Pharmacology, Biochemistry and Behavior 103 (2013) 501509

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Pharmacology, Biochemistry and Behavior

journal homepage: www.elsevier.com/locate/pharmbiochembeh

Long-term cognitive and neurochemical effects of bath salt designer drugs

methylone and mephedrone
Bjrnar den Hollander a,, Stanislav Rozov b, Anni-Maija Linden a, Mikko Uusi-Oukari c,
Ilkka Ojanper d, Esa R. Korpi a
a

Institute of Biomedicine, Pharmacology, Biomedicum Helsinki, Haartmaninkatu 8, FI-00014, University of Helsinki, Finland
Neuroscience Center and Institute of Biomedicine, Anatomy, Biomedicum Helsinki, Haartmaninkatu 8, FI-00014, University of Helsinki, Finland
Institute of Biomedicine, Pharmacology, Drug Development and Therapeutics, FI-20014, University of Turku, Finland
d
Hjelt Institute, Department of Forensic Medicine, Kytsuontie 11, FI-00014, University of Helsinki, Finland
b
c

a r t i c l e

i n f o

Article history:
Received 17 August 2012
Received in revised form 12 October 2012
Accepted 15 October 2012
Available online 22 October 2012
Keywords:
Methylone
Mephedrone
Cathinone
Monoamine
Neurotoxicity
Working memory

a b s t r a c t
Introduction/aims: The use of cathinone-derivative designer drugs methylone and mephedrone has increased
rapidly in recent years. Our aim was to investigate the possible long-term effects of these drugs on a range of
behavioral tests in mice. Further, we investigated the long-term effects of these drugs on brain neurochemistry in both rats and mice.
Methods: We treated animals with a binge-like regimen of methylone or mephedrone (30 mg/kg, twice daily
for 4 days) and, starting 2 weeks later, we performed behavioral tests of memory, anxiety and depression
and measured brain levels of dopamine (DA), serotonin (5-HT), their metabolites and norepinephrine
(NE). 5-HT and DA transporter (5-HTT and DAT) levels were also measured in rats by [3H]paroxetine and
[3H]mazindol binding.
Results: Mephedrone reduced working memory performance in the T-maze spontaneous alternation task but
did not affect neurotransmitter levels aside from a 22% decrease in striatal homovanillic acid (HVA) levels in
mice. Methylone had little effect on behavior or neurotransmitter levels in mice but produced a widespread
depletion of 5-HT and 5-HTT levels in rats.
Conclusions: Both methylone and mephedrone appeared to have a long-term effect on either behavioral or
biochemical gauges of neurotoxicity in rodents.
2012 Elsevier Inc. All rights reserved.

1. Introduction
Recent years have witnessed a rise in the use of a particular type
of stimulant designer-drugs primarily sold to consumers via the internet as bath salts or plant food in order to circumvent laws
banning their sale as products for human consumption (Vardakou
et al., 2011). A large number of different substances have been identied as constituents of bath salts, but among the most prevalent
Abbreviations: 5-HT, serotonin; 5-HTT, serotonin transporter; AMPH, D-amphetamine;
ANOVA, analysis of variance; CCD, charge coupled device; COMT, catechol-Omethyltransferase; DA, dopamine; DAT, dopamine transporter; EDTA, ethylenediaminetetraacetic acid; GFAP, glial brillary acidic protein; HPLC, high performance liquid chromatography; HVA, homovanillic acid; LLOQ, lower limit of
quantication; MAO-A, monoamine oxidase A; MAO-B, monoamine oxidase B;
MDMA, 3,4-methylenedioxymethamphetamine; METH, methamphetamine; NE,
norepinephrine; NET, norepinephrine transporter; SD, standard deviation; SEM,
standard error of the mean; TAAR1, trace amine associated receptor 1; TH, tyrosine
hydroxylase; VMAT-2, vesicular monoamine transporter 2.
Corresponding author at: Institute of Biomedicine, Pharmacology, Haartmaninkatu
8 (Biomedicum Helsinki), 00290 Helsinki, P.O. Box 63, 00014, University of Helsinki,
Finland. Tel.: +358 9 191 25356; fax: +358 9 191 25364.
E-mail address: bjornar.denhollander@helsinki. (B. den Hollander).
0091-3057/$ see front matter 2012 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.pbb.2012.10.006

drugs are mephedrone (4-methylmethcathinone) and methylone

(3,4-methylenedioxymethcathinone). The surge in popularity of
these drugs has led to a recent ban of these substances in both the
US and EU but this has done little to reduce their availability
(McElrath and O'Neill, 2011).
The effects of these substances are described by users as a mix of
AMPH, MDMA and cocaine, with many users rating them as better
than cocaine (A.R. Winstock et al., 2011). Mephedrone appears to
be highly reinforcing and users experience a strong desire to redose,
leading them to ingest large amounts of the drugs in binges that can
last several days (A. Winstock et al., 2011). This pattern of use leads
to a risk of potentially fatal overdoses, and many emergency room
visits as well as some deaths have been attributed to mephedrone
and methylone use (Maskell et al., 2011; Pearson et al., 2012; Wood
et al., 2011). Further, the use of these substances may also be associated with more subtle long-term effects on brain chemistry and cognition. Closer examination of the effects of these drugs is needed in
order to create an evidence-based public health strategy and inform
users of the possible risks associated with these substances.
Methylone and mephedrone show a strong structural (see Fig. 1)
and pharmacological similarity with MDMA and METH, respectively.

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B. den Hollander et al. / Pharmacology, Biochemistry and Behavior 103 (2013) 501509

H
N

Methamphetamine

MDMA

H
N

O
O

H
N

O
Mephedrone

Methylone

Fig. 1. Mephedrone and methylone share a strong structural similarity to METH and
MDMA, respectively.

possible long-term cognitive and neuropsychiatric effects of methylone

and mephedrone.
The present study aims to address some of these shortcomings in the
literature. Specically, our study comprised 2 experiments with the following aims: (1) to investigate the long-term effects of mephedrone
and methylone on mice using behavioral models of memory, anxiety and depression; and (2) to compare the long-term effects of
mephedrone, methylone and AMPH on brain monoamine levels in
mice and rats. Additionally, we assessed the effect of mephedrone
and methylone on 5-HTT and DAT levels in rats. We hypothesized
that mice treated with methylone and mephedrone would show
decreased performance on behavioral tests of memory and show
anxious and depressed behavior compared to mice treated with saline and that, furthermore, methylone and mephedrone would decrease brain monoamine levels compared to saline-treated control
animals and that the affected monoamine systems would differ between mice and rats.
2. Material and methods

In vitro studies in synaptosomes reveal that these drugs are nonselective substrates for the monoamine transporters DAT, 5-HTT and
NET, which lead to blockade of the reuptake and the enhancement
of the release of monoamines by reversing the ow of the transporter,
resulting in elevated synaptic neurotransmitter levels (Hadlock et al.,
2011; Lopez-Arnau et al., 2012; Sogawa et al., 2011).
These ndings are supported by another study which also demonstrated that a range of -keto amphetamines, including mephedrone
and methylone, inhibit the transporters and increase the release of
DA, 5-HT and NE with varying potencies. Unlike the non--keto
amphetamines, however, they exhibited an approximately 10-fold
lower afnity for the TAAR1 (Simmler et al., in press). In vivo studies
employing microdialysis in rats show a dose-dependent 2 to 9-fold increase in DA and 5-HT levels in the nucleus accumbens following
acute challenges with methylone or mephedrone (Baumann et al.,
2012; Kehr et al., 2011).
MDMA, METH and its primary active metabolite AMPH are neurotoxic and cause a long-lasting reduction in striatal and cortical DA
and/or 5-HT levels in rodents (Ali et al., 1994; Battaglia et al., 1987;
Friedman et al., 1998; O'Callaghan and Miller, 1994; O'Shea et al.,
2001; Sabol et al., 2001), while animals as well as human users of
these drugs show long-lasting and widespread decreases in 5-HTT
and DAT binding (Johanson et al., 2006; McCann et al., 1998a,b;
Reneman et al., 2001; Sekine et al., 2001; Semple et al., 1999). Furthermore, there is ample evidence linking the use of these substances with
decreased memory function and increases in neuropsychiatric symptoms like anxiety and depression in animal models as well as humans
(McCardle et al., 2004; McGregor et al., 2003; Moon et al., 2007;
Parrott et al., 2000; Piper and Meyer, 2004; Sprague et al., 2003;
Volkow et al., 2001). The structural and pharmacological similarities between MDMA/METH and methylone/mephedrone suggest the latter
drugs may also produce similar effects on neurochemistry and neuropsychological function.
A few reports on the toxicity of these substances have already been
published, but are not entirely consistent. For instance, Hadlock et al.
(2011) reported decreased hippocampal 5-HT levels one week after
mephedrone (4 10 or 25 mg/kg) in rats whereas Baumann et al.
(2012) found no effect of mephedrone or methylone (both 3 3 or
10 mg/kg) on monoamine levels 2 weeks after treatment. Angoa-Perez
et al. (2012) also found no effect of mephedrone (4 20 or 40 mg/kg)
on striatal DA levels or any other measure of neurotoxicity after one
week in mice. These inconsistencies can be partially due to differences
in the employed dosing-regimen and recovery period. However, there
might also be species differences, as is the case with MDMA, which primarily affects the DA system in mice, but selectively affects the 5-HT system in rats (Logan et al., 1988). Moreover, the focus so far has been on
biochemical measures of toxicity and no attention has been paid to the

2.1. Animals
The C57BL/J6 mice and Wistar rats used in this experiment were
obtained through Nova-Scanbur (Sollentuna, Sweden). All animals
were males and aged 8 weeks at arrival in the lab where they were
randomized and housed in 3 per cage with food pellets (Harlan BV.,
Horst, Netherlands) and tap water available ad libitum at standard
housing conditions (12-h lightdark cycle, lights on at 06:00 h; temperature, 2023 C; relative humidity, 5060%; and aspen chip beddings).
Animals were allowed 2 weeks to habituate to laboratory conditions
prior to the experiments. All animal tests were approved by the Laboratory Animal Committee of the Southern Finland Provincial Government.
2.2. Drugs and dose titration
Racemic mephedrone and methylone were acquired from the Department of Forensic Medicine, Hjelt Institute, University of Helsinki,
and analyzed for purity by gas chromatography and Fourier transform
infrared spectroscopy which determined both compounds were pure
(>95%) hydrochloride salts. D-Amphetamine sulphate was obtained
from GlaxoSmithKline. The drugs were dissolved in a saline solution
(0.9% NaCl) and administered intraperitoneally at a volume of 10 ml/kg
for mice or 1 ml/kg for rats. Control groups were administered equal
amounts of saline.
In pilot experiments in mice we tested doses of methylone and
mephedrone between 15 and 60 mg/kg while observing locomotor
activity and measuring body with a BAT-12 thermometer with rectal
probe (Physitemp Instruments Inc., Clifton, NJ, USA). Both drugs produced hyperlocomotion and slight hyperthermia at 15 mg/kg while
doses of 30 mg/kg produced a consistent 2 C increase in body temperature and more pronounced increases in locomotor activity. Body temperatures were measured every 15 5 min and reached a peak 45
5 min after drug administration. Higher doses of methylone (45 and
60 mg/kg) further increased hyperthermia and caused lethality, with
100% lethality within 1 h at 60 mg/kg. Higher mephedrone doses
(45 and 60 mg/kg) paradoxically caused hypothermia and produced
no overt hyperlocomotion as the lower doses did. Furthermore, no
lethality was observed at doses of up to 60 mg/kg of mephedrone.
Due to the unpredictable and paradoxical effects of higher doses
we opted to use the 30 mg/kg dose in the current experiments.
2.3. Experimental design
For the behavioral study, mice were treated with a binge-like regimen of mephedrone and methylone (30 mg/kg twice daily for 4 consecutive days) and tested on a range of behavioral tasks 28 weeks

B. den Hollander et al. / Pharmacology, Biochemistry and Behavior 103 (2013) 501509

following the nal treatment. For the biochemical study, we treated

rats and mice with the same binge-like regimen of mephedrone or
methylone and for the HPLC analysis experiments we also treated
separate groups of mice and rats with AMPH as a positive control,
as this drug is known to deplete monoamine levels in rodents
(Bowyer et al., 1998; O'Callaghan and Miller, 1994). Core body temperature was recorded on each day of dosing 45 5 min after the
drug treatments in a similar fashion as described above for the pilot
experiments. AMPH was administered at a dose of 30 mg/kg for mice
and 12.5 mg/kg for rats, both twice daily for 4 consecutive days. These
dose levels are sufcient to induce substantial monoamine depletion
based on existing literature (O'Callaghan and Miller, 1994; Wan et al.,
2000). After the drug treatments the animals were allowed 2 weeks to
recover before they were sacriced and brain monoamine levels in the
frontal cortex, striatum and hippocampus were assessed with HPLC. Additionally, 5-HTT levels in the frontal cortex and hippocampus as well as
DAT levels in the striatum were assessed by means of [3H]paroxetineand [3H]mazindol-binding.
2.4. Behavioral experiments
We assessed the effect of the binge-like mephedrone and methylone
treatment on behavioral tests of anxiety (elevated plus-maze), spatial
working memory (T-maze spontaneous alternation), long-term spatial
memory (Morris water maze), and depressive behavior (tail suspension
test) as well as locomotor activity/habituation in novel arena in mice essentially as we have described earlier (Leppa et al., 2011; Linden et al.,
2008; Procaccini et al., 2011). All behavioral tests were performed
blinded to drug treatments, between 2 and 8 weeks after the nal
drug treatment, using the same animals with at least 4 days interval between each test in the order described below. The elevated plus-maze
was performed as the rst test, 2 weeks after the nal drug treatments.
The T-maze was performed after 3 weeks; the locomotor activity/
habituation test after 4 weeks; the Morris water maze after 5 weeks;
and the tail suspension test was performed as the last test, starting
7 weeks after the last drug treatments.
2.4.1. Elevated plus-maze
This test was performed as the rst test, before any other tests. The
apparatus was made of gray plastic and elevated 50 cm from the oor
level. It consisted of a central platform (55 cm), from which two
open arms (540 cm with a 0.7 cm ledge) and two enclosed arms
(54020 cm) extended (Leppa et al., 2011; Lister, 1987). The light intensity was set at 50 lx on the open arms and 20 lx in the closed arms.
The mice were placed individually on the central platform facing an
open arm and allowed free exploration of the maze for 5 min with
their behavior being recorded using a video tracking system with a CCD
video camera above the plus maze. The position and movements of the
center of the animal's surface area were analyzed automatically using
EthoVision software (Noldus Information Technology, Wageningen,
Netherlands). The central area was extended to include the rst
2 cm of each arm. An arm entry was recorded when the center of
the mouse entered the arm from the central part. The plus-maze
was cleaned with a water-moistened paper towel and dried after
each mouse. The mice were returned to their home cage when all
mice from the same cage were tested.
2.4.2. T-maze spontaneous alternation
A T-maze was used to study working memory (Gerlai, 1998; Linden
et al., 2007). The T-maze was made of gray plastic and consisted of three
arms (50 10 cm; walls, 15-cm height). A door separated a 12-cm compartment of the start arm, and two other doors separated 24-cm compartments of the other arms. The mouse was placed in the start
compartment with the door closed for 5 s, after which the door was
opened, and the latency for the mouse to visit the left arm (a door to
the right arm was closed) and to return to the start arm was recorded

503

(test trial). If the test trial took longer than 5 min, the mouse was not accepted for the spontaneous alternation test because of too low exploratory activity. After the test trial, to test spontaneous alternation, the
mouse was rst conned in the start arm for 5 s, and then the door
was opened to allow the mouse to visit either the left or right arm.
After entering either arm, the opposite arm was closed. Every time the
mouse returned to the start arm after entering either arm, it was conned for 5 s in the start arm, after which the doors were again opened
for the mouse to visit either arm. The spontaneous alternation was measured for 15 min. If the animal performed less than ve trials, it was excluded from the study due to low exploratory activity. A video camera
was located above the T-maze, and the experimenter observed the behavior through a video monitor system and remotely operated the
doors from an adjacent room.
2.4.3. Exposure to novel cages
Locomotor activity was analyzed for 2 h in empty plastic rectangular
cages (40 30 20 cm). Horizontal movements of eight mice, placed in
visually isolated cages, were simultaneously recorded using a CCD camera above the cages and analyzed automatically by EthoVision software.
2.4.4. Morris water maze
Spatial learning and memory were assessed with the Morris water
maze test (Linden et al., 2007). Mice were trained during 4 days
(6 trials/day, with a 10-min inter-trial interval) to nd a hidden platform (14 14 cm) submerged 1 cm below the surface of a 120-cm diameter pool of water. Visual markers were placed on the walls of the
experiment room to facilitate spatial learning. The pool was divided
into 4 equal quadrants. The water temperature was 20 2 C and
the water was opacied with milk powder to prevent the mice from
seeing the platform. During training the platform was constantly in
the same quadrant of the maze, and the quadrant in which the mice
were placed varied from trial to trial in a pseudorandom manner.
Trial cut-off time was 3 min and if the mouse did not nd the platform
during a trial, it was gently guided onto it. The mice were towel-dried
after each trial and placed on a heater pad (38 C) for 1 min before
returning them to home cage. During each learning trial the escape latency was recorded. On day 5, a 1-min probe trial was performed in
which the platform was removed and the time the mice spent in the
platform quadrant (Q1) as well as the other quadrants (Q24) was
recorded using EthoVision software. On day 6, reversal learning took
place. The platform was moved to the opposite quadrant (Q3) and the
mice were given 5 trials to learn the new platform location. On day 7
a second probe trial was performed. Finally, on the last day, latencies
for reaching a visible platform placed in novel positions were tested
twice to determine visual capabilities of the mice.
2.4.5. Tail suspension test
In the tail suspension test (Procaccini et al., 2011; Steru et al., 1985),
the mice were attached for 6 min with adhesive tape by their tails to a
vertical metal bar elevated 1520 cm. The behaviors of three visually
isolated mice were recorded simultaneously with a Flip Video camera
(Cisco Systems, Irvine, CA, USA), and individually analyzed off-line by
scoring immobility using data acquisition software (Ethograph software
2.06; RITEC, St Petersburg, Russia). The total duration of immobility and
the latency to the rst immobility were analyzed for the last 4 min, excluding the rst 2 min. The mouse was considered to be immobile
when it only displayed quiet movements of the forepaws and hindpaws
without struggling.
2.5. Biochemical experiments
Two weeks following the nal drug treatment a separate batch of
mice not exposed to behavioral experiments as well as the rats were
killed by decapitation following CO2 anesthesia. The brains were quickly
removed and dissected according to the method of Chiu et al. (2007)

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B. den Hollander et al. / Pharmacology, Biochemistry and Behavior 103 (2013) 501509

into frontal cortex, striatum and hippocampus. After dissection the samples were put in plastic tubes, frozen on dry ice and stored at 80 C
until analysis.

3. Results

2.5.1. Measurement of neurotransmitter levels

For analysis of neurotransmitter levels samples were prepared by
sonication in 10 volumes of 2% perchloric acid, centrifuged for 30 min
at 15,000 g after which 10 l of ltered supernatant was injected
into HPLC system equipped with a Waters Concorde electrochemical
detector set to a potential +0.80 V, column oven and a column Gemini
C18 5 m 1504.60 mm (Phenomenex, Torrance, CA, USA). The mobile
phase consisted of puried water with 8% methanol, 50 mM citric acid,
1.5 mM 1-octanesulfonic acid, 0.05 mM EDTA and 50 mM phosphoric
acid. Column temperature was set at 37 C and a ow rate of 1 ml/min.
System control, data acquisition and analysis were performed using Waters Empower software (Waters, Milford, MA). No internal standard was
used. Concentrations of the neurotransmitters and metabolites were calculated from standard curves which were linear from 10 nM to 1 M.
LLOQ was 10 nM or 0.1 pmol/g of wet tissue. The actual concentration
of the chemicals in wet tissue was calculated by multiplying the value
from the prepared sample by 10. The retention times for NE, DOPAC,
DA, 5-HIAA, HVA and 5-HT were 3.65, 5.47, 7.45, 9.16, 12.11 and
19.04 min, respectively. The samples were standardized based on their
wet weight.

In mice, body temperature was signicantly affected by acute drug

treatments (F3, 20 = 30.56, p b 0.0005). Post-hoc comparison indicated
that mephedrone, methylone and AMPH all produced an increase in
body temperature compared to saline-treated mice (Fig. 2A). Also in
rats a signicant effect of treatment on body temperature was found
(F3, 21 = 6.62, p b 0.01). Post-hoc comparison showed that methylone
and AMPH, but not mephedrone, caused a signicant rise in body
temperature compared to saline-treated rats (Fig. 2B). An ANOVA
revealed no effect of treatment day on body temperature in mice
(F3, 92 = 0.07, p > 0.05) or rats (F3, 96 = 0.14, p > 0.05), indicating that
there was no tolerance or sensitization to the hyperthermic response.

2.6. Statistics
All data from body temperature measurements, transporterbinding, HPLC analysis and most behavioral experiments were tested
for normality and homogeneity of variances using the Kolmogorov
Smirnov and Levene's test and analyzed using a one-way ANOVA with
Fisher's LSD test for post-hoc comparisons or KruskalWallis one-way
ANOVA with post-hoc pairwise comparisons where appropriate. The
data from the Morris water maze and locomotor activity tests were analyzed using a repeated measures ANOVA with GreenhouseGeisser
correction. All data were analyzed using SPSS Statistics Package v. 20
(IBM, Armonk, NY, USA) and Prism v. 5.00 (GraphPad Software, Inc.,
La Jolla, CA, USA) and presented as mean SEM.

3.2. Behavioral experiments

Results from all behavioral experiments are shown in Table 1 and
Figs. 3 and 4.
3.2.1. Elevated plus maze
An ANOVA revealed no signicant effect of treatment on the total
time spent in open arms (F2, 27 = 0.21, p > 0.05), the total number of
entries into open arms (F2, 27 = 1.06, p > 0.05), the latency to the
rst entry into an open arm (F2, 27 = 0.23, p > 0.05) or the total distance moved (F2, 27 = 0.07, p > 0.05).

Body temperature (C)

40

***

***

38

36

34

Saline

Body temperature (C)

2.5.2. Measurement of 5-HTT and DAT

Analysis of [3H]paroxetine binding to 5-HTT in the rat frontal cortex
and hippocampus and [ 3H]mazindol binding to DAT in rat striatum was
performed according to the methods described previously (Battaglia et
al., 1987; Hrdina et al., 1990; Robledo et al., 2004). Briey, brain tissue
samples were placed in a tube containing 10 ml of 50 mM TrisHCl
buffer (pH 7.4) containing 120 mM NaCl and 5 mM KCl (incubation
buffer), and homogenized using a Polytron at the lowest setting for
30 s. The tubes were centrifuged at 48,000 g for 10 min, washed, and
resuspended to a volume of 15 mg wet weight/ml. Total [ 3H]paroxetine
binding was assessed by incubating the samples in tubes containing
4 nM [ 3H]paroxetine for 2 h at 22 C. Non-specic binding was
assessed in separate tubes by the addition of 20 M citalopram to
the incubation buffer to block [ 3H]paroxetine binding to 5-HTT. To
determine total [ 3H]mazindol binding the samples were incubated
for 1 h at 4 C in tubes containing 6 nM [ 3H]mazindol in the presence
of 0.3 M desipramine to block [ 3H]mazindol binding to NE transporters. Non-specic binding was assessed in separate tubes by the addition of 100 M nomifensine to the incubation buffer to block [3H]
mazindol binding to DAT. Following incubation, the tubes were ltered
through Whatman GF/B lters pre-soaked with 0.05% polyethylenimine
and washed with 15 ml of buffer. The lters were then added to 3 ml
Optiphase HiSafe 3 scintillation uid (PerkinElmer, Turku, Finland)
and counted at 47% efciency in a scintillation spectrometer. Finally,
sample protein content was determined using the Bio-Rad Protein
Assay Kit.

3.1. Body temperature during drug treatments

Mephedrone

Methylone

40

***
38

36

34

Saline

Mephedrone

Methylone

Fig. 2. Body temperatures in mice (A) and rats (B) measured after the rst drugtreatments on the rst day of drug treatments. Measurements were taken 45 5 min
after drug administration, at the time of the peak hyperthermic response. In mice,
both methylone and mephedrone (both 30 mg/kg) caused an increase in body temperature compared to saline-treated mice. In rats, only methylone produced signicant
hyperthermia. Data are expressed as mean SEM. ***pb 0.001. Mice N = 1324 per
group. Rats N= 67 per group.

B. den Hollander et al. / Pharmacology, Biochemistry and Behavior 103 (2013) 501509

505

Table 1
Summary of results from behavioral experiments.
Test

Condition

Elevated plus
maze

Time in open arm

(s)
Saline
37 35
Mephedrone 40 26
Methylone
31 31
Total distance
moved (m)
Saline
206 38
Mephedrone 215 47
Methylone
202 67
Escape latency
Day 1 (s)

Locomotor
activity

MWM
training

Results

Saline
92 50
Mephedrone 82 83
Methylone
72 72
MWM probe
Time in quadrant
Q1 (s)
Saline
21 7
Mephedrone 22 5
Methylone
21 7
Time immobile
Tail
(s)
suspension
test
Saline
133 41
Mephedrone 158 18
Methylone
154 26

Time in closed Total distance

arm (s)
moved (m)
196 44
13 3
185 52
12 3
190 49
12 4
Mean velocity (cm/s)
2.88 0.53
2.98 0.65
2.80 0.94
Day 2 (s)
40 40
20 9
52 54

Day 3
(s)
14 7
11 1
16 6

Day 4
(s)
10 2
11 3
14 7

Q2 (s)
Q3 (s) Q4 (s)
10 5
10 7 19 8
13 4
84
18 4
13 6
10 4 16 5
Latency to rst immobility (s)
79
11
43

Animals were treated with mephedrone or methylone (both 30 mg/kg twice daily for
4 days) and behavioral tests were performed 28 weeks after treatments. Data are
expressed as mean SD. N = 712 per group. No signicant differences were seen between groups in these tests. See Figs. 3 and 4 for the results from the T-maze spontaneous alternation test and Morris water maze (MWM) reversal probe trial where
signicant differences were observed.

3.2.2. T-maze
All the mice tested completed the test trial within 5 min, performed
at least 5 trials and were included in the analysis. An ANOVA showed a
signicant effect of treatment on the rate of spontaneous alternations in
the T-maze (F2, 23 = 3.65, p b 0.05). Post-hoc comparisons revealed that
mephedrone-treated mice had a signicantly lower rate of spontaneous
alternations compared to saline-treated mice whereas the methylonetreated mice did not differ signicantly from saline controls (Fig. 3).
3.2.3. Locomotor activity and habituation
The 12 time points each representing the distance moved or velocity in the previous 10 min were analyzed using a repeated measures ANOVA with GreenhouseGeisser correction and treatment
as between-subjects factor. This demonstrated a signicant decrease
in the distance moved as time passed (F4, 104 = 95.92, p b 0.0005), but no
effect of treatment (F2, 27 = 0.16, p > 0.05) and no treatment time
point interaction (F8, 104 =1.88, p > 0.05). The mice's average velocity
also decreased signicantly with time (F4, 105 = 96.56, p b 0.0005), but
was not affected by treatment (F2, 27 = 0.15, p >0.05), and showed no
treatment time point interaction (F8, 105 = 0.83, p > 0.05).
3.2.4. Morris water maze
A repeated measures ANOVA with GreenhouseGeisser correction
of the four training days with condition as between-subjects factor
revealed a signicant effect of training day on the latency to discover
the hidden platform (F2, 41 = 36.58, p b 0.0005). There was no main effect of treatment (F2, 22 = 0.48, p > 0.05) or treatment training day
interaction (F4, 41 =1.07, p>0.05) indicating that drug treatment did
not affect the ability of mice to learn the hidden platform location. During
the subsequent probe trial an ANOVA demonstrated no effect of treatment on time spent in Q1 (F2, 22 =1.37, p>0.05), Q3 (F2, 22 =0.58,
p>0.05) or Q2+3 (F2, 22 =0.64, p>0.05). There was also no effect of
treatment on reversal learning (F2, 22 =1.35, p>0.05). However, an
ANOVA of the probe trial data following reversal learning indicated a

Fig. 3. The spontaneous alternation ratio of mice in the T-maze was reduced to chance
level 2 weeks after a 4-day binge treatment with mephedrone but was unaffected by
methylone (both twice daily 30 mg/kg), compared to saline-treated controls. Data
are expressed as mean SEM. *p b 0.05. N = 710 per group.

signicant effect of treatment on the time spent in the old platform

quadrant Q1 (F2, 22 =7.14, pb 0.005), as well as in the new platform
quadrant Q3 (F2, 22 =3.76, pb 0.05), while the time spent in Q2+4 was
unaffected (F2, 22 =1.44, p>0.05). Post hoc comparisons showed that
both methylone- and mephedrone-treated mice spent signicantly less
time in the old platform location and that methylone, in addition,
increased the time spent in the new platform location, compared to
saline-treated mice (Fig. 4). Visual acuity was not affected by treatment
(F2, 22 =0.92, p>0.05).
3.2.5. Tail suspension test
An ANOVA showed no effect of treatment on the total time spent
immobile (F2, 19 = 1.55, p > 0.05), and there was also no signicant
difference in the latency to the rst occurrence of immobility
(F2, 19 = 1.84, p > 0.05).
3.3. Biochemical experiments
3.3.1. Neurotransmitter levels
The effects of drug treatments on mouse brain monoamine levels in
the frontal cortex, striatum and hippocampus are shown in Table 2.

Fig. 4. The effect of a 4-day binge treatment of methylone or mephedrone (both twice
daily 30 mg/kg) on the probe trial following reversal learning in the mice Morris water
maze test in mice. Methylone decreased the time spent in the old target quadrant (Q1)
and increased the time spent in the new target quadrant (Q3) compared to
saline-treated mice. Mephedrone also reduced the time spent in the old target quadrant compared to saline-treated mice. The time spent in the anking non-target quadrants was not affected by either treatment. Data are expressed as mean SEM.
*p b 0.05; **p b 0.01. N= 79 per group.

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B. den Hollander et al. / Pharmacology, Biochemistry and Behavior 103 (2013) 501509

Table 2
Neurotransmitter content in the mouse brain.
Region

Frontal ctx.

Striatum

Hippocampus

Condition

Saline
Mephedrone
Methylone
AMPH
Saline
Mephedrone
Methylone
AMPH
Saline
Mephedrone
Methylone
AMPH

Neurotransmitter content (nmol/g wet tissue)

5-HT

5-HIAA

DA

DOPAC

HVA

NE

1.8 0.8
1.7 0.3
1.6 0.2
1.8 0.5
2.2 0.6
2.2 0.5
2.6 0.7
2.8 0.4
2.1 1.0
1.5 0.2
1.6 0.2
1.5 0.3

1.7 0.4
1.6 0.2
1.4 0.4
2.0 0.3
2.0 0.6
1.7 0.2
2.1 0.5
2.3 0.5
2.6 0.4
2.6 0.2
2.7 0.2
2.2 0.4

0.3 0.3
0.3 0.2
1.1 0.8
0.1 0.1
31.5 4.6
30.7 2.9
33.0 6.8
10.7 5.5
0.2 0.1
0.1 0.1
0.2 0.0
0.1 0.0

0.4 0.3
0.3 0.1
1.0 0.7
0.3 0.2
11.1 2.3
7.6 3.1
8.4 3.2
9.5 3.0
0.2 0.0
0.2 0.0
0.2 0.1
0.2 0.0

0.5 0.2
0.4 0.1
0.7 0.2
0.4 0.2
4.9 0.9
3.8 0.3
4.3 0.8
4.9 0.8
0.2 0.0
b LLOQ
0.1 0.0
b LLOQ

1.6 0.5
1.5 0.2
1.4 0.2
1.5 0.4
1.1 0.5
1.4 0.4
1.8 0.7
1.7 1.0
1.9 0.7
1.5 0.2
1.5 0.2
1.9 0.7

Data shows average content (nmol/g wet tissue) of DA, 5-HT, their metabolites and NE in the frontal cortex, striatum and hippocampus 2 weeks following a 4-day binge treatment
with mephedrone, methylone (both twice daily 30 mg/kg) or AMPH (twice daily 12.5 mg/kg). b LLOQ means the value was below the lower limit of quantication (b0.1 pmol/g
wet tissue). Data are expressed as mean SD. N= 125 per group.
p b 0.05.
p b 0.001.

Neither mephedrone nor methylone caused any signicant changes in

neurotransmitter levels aside from a 22% decrease in striatal HVA levels
in mephedrone-treated mice. As expected, AMPH produced a signicant 65% reduction of striatal DA levels compared to saline controls.
Brain monoamine levels following drug treatments in rats are
presented in Table 3. Mephedrone resulted in no signicant changes
in neurochemistry in any brain region. Methylone, however, had a
profound impact on 5-HT levels in the rat brain: we observed a 44%,
40% and 38% decrease in 5-HT levels in the frontal cortex, striatum
and hippocampus, respectively. Additionally, 5-HIAA levels were reduced by 28% (striatum) and 15% (hippocampus). As expected, AMPH
has a strong effect on monoamine levels. Compared to saline treated
rats, AMPH lowered striatal DA by 45% and reduced 5-HT levels in the
frontal cortex and hippocampus by 33% and 28%, respectively. Additionally, striatal 5-HIAA levels were reduced by 24%.

3.3.2. 5-HTT and DAT binding

An ANOVA revealed a signicant effect of treatment on [3H]paroxetine
binding to 5-HTT in the frontal cortex (F2, 17 =3.94, pb 0.05) as well as the
hippocampus (F2, 15 = 4.21, p b 0.05). Post hoc testing showed this effect
was due to a 27% lower binding in the frontal cortex and 36% lower
binding in the hippocampus of methylone-treated rats, compared to

saline controls (Fig. 5). No signicant effect of treatment was found on

[ 3H]mazindol binding to striatal DAT (F2, 15 = 0.09, p > 0.05) (Fig. 5).
4. Discussion
Our study shows that mephedrone is capable of producing a
long-term reduction in working memory performance at a dose that
does not produce any apparent long-term changes in brain chemistry
in mice. However, anxiety- and depression-related behaviors do not
appear to be affected by either methylone or mephedrone. Furthermore, we show there are species differences in the sensitivity to
long-term neurochemical effects of methylone, as it produces widespread depletion of 5-HT and 5-HT transporter levels in rats, whereas
no effects on 5-HT levels were seen in mice. Mephedrone, conversely,
does not appear to cause any overt depletion of brain monoamine
levels in rodents aside from a small decrease in the DA metabolite
HVA in the mouse striatum. As expected, AMPH caused a reduction
in striatal DA levels in both mice and rats and reduced 5-HT levels
in rats.
These results are important as they highlight the fact that
mephedrone can affect cognitive function even though no apparent
changes are seen in monoamine levels. The fact that no large longterm neurochemical changes were observed following mephedrone

Table 3
Neurotransmitter content in the rat brain.
Region

Condition

Frontal ctx.

Saline
Mephedrone
Methylone
AMPH
Saline
Mephedrone
Methylone
AMPH
Saline
Mephedrone
Methylone
AMPH

Striatum

Hippocampus

Neurotransmitter content (nmol/g wet tissue)

5-HT

5-HIAA

DA

DOPAC

HVA

NE

3.0 0.5
2.5 0.5
1.7 0.3
2.0 0.5

1.8 0.3
1.7 0.3
1.4 0.4
1.7 0.2
2.1 0.3
1.9 0.2
1.5 0.2
1.6 0.3
1.8 0.3
1.9 0.1
1.4 0.3

0.3 0.1
0.4 0.3
0.2 0.1
0.3 0.1
39.1 7.9
36.0 14.7
37.0 10.0
21.5 10.3
0.1 0.1
0.1 0.0
0.1 0.1
0.1 0.0

0.3 0.1
0.2 0.1
0.2 0.1
0.2 0.1
12.5 5.3
12.1 2.5
12.9 2.8
9.7 4.1
0.1 0.0
0.1 0.0
0.1 0.0
0.1 0.0

0.2 0.1
0.2 0.1
0.2 0.1
0.2 0.1
3.8 0.8
3.7 0.9
3.8 0.6
3.1 1.1
b LLOQ
b LLOQ
b LLOQ
b LLOQ

1.5 0.1
1.5 0.3
1.3 0.1
1.4 0.2
1.6 1.1
1.2 0.3
1.3 0.6
1.5 0.4
1.7 0.3
1.6 0.4
1.5 0.3
1.5 0.3

2.0 0.8
1.5 0.4
1.2 0.4
1.1 0.4
2.1 0.3
1.7 0.4
1.3 0.2
1.5 0.4

1.7 0.3

Data shows average content (nmol/g wet tissue) of DA, 5-HT, their metabolites and NE in the frontal cortex, striatum and hippocampus 2 weeks following a 4-day binge treatment
with mephedrone, methylone (both twice daily 30 mg/kg) or AMPH (twice daily 12.5 mg/kg). b LLOQ means the value was below the lower limit of quantication (b0.1 pmol/g
wet tissue). Data are expressed as mean SD. N= 57 per group.
p b 0.05.
p b 0.01.
p b 0.001.

B. den Hollander et al. / Pharmacology, Biochemistry and Behavior 103 (2013) 501509

Fig. 5. The effect of 4-day binge treatments of methylone or mephedrone (both twice
daily 30 mg/kg) on [3H]paroxetine binding to 5-HTT in the rat frontal cortex and hippocampus as well as [3H]mazindol binding to striatal DAT. Data is shown as percentage
of control values and represents the mean SEM. N = 67 per group. Control values
were: frontal cortex, 52776; hippocampus, 325120; striatum, 236109 fmol/mg
of protein.

binge-dosing indicates that the effects of this drug are subtle when
compared to METH or AMPH which causes widespread depletion of
brain DA levels in rodents (Fukumura et al., 1998; Kita et al., 1998;
O'Callaghan and Miller, 1994). This stresses the importance of assessing
behavioral as well as biochemical tests of toxicity when screening new
psychoactive drugs of abuse. Moreover the results from our monoamine
analysis demonstrate that there are clear species differences in the sensitivity to the toxic effects of methylone. This might help explain some current and future discrepancies between results as both mouse and rat
models have been employed by different investigators.
Our results are in agreement with other recent reports regarding
methylone and mephedrone toxicity. Angoa-Perez et al. (2012) also
treated C57BL/6 mice with mephedrone and found no biochemical
evidence of toxicity such as changes in DA, TH and GFAP levels or
microglial activation. Also in rats, mephedrone was shown to have
no effect on monoamine levels 2 weeks after treatment (Baumann
et al., 2012) or 7 weeks after treatment (Motbey et al., 2012). On the
other hand, Hadlock et al. (2011) reported a decrease in hippocampal
5-HT levels. A possible reason for this discrepancy is that they measured
monoamine levels 1 week after treatment instead of 2 weeks in the
current experiments, or that the toxicity was exacerbated due to the
high ambient temperature (27 C). Regarding methylone we are
only aware of one report, which reported no changes in monoamine
levels two weeks following treatment in rats (Baumann et al., 2012).
This is in contrast to our nding of lowered 5-HT levels and is most likely due to the fact that the doses employed in that study (3 or 10 mg/kg,
3 times every 2 h) were much lower than in the current study.
Baumann et al. (2012) suggested that the reason why methylone failed
to affect 5-HT levels could be related to its low potency at the VMAT-2
(Cozzi et al., 1999). Although this could be the case, our results demonstrate that this reduced potency does not prevent methylone from depleting 5-HT levels at higher doses.
It has been shown that mephedrone and methylone elicit conditional place preference, increase locomotor activity and cause clear
behavioral changes in a functional observational battery during acute administration (Lisek et al., 2012; Marusich et al., 2012). Just very recently a
report (Motbey et al., 2012) was published demonstrating residual behavioral effects of mephedrone. Our nding of reduced working memory
function in mice is of particular interest as Motbey et al. (2012) reported
decreased memory performance on a novel object recognition test in
rats 5 weeks following a high-dose regimen (30 mg/kg once daily
for 10 days) of mephedrone. Additionally, there is also evidence of
mephedrone affecting memory in humans. One study (Freeman et
al., 2012) reported impaired working memory (spatial N-back task
performance) in individuals under the inuence of mephedrone.

507

Although their impairment was transient and seemingly recovered

with abstention from the drug, the possibility remains that more frequent or high-dose use, such as mimicked in our binge-dosing model,
would produce more long-lasting impairments in working memory.
The question remains what the exact relationship is between the effects
of mephedrone on memory function and neurochemistry. Repeated
METH-treatment causes impairment of working memory function as
well as depletion of striatal DA levels (Lee et al., 2011; Miller and
O'Callaghan, 1996). However, it should be noted that the T-maze spontaneous alternation test employed in our study is also subject to inuence by factors not related to working memory, such as attention and
novelty preference, which could also have been affected by drug treatments (Hughes, 2004). Although mephedrone did not deplete striatal
DA levels we did observe a decrease in striatal HVA levels in mice.
This points towards a decrease in the HVA/DA ratio and suggests a
blockade of DA metabolism (Zambrano and Reyes-Mugica, 2002).
MDMA inhibits MAO with a preference for MAO-A over MAO-B
(Leonardi and Azmitia, 1994). A strong ability of mephedrone to inhibit
MAO-B, possibly in combination with inhibition of COMT could form an
explanation for the reduction in HVA without an effect on 5-HIAA levels.
However, since this effect was observed 2 weeks after the nal drug administration, any changes in enzyme levels or function are more likely
to be a result of drug-induced long-term changes in gene expression
or other intracellular mechanisms rather than due to direct inhibition
of enzyme function by mephedrone. We also cannot say if this alteration in DA metabolism is related to the observed effects on memory.
Since we only measured monoamine levels, the observed behavioral effects could also be due to other changes in the monoamine systems,
such as receptor levels. Furthermore, the possibility remains that the
behavioral effects are mediated by drug-induced changes in entirely
different neurotransmitter systems which were also not measured in
this study. Finally, the measurement of monoamine levels took place
2 weeks following the last drug treatments, but the behavioral tests
were performed up to 8 weeks after the nal treatment. It is therefore
possible that recovery took place during this time which reduced the
likelihood of observing behavioral changes.
The ability of methylone to produce selective depletion of 5-HT in
rats but not in mice is similar to MDMA, although MDMA also depletes
DA in mice (Battaglia et al., 1987; O'Callaghan and Miller, 1994), something we did not observe with methylone. One reason for the observed
difference in toxicity between methylone and mephedrone in rats
might be related to hyperthermia as methylone, but not mephedrone,
caused a signicant rise in body temperature. Hyperthermia is known
to play an important role in mediating the depletion of monoamines
after MDMA and METH treatments (Malberg and Seiden, 1998; Tata
et al., 2007). On the other hand, in mice, both drugs produced a similar
degree of hyperthermia but did not affect monoamine levels. Other explanations for the species difference in sensitivity to toxicity include the
formation of different metabolites with varying degrees of toxicity or
varying abilities of the drug to inhibit the 5-HT synthesizing enzyme
TH (Easton and Marsden, 2006; Logan et al., 1988).
In the Morris water maze we observed an improved performance
of both drug-treated groups at nding the hidden platform during the
reversal probe trial. This was contrary to our hypothesis that these
drugs would have a long-lasting negative impact on memory function. This could be either due to increased ability to learn the new location but also because the drug-treated groups more quickly forgot
the location of the old platform. No effects were seen on learning or
recall during normal training, suggesting that the long-term effect
of these drugs on spatial learning and recall is limited. Interestingly,
improvement in visuo-spatial associative memory was also recently
reported during an acute, low-dose (0.32 mg/kg) mephedrone challenge in macaques (Wright et al., 2012). It should also be noted that
while the measurement of monoamine levels took place 2 weeks after
drug treatments, the behavioral tests were performed up to 8 weeks
after the treatment. It is therefore possible that recovery took place

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B. den Hollander et al. / Pharmacology, Biochemistry and Behavior 103 (2013) 501509

during this time which reduced the likelihood of observing behavioral

changes.
A major limitation of this study is that we were only able to perform behavioral experiments on mice and not on rats. The fact that
methylone lowered 5-HT levels in rats suggests that the behavioral
effects of this drug might also be more severe in this species. MDMA
is toxic to the 5-HT system in both rats and humans, whereas it affects
the DA system in mice (Logan et al., 1988; Reneman et al., 2001). For
this reason the rat is generally considered to be a better model organism than mouse. It is currently not clear whether rats are also a better
model for mephedrone and methylone neurotoxicity, as neither the
behavioral nor neurochemical long-term effects of these drugs have
been characterized in humans. It should therefore be noted that the
behavioral and cognitive effects of these drugs may be different, and
possibly more severe, in rats and humans. Another important limitation is that the behavioral experiments, unlike the monoamine assay,
were not performed with a reference compound such as AMPH. It is
therefore not possible to directly compare the behavioral effects of
mephedrone and methylone to those of a substance with known longterm behavioral/cognitive effects. A further limitation is that only one
dose level (30 mg/kg) was employed. However, the employed dose
was established in pilot experiments aimed at determining a dose
which produced clear hyperthermia but no lethality and, further, is in a
similar range as those used in other studies (Angoa-Perez et al., 2012;
Hadlock et al., 2011) Finally, in mice we only measured monoamine
levels and not the 5-HTT or DAT binding, as it would have required too
many mice to produce enough tissue to reliably assess transporter levels
using the ligand binding assay. However, since 5-HT and DA levels were
unaffected in mice, the transporter protein levels would most likely not
have been altered either (Yamamoto et al., 2010).
Further investigation is needed to determine if the effect of
mephedrone on working memory is due to changes in the DA system, such as altered metabolism, or if it is mediated by different
mechanisms. It is of interest also to determine the effects of these
substances on human neurochemistry and cognition in order to draw
more rm conclusions about the effects of these drugs in humans and
the validity of the rat and mouse models.
5. Conclusions
It appears that both methylone and mephedrone can cause longterm changes in either neurochemistry or cognitive function in rodents. We cannot yet say with certainty whether similar changes also
occur in human users of these drugs, but it certainly cannot be ruled
out as animal models of binge MDMA and METH use closely mimic similar changes seen in human users. Peer educators should be made aware
of the possible long-term effects associated with the use of cathinonederivatives such as methylone and mephedrone in an effort to inform
users that these previously legal highs are not risk-free.
Acknowledgments
The authors wish to thank the Finnish Cultural Foundation SKR
and the Helsinki Biomedical Graduate Program for their nancial support. We also thank Heidi Pehkonen for her help in performing the
experiments and Pertti Panula for the use of HPLC reagents.
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