT) measurements. Intracerebroventricular pre‐treatment (21 to 28 days earlier) with 5,7‐dihydroxytryptamine (150 µg) or 6‐hydroxydopamine (300 µg) was used to examine the impact of 5‐HT or dopamine depletion on mephedrone‐induced changes in temperature and activity. A final study examined the influence of i.p. pre‐treatment (−30 min) with the 5‐HT1A receptor antagonist WAY‐100635 (0.5 mg/kg), 5‐HT1B receptor antagonist GR 127935 (3 mg/kg) or the 5‐HT7 receptor antagonist SB‐258719 (10 mg/kg) on mephedrone‐induced changes in locomotor activity and rectal temperature. Mephedrone caused rapid‐onset hyperactivity, hypothermia (attenuated on repeat dosing) and increased striatal dopamine and 5‐HT release following each injection. Mephedrone‐induced hyperactivity was attenuated by 5‐HT depletion and 5‐HT1B receptor antagonism, whereas the hypothermia was completely abolished by 5‐HT depletion and lessened by 5‐HT1A receptor antagonism. These findings suggest that stimulation of central 5‐HT release and/or inhibition of 5‐HT reuptake play a pivotal role in both the hyperlocomotor and hypothermic effects of mephedrone, which are mediated in part via 5‐HT1B and 5‐HT1A receptors.


INTRODUCTION
The synthetic cathinone derivative 4-methylmethcathinone (mephedrone) was first synthesized in 1929 and became popular amongst recreational users at the beginning of the 21st century as a legal high (Green et al. 2014). Although mephedrone has been implicated in a number of deaths and became illegal in Europe and the United States between 2010 and 2012 Gershman & Fass 2012), it remains widely available for illicit use (Kelly et al. 2013;Yamamoto et al. 2013;Elliott & Evans 2014), and users report similar psychoactive effects to 3,4-methylenedioxymethamphetamine (MDMA). Mephedrone is a high-affinity substrate for the monoamine reuptake transporters for dopamine, noradrenaline and 5-hydroxytryptamine (5-HT). Once transported into the cell, mephedrone stimulates neurotransmitter release and disrupts vesicular storage by interaction with the vesicular monoamine transporter and can also stimulate non-exocytotic release by reversing the monoamine transporter flux (Simmler et al. 2013). Consistent with this, systemic mephedrone administration to freely moving rats elevates extracellular levels of dopamine, and to a greater extent 5-HT, in the nucleus accumbens (Kehr et al. 2011;Baumann et al. 2012;Wright et al. 2012).
Multiple re-dosing is common with mephedrone users attempting to maintain the desired effects of this shortacting drug, and while a typical recreational dose is often between 100-200 mg, individuals may re-dose and ingest up to 4 g in a single session (Schifano et al. 2011;Winstock et al. 2011). Most studies show the acute effect of a single injection, or self-administration of mephedrone in the rat is hypothermia (Aarde et al. 2013;Miller et al. 2013;Shortall et al. 2013a), but hyperthermia has also been reported following rapid repeated dosing (Hadlock et al. 2011;Baumann et al. 2012). Given the established association of hyperthermia with life-threatening adverse effects of MDMA (Docherty & Green 2010), it is essential to see if there might be a similar adverse risk with repeated mephedrone. The current study therefore examined the temporal profile of the temperature and locomotor response to short-term repeated mephedrone and established the involvement of serotonergic and dopaminergic neurons in these changes because of their known role in the effects of MDMA.
In the current study, rats received three intraperitoneal (i.p.) injections of mephedrone (10 mg/kg) at 2 h intervals. Previous calculations suggest that this dose and route of mephedrone administration would produce similar plasma exposure to that occurring in many recreational users (Green et al. 2014). However, as pharmacokinetic studies of mephedrone have not been performed in man and there is wide variation in use of single or repeated recreational dose schedules, firm conclusions of the translatable accuracy of this dose cannot be made. Importantly, 10 mg/kg i.p. produces robust but sub-maximal physiological and behavioural changes in the rat (Wright et al. 2012;Shortall et al. 2013a;Shortall et al. 2013b), thereby enabling detection of either enhanced or attenuated temperature and locomotor effects following repeated injection (Green et al. 2014). In the current study, all experiments were performed at ambient temperature as Wright et al. (2012) observed that mephedrone produced a comparable hypothermia and increase in locomotor activity when recorded at normal (23°C) and elevated (27°C) ambient room temperature in Wistar rats.
The current repeat dosing studies used continuous radiotelemetry to accurately and repeatedly record locomotor activity and core body temperature over a prolonged period in the same animal, without repeated insertion of a rectal probe, which would confound assessment of activity, at a consistent dose interval to previous preclinical studies using MDMA or mephedrone (Baumann et al. 2008;Rodsiri et al. 2011;Baumann et al. 2012).
Because mephedrone causes hyperlocomotion (Shortall et al. 2013b) and the striatum plays a role in motor activity (Schultz 2000), extracellular dopamine and 5-HT efflux from this region were measured by in vivo microdialysis to examine whether neurotransmitter release correlated with the behavioural effects.
Previous pharmacological studies suggest the involvement of dopamine in mephedrone-induced hypothermia (Shortall et al. 2013a), so we further examined the contribution of serotonergic and dopaminergic neurons to the behavioural effects of mephedrone. Intracerebroventricular (i.c.v.) pre-treatment with selective neurotoxins (5,7-dihydroxytryptamine (5,7-DHT) and 6-hydroxytryamine, 6-OHDA, respectively) was used to determine the impact of 5-HT or dopamine depletion on the thermoregulatory and locomotor stimulant effects of repeated mephedrone measured using radiotelemetry. After identifying a role of 5-HT in mephedrone-induced hyperactivity and hypothermia, a final acute study investigated the involvement of specific 5-HT receptors by assessing the impact of selective 5-HT 1A , 5-HT 1B or 5-HT 7 receptor antagonists on acute mephedrone-induced hyperlocomotion or hypothermia. These receptors were chosen because of their known role in locomotion and/or thermoregulation in the rat and to permit comparisons with the published effects of MDMA. Radiotelemetry was not used in these studies in accordance with the three Rs principle that invasive surgical implantation was unnecessary for acute measurement. This is the first study to concomitantly examine the effects of repeated mephedrone on hyperactivity, hypothermia and striatal dopamine efflux in short time periods (to provide a good temporal resolution) and establish the differential role of dopamine and 5-HT in mephedroneinduced hyperactivity and hyperthermia for comparison with the established effects of repeated MDMA injection.

Animals
Experimentally naïve young adult male Lister hooded rats (190-300 g; Charles River UK) were used in all experiments. Rats were housed in groups of four prior to surgery and in individual cages post-surgery, under constant housing conditions (12 hours light : dark cycle with lights on at 07.00 hours, ambient temperature 21 ± 2°C and relative humidity 55 ± 10%). Food and water were freely available, and wet mash was provided for 5 days post-surgery.
The drug doses and behavioural schedule used were chosen to comply with the three Rs of humane animal testing. All experiments were conducted in accordance with the Animals (Scientific Procedures) Act, 1986 and Animal Research: Reporting of In Vivo Experiments guidelines with approval of University of Nottingham Local Ethical Committee.

Radiotelemetry
Radiotelemetry was conducted as previously described (Rodsiri et al. 2011). Sterile radio-transmitters (Model TA 10TA-F20, DataScience International, St. Paul, MN, USA) were surgically implanted into the peritoneal cavity under isoflurane anaesthesia. Post-operative analgesia was administered for 3 days (Rimadyl (carprofen), Pfizer; 4 mg/kg, subcutaneous), and rats were allowed to recover for 9 days, then transferred to the procedure room 24 hours prior to testing to habituate. During testing, core body temperature and activity were continuously monitored in the home cage at ambient room temperature (19.9-20.9°C), using receivers (RPC-1) and A.R.T. v4 acquisition software (DataScience International, St. Paul, MA, USA). Rats (n = 5 per treatment group) received three i.p. injections of either saline vehicle (1 ml/kg) or mephedrone (10 mg/kg) at 2-hour intervals. Data were collected for 10 seconds every 2 minutes starting 60 minutes prior to the first injection, grouped into 20-minute epochs and expressed as mean ± standard error of the mean (SEM) activity counts. The change in body temperature (°C) from the baseline reading at 0 minute immediately prior to drug injection and the total cumulative activity counts in the 120 minutes following each injection were calculated and presented as mean ± SEM.

Microdialysis
As our Animals (Scientific Procedures) Act, 1986 project licence did not permit radiotelemetry and microdialysis to be performed in the same animal, microdialysis was measured in a separate cohort of rats using an identical protocol as previously described (Rodsiri et al. 2011). A CMA12 polyurethane guide cannula (CMA Microdialysis AB, Kista, Sweden) was implanted above the striatum using stereotaxic coordinates anterior-posterior +0.48, medial-lateral ±3.0, dorsal-ventral À3.6 from Bregma (Paxinos & Watson 1997) under isoflurane anaesthesia. Seven days post-surgery, rats were briefly anaesthetized (isoflurane/O 2 /N 2 O) to insert a microdialysis probe (CMA12, 4 mm polyarylethersulpone membrane, 500 μm outer diameter, 3 μl internal volume with a 20 kDa molecular cut-off; CMA Microdialysis AB) and each rat then placed in a circular arena (50 cm diameter, 45 cm height) with sawdust bedding and food and water freely available. The probe was connected to a microinfusion pump (Harvard Apparatus, Holliston, MA, USA) using FEP tubing (Instech Laboratories Inc, Plymouth Meeting, PA, USA) via a liquid swivel (Instech 375/22, Instech Laboratories Inc) to allow unrestricted movement and perfusion with artificial cerebrospinal fluid (125 mM NaCl, 13.5 mM NaHCO 3 , 1.25 mM KCl, 0.22 mM NaH 2 PO 4 , 0.9 mM Na 2 HPO 4 , 0.3 mM Na 2 SO 4 , 0.5 mM MgCl 2 , 0.5 mM CaCl 2 2H 2 O, pH 7.4) at 1 μl/min. The following day, rats (n = 10 per treatment group) received three injections at 2-hour intervals of saline vehicle (1 ml/kg) or mephedrone (10 mg/kg i.p.), and samples were collected every 20 minutes into microtubes containing 5 μl of 0.1 M perchloric acid with 0.03% sodium metabisulfite. Samples were immediately stored on dry ice and then at À80°C until analysis by high performance liquid chromatography-electrochemical detection. After a collection of the final microdialysis sample, rats were euthanized with pentobarbital. Brains were rapidly removed and stored in 4% paraformaldehyde until sectioned (150-μm coronal slices) using a vibrotome (Campden Instruments Ltd, Loughborough, UK). Location of the probe in the striatum was confirmed under a light microscope using Paxinos & Watson (1997).

Dopamine and 5-hydroxytryptamine depletion
In a third group of rats, bilateral i.c.v. injection of a monoamine neurotoxin (5,7-DHT or 6-OHDA) was performed under isoflurane anaesthesia as previously described (King et al. 2009). All rats received desipramine (15 mg/kg, i.p., 30-minute pre-treatment) to protect noradrenergic neurons prior to 5 μl of 0.2% w/v ascorbic acid vehicle, 75 μg/5 μl of 5,7-DHT or 150 μg/5 μl of 6-OHDA into each lateral ventricle (anterior-posterior À0.8, medial-lateral ±1.5, dorsal-ventral À3.8 from Bregma (Paxinos & Watson 1997) at a rate of 5 μl/minute. These doses were chosen as they reportedly produce a similar degree of depletion ( Twenty-one days post-surgery, each rat (n = 8 per treatment group) received three injections of saline vehicle (1 ml/kg) or mephedrone (10 mg/kg i.p.) at 2-hour intervals, with radiotelemetry measurements as described previously. Using a cross-over design, rats received the opposite treatment during repeat monitoring 28 days post-surgery to minimize inter-individual responses to drug treatment or the lesion.

Neurochemical detection by high performance liquid chromatography-electrochemical detection
Seven days after radiotelemetry recording (35 days after i.c.v. injection), rats were killed by concussion followed by immediate decapitation, and the hypothalamus, right striatum, frontal cortex and hippocampus were collected on a refrigerated table (4°C), flash frozen in liquid nitrogen and stored at À80°C until analysis of dopamine, 5-HT and their major metabolites by high performance liquid chromatography-electrochemical detection, as previously described (Shortall et al. 2013b). Samples were thawed, weighed and sonicated for 30 seconds in 800 μl 0.05 M perchloric acid containing 1 μM sodium metabisulfite, centrifuged (17 400xg, 4°C for 20 minutes; Harrier 18/80: MSE Scientific Instruments, London, UK), and the supernatant filtered (0.45 μM syringe tip filter, Kinesis Ltd, Saint Neots, UK). Monoamines were separated using a Targa C18 3μm column (100 × 2.1 mm; Phenomenex, Cheshire, UK) and detected using an Antec VT-03 cell with a glassy carbon 2 mm working electrode set to +0.59 V with an in situ Ag/AgCl reference electrode. This system was also used to quantify extracellular dopamine and 5-HT in microdialysis samples. In addition, noradrenaline levels were measured in the same regions in 5,7-DHT and 6-OHDA pre-treated rats using a modified HPLC protocol (a mobile phase of 20 mM KH 2 PO 4 /Na acetate, 8 mM KCl, 0.1 mM EDTA, 1 mM OSA, containing 10% methanol, pH 4.07).

Locomotor activity
Rats were placed in individual Perspex arenas and allowed to habituate for 60 minutes prior to the first injection. LMA was continuously recorded (in 5-minute time bins) for 30 minutes after the first and 60 minutes after the second injection using a Photobeam Activity System (San Diego Instruments, San Diego, CA, USA) to record ambulation and rears.

Rectal temperature
In acute drug studies, rats were placed in individual Perspex arenas and basal temperature measured 40 minutes prior to the first injection to allow habituation to the recording procedure, which involved insertion of a rectal probe (Portec Instrumentation, Bedfordshire, UK) to a depth of 6.5 cm for approximately 20 seconds. Rectal temperature was measured immediately prior to each injection and then at 20-minute intervals for the next 2 hours.

Statistical analysis
Analyses were performed using GraphPad Prism v6.02 or SPSS v21 software. Radiotelemetry data were analysed by two-way repeated measures analysis of variance (ANOVA, with drug treatment and time as between and within factors, respectively) where rats received vehicle or mephedrone alone, or four-way repeated measures ANOVA (applied separately to 5,7-DHT and 6-OHDA groups, with i.c.v. injection and drug as between factors and time and week as within factors) where they also received i.c.v. injections. Dopamine microdialysis data were analysed by twoway repeated measures ANOVA (with drug treatment and time as between and within factors, respectively). 5-HT microdialysis data were analysed by one sample t-test against the pre-injection basal value as vehicle values fell below the limit of detection after 40 minutes. HPLC data were analysed by one-way ANOVA where rats received vehicle or mephedrone alone, or two-way ANOVA where they also received i.c.v. injections. Acute LMA and rectal temperature data were analysed by three-way repeated measures ANOVA (with 5-HT receptor antagonist pre-treatment and mephedrone treatment as between factors and time as the within factor). Total cumulative activity counts were analysed by two-way ANOVA (with pre-treatment and treatment as between factors). Bonferroni multiple comparisons post hoc test was used where appropriate, and P < 0.05 was considered statistically significant. All data are presented as mean ± SEM.

Locomotor activity
Mephedrone increased activity above vehicle control levels for 40 minutes after the first injection and 80 minutes after the second and third injections, such that there was a drug × time interaction (F (18,144) = 3.43, P < 0.001, Fig. 1a). The response to vehicle appeared to diminish with each consecutive administration, whereas the magnitude of the mephedrone-induced increase was similar after each injection.
Analysis of total cumulative activity in the 2 hours following each injection confirmed that mephedrone caused a reproducible hyperactivity on each occasion, with no significant difference between injections (First: 580 ± 56; Second: 567 ± 98; Third: 416 ± 115 counts/2 hours). The peak response (increase compared with each pre-injection value) was also similar (7.2 ± 2.2; 8.2 ± 3.3; 7.9 ± 2.7). However, in vehicle-treated rats, the total decreased from 197 ± 105 following the first to 61 ± 15 after the third administration (P < 0.05), suggesting some habituation to injection, which was not observed following mephedrone. This was reflected by a drug × injection number interaction (F (2,16) = 3.87, P < 0.05), which was attributed to the change in the vehicle rather than mephedrone response.

Core body temperature
There were no between-group differences in temperature (recorded simultaneously with locomotor activity) in the 60 minutes prior to injection (data not shown), with baseline values (at the time of the first injection) being 37.8 ± 0.2°C in rats due to receive vehicle and 37.9 ± 0.1°C in those due to receive mephedrone. Following injection there was a drug x time interaction: (F (18,144) = 4.26, P < 0.001, Fig. 1b), and although mephedrone decreased body temperature to a greater extent than vehicle from 40-60 minutes after the first injection only, the maximum temperature change from baseline following each consecutive mephedrone injection was similar, being À1.3°C, À1.4°C and À1.2°C following the first, second and third injections, respectively. However, temperature did not return to baseline between injections, and the magnitude of each further decrease (compared with immediate preinjection values; at T 0 , T 120 and T 240 ) was attenuated (First: À1.3 ± 0.3°C; Second: À0.6 ± 0.3°C; Third: À0.2 ± 0.2°C reaching significance for the last injection; P < 0.05 from the first response) suggesting tolerance occurred.

In vivo striatal dopamine and 5-hydroxytryptamine efflux
In a separate group of rats to those used for radiotelemetry, there were no between-group differences in basal extracellular dopamine levels in the 60 minutes prior to the first injection (7.32 ± 1.65 pmol/ml in rats due to receive vehicle and 5.08 ± 0.85 pmol/ml in those due to receive mephedrone). Following injection, there was a drug × time interaction (F (18,319) = 3.55, P < 0.001, Fig. 1c). Mephedrone rapidly increased extracellular dopamine levels above vehicle control for 40 minutes after the first and third injections and 60 minutes after the second, but dopamine levels returned to near basal between injections. Thus, each injection produced a similar magnitude (First: 298%, Second: 520%, Third: 435% peak change from baseline) and time course of elevation in extracellular striatal dopamine.
Basal extracellular levels of 5-HT were close to the detection limit but equivalent in both groups when measured immediately prior to the first injection (0.295 ± 0.12 and 0.323 ± 0.07 pmol/ml in control and mephedrone groups, respectively). In vehicle-treated rats, post-injection 5-HT levels remained either close to or below the detection limit, and the pre-injection value has been used to calculate the percentage increase (Fig. 1d). The first mephedrone injection failed to elevate extracellular 5-HT, but the two subsequent injections produced statistically significant increases (P < 0.05 to P < 0.01, versus mean baseline 60 minutes after the second [458% peak change from baseline] and 40 minutes after the third injection [351% peak change from baseline]).

Ex vivo monoamine content
There was no significant effect of repeated mephedrone administration on tissue levels of dopamine, 5-HT or their major metabolites in the hypothalamus, striatum, hippocampus or frontal cortex measured 7 days after radiotelemetry recording (data not shown).
Consistent with the previous experiment, total cumulative activity in the 2 hours following each injection confirmed mephedrone-induced hyperactivity, with a drug × injection number interaction (F (2,36) = 5.31, P < 0.01, Table 1). 5,7-DHT completely prevented the response to the first mephedrone injection and attenuated that to the third (P < 0.01 versus sham control mephedrone response). In contrast, 6-OHDA-treated rats continued to exhibit an increase in cumulative activity following each mephedrone injection, which did not differ from the response in mephedrone-treated sham controls.

Core body temperature
Basal core body temperatures prior to the first injection on each test day (recorded simultaneously with locomotor activity in the same sham and lesioned rats) were equivalent, being 37.2 ± 0.2°C and 37.5 ± 0.2°C in sham controls, 36.9 ± 0.2°C and 37.2 ± 0.2°C in 5,7-DHT and 37.5 ± 0.2°C and 37.4 ± 0.2°C in 6-OHDA rats prior to injection of vehicle or mephedrone, respectively. The maximum temperature change from baseline following each mephedrone injection in sham controls (À1.2°C, À1.2°C and À1.0°C following the first, second and third injections, respectively) was equivalent, but in agreement with the first study, the maximum temperature decrease (compared with each pre-injection value) was attenuated following both the second and third (P < 0.05) compared with the first injection (First: À1.2 ± 0.2°C; Second: À0.3 ± 0.1°C; Third: À0.1 ± 0.2°C).

Ex vivo neurochemistry
Dopamine, 5-HT and noradrenaline levels in the hypothalamus, right frontal cortex, hippocampus and striatum were measured 35 days after neurotoxin administration to confirm selective monoamine depletion. As expected, the serotonergic neurotoxin, 5,7-DHT, significantly reduced 5-HT to 46% of control in the frontal cortex (P < 0.001), 13% in the hippocampus (P < 0.01), 42% in the hypothalamus (P < 0.001) and 66% in the striatum, although the latter did not reach significance because of high individual variation (Table 2). In contrast, 6-OHDA reduced dopamine to 52% of control in the striatum (P < 0.001) and 56%, 80% and 86% of control in the frontal cortex, hippocampus and hypothalamus, respectively, although the depletion in these areas was not statistically significant (Table 2). However, the 6-OHDA-induced decrease in striatal dopamine was accompanied by a significant reduction in hippocampal 5-HT (F (2,20) = 7.19, P < 0.01) as well as decreased noradrenaline levels in the hypothalamus and hippocampus (F (2,21) = 9.53, P < 0.001), but noradrenaline levels were unchanged in the other regions examined.

EFFECT OF 5-HT 1 A , 5-HT 1 B AND 5-HT 7 RECEPTOR ANTAGONISTS ON ACUTE MEPHEDRONE-INDUCED HYPERACTIVITY AND DECREASES IN RECTAL TEMPERATURE
In a final study, separate groups of rats were pre-treated i.p. with the 5-HT 1A receptor antagonist, WAY-100635, the 5-HT 1B receptor antagonist, GR 127935, or the 5-HT 7 receptor antagonist, SB-258719, to investigate the role of specific 5-HT receptors in mephedrone-induced hyperactivity and hypothermia.

Locomotor activity
None of the three 5-HT receptor antagonists had any effect on activity counts following their injection (data not shown). The predominant locomotor stimulant effect of mephedrone in vehicle pre-treated rats was a prolonged increase in ambulatory activity (P < 0.05-0.001, accompanied by a smaller increase in fine movement without increased rearing consistent with previous studies by our group (Shortall et al. 2013b) and the current telemetry data. It was briefly attenuated by WAY-100653 (Fig. 3a) at 15-minute post-injection and more substantially attenuated by GR 127935 (Fig. 3b) from 15-35-minute post-mephedrone injection, but completely unaffected by SB-258719 (Fig. 3c). Consistent with the time-course data, total cumulative ambulation in Table 2 Effect of i.c.v. administration of 6-OHDA or 5,7-DHT on brain tissue dopamine, 5-HT and noradrenaline levels 5 weeks postsurgery.

DISCUSSION
This study investigated the effects of repeated mephedrone injection on core body temperature, locomotor activity and striatal dopamine and 5-HT release in the rat and examined the role of dopamine and 5-HTcontaining neurons in mephedrone-induced changes in body temperature and activity. This is one of a few studies to use radiotelemetry to obtain a high temporal resolution of changes appropriate for the short-duration responses (Miller et al. 2012;Wright et al. 2012;Aarde et al. 2013). The main findings were as follows: (1) while hyperactivity and increased extracellular striatal dopamine seen after the first mephedrone injection were similar in magnitude and time course to those following the second and third injections, the hypothermia was attenuated with repeated dosing; (2) extracellular striatal 5-HT overflow was more variable but was enhanced when second and third injections were given when compared with the first response; (3) 6-OHDA did not affect hyperactivity but reduced the duration of the hypothermic response; (4) 5,7-DHT administration and 5-HT 1B receptor antagonism attenuated mephedrone-induced hyperactivity; (5) 5,7-DHT administration completely abolished, and 5-HT 1A receptor antagonism attenuated mephedroneinduced hypothermia. Importantly, some of these observed effects contrast with those reported with MDMA, Comparison of the effect of (a) the 5-HT 1A receptor antagonist WAY-100635, (b) the 5-HT 1B receptor antagonist GR 127935, and (c) the 5-HT 7 receptor antagonist SB-258719 on saline vehicle (1 ml/kg) or mephedrone (10 mg/kg)-induced change in locomotor activity following a single injection in adult male Lister hooded rats (n = 8 per treatment group). Saline vehicle (1 ml/kg), WAY-100635 (0.5 mg/kg), GR 127935 (3 mg/kg) or SB-258719 (10 mg/kg) was injected À30 min, before saline or mephedrone at time = 0 min. All data are presented as mean ± SEM. Line indicates significance at indicated time points. * P < 0.05, *** P < 0.001 vehicle + mephedrone versus vehicle + vehicle; † P < 0.05, † † P < 0.01, † † † P < 0.001 antagonist + mephedrone versus vehicle + vehicle; † P < 0.05, † † P < 0.01, † † † P < 0.001 antagonist + mephedrone versus antagonist + vehicle, ‡ P < 0.05, ‡ ‡ P < 0.05 antagonist + mephedrone versus vehicle + mephedrone, Bonferroni post hoc following three-way repeated measures ANOVA suggesting differing possible adverse effects following recreational use. Mephedrone has a high affinity for rat dopamine and 5-HT transporters as well as the 5-HT 2A and 5-HT 2C receptors, and α 1A and α 2A adrenoceptors Eshleman et al. 2013;Simmler et al. 2013). It increases extracellular dopamine and to an even greater extent 5-HT in the nucleus accumbens (Kehr et al. 2011;Baumann et al. 2012;Wright et al. 2012;Eshleman et al. 2013). The current study, in contrast, suggests that in the striatum, the percentage increase in 5-HT and dopamine is rather similar, at least after the second and third doses.
The repeated dose given in the current study (10 mg/ kg) did not produce any neurotoxic loss of brain regional dopamine or 5-HT measured 7 days post-injection. This is in marked contrast to MDMA, where a repeated dose schedule that releases striatal dopamine also produces significant long-term neurotoxic 5-HT depletion in the rodent (Green et al. 2003), but similar to methcathinone where a much larger dose than that needed to elicit behavioural changes is required to obtain neurotoxicity (Sparago et al. 1996). Although hypothermia protects against MDMA neurotoxicity (Malberg & Seiden 1998), a previous study in which mephedrone produced hyperthermia in the rat also failed to detect any neurotoxic loss of post-mortem brain monoamines two weeks after a repeated dosing schedule similar to that used in the current study (Baumann et al. 2012). These data therefore suggest that rapid repeated mephedrone administration is less likely to produce monoamine neurotoxicity than MDMA.
Mephedrone induces hyperactivity in rodents following both acute and intermittent administration (Kehr et al. 2011;Angoa-Perez et al. 2012;Baumann et al. 2012;Marusich et al. 2012;Wright et al. 2012;Shortall et al. 2013b). Mephedrone (0.5 to 30 mg/kg i.p. or subcutaneous) has consistently been shown to elicit hyperactivity in a variety of rat strains, when given during both the light (Lisek et al. 2012;Gregg et al. 2013;Shortall et al. 2013b) or dark (Motbey et al. 2012;Miller et al. 2013) phase of the circadian cycle. Because significant hyperactivity was found irrespective of circadian phase, the current study was conducted in the light phase to enable comparison with the many studies on MDMA, including our own, which use this protocol. In the current study, repeated 'binge-style' mephedrone administration caused reproducible hyperactivity after each injection, the onset of which occurred within minutes of injection but returned to baseline levels within 1 hour. The time courses for both the striatal dopamine release and the hypothermia are consistent with a previous study using a single systemic injection (Shortall et al. 2013b). It is noteworthy that the peak plasma level of Comparison of the effect of (a) the 5-HT 1A receptor antagonist WAY-100635, (b) the 5-HT 1B receptor antagonist GR 127935 and (c) the 5-HT 7 receptor antagonist SB-258719 on saline vehicle (1 ml/kg) or mephedrone (10 mg/kg) induced change in rectal temperature following a single injection in adult male Lister hooded rats (n = 8 per treatment group). Saline vehicle (1 ml/kg), WAY-100635 (0.5 mg/kg), GR 127935 (3 mg/kg) or SB-258719 (10 mg/ kg) were injected À30 min before saline or mephedrone at time = 0 minute. Rectal temperature was measured at À30-minute and at 20-minute intervals from 0 to 120 minutes, and data are expressed as change in temperature (°C, mean ± SEM) from the reading taken at 0 minute. *** P < 0.001 vehicle + mephedrone versus vehicle + vehicle; † P < 0.05, † † † P < 0.001 antagonist + mephedrone versus vehicle + vehicle; † P < 0.05, † † P < 0.01, † † † P < 0.001 antagonist + mephedrone versus antagonist + vehicle, Bonferroni post hoc following three-way repeated measures ANOVA