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ORIGINAL ARTICLE

A Single Neurotoxic Dose of Methamphetamine Inducesa Long-Lasting Depressive-Like Behaviour in Mice

Carlos D. Silva • Ana F. Neves • Ana I. Dias • Hugo J. Freitas • Sheena M. Mendes •

Ines Pita • Sofia D. Viana • Paulo A. de Oliveira • Rodrigo A. Cunha •

Carlos A. Fontes Ribeiro • Rui D. Prediger • Frederico C. Pereira

Received: 30 May 2013 / Revised: 22 August 2013 / Accepted: 1 September 2013! Springer Science+Business Media New York 2013

Abstract Methamphetamine (METH) triggers a disrup-tion of the monoaminergic system and METH abuse leads

to negative emotional states including depressive symp-

toms during drug withdrawal. However, it is currentlyunknown if the acute toxic dosage of METH also causes a

long-lasting depressive phenotype and persistent mono-

aminergic deficits. Thus, we now assessed the depressive-like behaviour in mice at early and long-term periods fol-

lowing a single high METH dose (30 mg/kg, i.p.). METH

did not alter the motor function and procedural memory ofmice as assessed by swimming speed and escape latency to

find the platform in a cued version of the water maze task.

However, METH significantly increased the immobilitytime in the tail suspension test at 3 and 49 days post-

administration. This depressive-like profile induced by

METH was accompanied by a marked depletion offrontostriatal dopaminergic and serotonergic neurotrans-

mission, indicated by a reduction in the levels of dopamine,

DOPAC and HVA, tyrosine hydroxylase and serotonin,

observed at both 3 and 49 days post-administration. Inparallel, another neurochemical feature of depression—

astroglial dysfunction—was unaffected in the cortex and

the striatal levels of the astrocytic protein marker, glialfibrillary acidic protein, were only transiently increased at

3 days. These findings demonstrate for the first time that a

single high dose of METH induces long-lasting depressive-like behaviour in mice associated with a persistent dis-

ruption of frontostriatal dopaminergic and serotonergic

homoeostasis.

Keywords Methamphetamine ! Depression ! Tail

suspension test ! Monoaminergic disruption ! Frontalcortex ! Striatum

Introduction

Methamphetamine (METH), while being a significant

drug problem in North America and East and Southeast

Asia since the past decade, has become a more prominentpart of the European drug scene (European Monitoring

Centre for Drugs and Drug Addiction 2012). ChronicMETH abuse leads to neurotoxicity that has been asso-

ciated with cognitive, mood and motor impairments

(Gouzoulis-Mayfrank and Daumann 2009; Carvalho et al.2012). Post-mortem brain analysis from chronic METH

users has revealed reduced levels of dopaminergic nerve

terminal markers [dopamine (DA), tyrosine hydroxylase(TH), and dopamine transporter (DAT)] in the striatum

(Wilson et al. 1996; Moszczynska et al. 2004; Kitamura

et al. 2007) and reductions in serotonin transporter (5-HTT) levels in the orbitofrontal and occipital cortices

(Kish et al. 2009). These neuropathological changes are

C. D. Silva ! A. F. Neves ! A. I. Dias ! H. J. Freitas !S. M. Mendes ! I. Pita ! S. D. Viana ! C. A. Fontes Ribeiro !F. C. Pereira (&)Laboratory of Pharmacology and Experimental Therapeutics,IBILI, Faculty of Medicine, University of Coimbra, Subunit1 – Polo 3, Azinhaga de Santa Comba, Celas,3000-548 Coimbra, Portugale-mail: fredcp@ci.uc.pt

P. A. de Oliveira ! R. D. PredigerDepartamento de Farmacologia, Universidade Federal de SantaCatarina, Florianopolis, SC, Brazil

R. A. CunhaCenter for Neuroscience and Cell Biology (CNC), Universityof Coimbra, Coimbra, Portugal

123

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DOI 10.1007/s12640-013-9423-2

Author's personal copy

probably associated with the psychiatric features seen in

METH chronic users such as aggressiveness, social iso-lation, psychosis, mood disturbances, and psychomotor

dysfunction (Semple et al. 2005; Scott et al. 2007; Darke

et al. 2008; Homer et al. 2008). In particular, abstinentMETH abusers show a reduced brain 5-HTT density and

regional cerebral metabolic abnormalities associated with

depressive symptoms (London et al. 2004; Sekine et al.2006; Panenka et al. 2012). The pro-depressive conse-

quences of METH usage may also involve the dopami-nergic system since clinical data have evidenced that

negative emotional symptoms may also be related to

dopaminergic dysfunction (Rowe et al. 1998; Segman andShalev 2003) in dopamine-rich brain regions such as the

striatum and prefrontal cortex, in accordance with the

functional and structural abnormalities found in thesebrain regions in major depressive disorder (Bora et al.

2012). In addition, dysfunction of astrocytes has been also

consistently noted in depressive disorders as well as inrodent models of depressive-like behaviour (Rajkowska

and Stockmeier 2013), as typified by changes in glial

fibrillary acidic protein (GFAP), the principle componentof astrocytic cytoskeletal intermediate filament used as a

marker of astrogliosis (Pekny and Nilsson 2005).

However, the study of the mood behavioural and neu-rochemical long-term consequences of METH consump-

tion is still poorly characterized. In this context, it was

recently showed that rats exhibited a depressive-like stateduring early withdrawal after compulsive METH intake

(Jang et al. 2013), but the behavioural phenotype of a

single high dose of METH is unknown. This single highMETH dose recapitulates the detrimental effects found in

METH users including hyperthermia, striatal dopaminergic

and astroglial dysfunction (Cappon et al. 2000; Imam andAli 2001; Xu et al. 2005; Zhu et al. 2005; Krasnova and

Cadet 2009; O’Callaghan et al. 2008; Kitamura et al. 2010;

Sailasuta et al. 2010; Pereira et al. 2012) and is particularlyrelevant to mimic the large doses ingested by naıve non-

tolerant users (Davidson et al. 2001; Krasnova and Cadet

2009).We now investigated the depressive-like phenotype in

mice following a single high METH injection at early

(3 days) and late (49 days) periods post-administration.The present results show that a high dose of METH

(30 mg/kg, i.p.) induces long-lasting depressive-like

responses in mice evaluated in the tail suspension testwithout evident impairment of motor function and striatal-

dependent memory. This METH-induced long-term

depressive-like phenotype was accompanied by a paralleldepletion of frontostriatal dopaminergic and serotonergic

neurotransmission, without persistent changes of GFAP

levels.

Materials and Methods

Animals

Male adult C57BL/6J mice (3–4 months old; 20–28 g;Charles River Laboratories, Barcelona, Spain) were housed

four per cage, under controlled environmental conditions

(12 h light/dark schedule, at room temperature of23 ± 1 "C, with food and water supplied ad libitum). All

experiments were approved by the Institutional Animal

Care and Use Committee from Faculty of Medicine,Coimbra University, and were performed following the

European Community directive (2010/63/EU). The animal

procedures were performed in strict accordance with the‘‘Guide for the Care and Use of Laboratory Animals’’

(Institute of Laboratory Animal Resources, National

Academy Press, 1996).

Drugs and Chemicals

We were issued permission to import METH!HCl from

Sigma-Aldrich (St. Louis, MO, USA) by INFARMED,

Portugal (National Authority of Medicines and HealthProducts). Standards for DA, 3,4-dihydroxyphenylacetic

acid (DOPAC), homovanillic acid (HVA), serotonin (5-

HT) were purchased from Sigma-Aldrich. The other usedchemicals (ultrapure and pro analysis quality) were pur-

chased from Sigma-Aldrich and Merck AG (Darmstadt,Germany).

Drug Administration and Temperature Monitoring

Animals were injected intraperitoneally with a single dose

of METH (30 mg/kg) or with saline solution (0.9 % NaCl;SAL) in a volume of 0.1 mL/10 g of body weight. This

METH regimen is representative of an acute toxic dosing

(ATD); as suggested by Davidson et al. (2001), andextensively discussed by Krasnova and Cadet (2009), this

regimen provides the following experimental advantages:

(1) it has an excellent epidemiological translational valuesince it recapitulates the high brain levels on first pass

extraction occurring after acute high intravenous or

smoked METH; (2) it mimics the large doses taken byhuman METH abusers, which can reach several grams per

day; (3) it is a good model of potential effects of an

overdose in naive non-tolerant users; (4) it offers greaterexperimental control over variables. Finally, this single

high dose METH protocol employed in this study has been

successfully used by us and others (Goncalves et al. 2010;Pereira et al. 2006, 2012; Xu et al. 2005; Tulloch et al.

2011; Zhu et al. 2005).

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Body temperature was assessed with a rectal probe

(BAT-12, Physitemp Instruments Inc., Clifton, NJ, USA)every 30 min following injection, up to 4 and at 24 h post-

injection. Animals treated with METH displayed hyper-

thermia starting at 30 min, peaking at 1 h (peak tempera-ture for controls and METH-treated: 36.8 ± 0.2 vs

39.1 ± 0.2 "C, n = 7–8; P \ 0.001) and returning to

normal values at 4 h. Twenty-four hours after METHinjection the body temperature remained normal. This

transient hyperthermia is consistent with previous findingsdescribed by our group (Pereira et al. 2012) and others (Xu

et al. 2005) using the same METH dose. In addition, this

model reduces the inherent complexity present in repeateddosage regimens and reduces the occurrence of seizures

and high mortality seen with multiple doses regimen

(Davidson et al. 2001; Miller and O’Callaghan 2003). Infact, in the present study, all animals survived this dosing

regimen and none showed convulsions or weight

reductions.

Behavioural Tests

During the first 4 or 49 days after i.p. injection of METH

(30 mg/kg) or SAL, the behavioural tests were conducted

in three independent cohorts of animals. The rationale forchoosing these two time-points was prompted by previous

studies showing that a single-day METH regimen triggered

a dopaminergic neurotoxicity at 3 days (30 mg/kg METH;Pereira et al., 2012) and at 48 days following METH

treatment (12.5 mg/kg METH, 4 times, 2 h apart) (Fried-

man et al. 1998). All tests were carried out between 9:00and 17:00 h in a sound-attenuated room under low-inten-

sity light (12 lx) and they were scored by the same rater in

an observation room where the mice had been habituatedfor at least 1 h before the beginning of the tests. Behaviour

was monitored through a video camera positioned above

the apparatuses and the images were later analysed with theANY Maze video tracking (Stoelting Co., Wood Dale, IL,

USA) by an experienced researcher who was unaware of

the experimental group of the animals tested. The first setof animals [SAL (n = 7) and METH (n = 8)] performed

tail suspension at 3 days post-treatment. The second set of

animals (n = 8 per group) performed Morris water mazeon days 1–4 following treatment. The third set of animals

(n = 8 per group) performed tail suspension test at 49 days

following treatments.

Tail Suspension Test

The tail suspension test has become one of the most widely

used tests for assessing antidepressant-like activity in mice:

it is based on the fact that animals subjected to the short-term inescapable stress of being suspended by their tail will

develop an immobile posture (Steru et al. 1985). In brief,

mice were suspended 50 cm above the floor by adhesivetape placed approximately 1 cm from the tip of the tail.

The immobility time was recorded during a 6-min period

by an observer blind to the drug treatment. Mice wereconsidered immobile only when they hung passively and

completely motionless.

Water Maze Test: Procedural Memory Version

Tests were performed in a circular swimming pool made of

grey-painted fibreglass, 1.2 m inside diameter, 0.8 m high,

which was filled to a depth of 0.6 m with water maintaineda 23 ± 2 "C. The target platform (10 9 10 cm2) of trans-

parent acrylic resin was submerged 1–1.5 cm beneath the

water surface and it was cued by a 7-cm diameter whiteball attached to the top of the platform and protruding

above the water. Starting points were marked on the out-

side of the pool as north (N), south (S), east (E), and west(W). Four distant cues (55 9 55 cm2) were placed 30 cm

above the upper edge of the water tank and the position of

each symbol marked the midpoint of the perimeter of aquadrant (circle = NE quadrant, square = SE quadrant,

cross = SW quadrant, and diamond = NW quadrant). A

monitor and a video-recording system were installed in anadjacent room. The animals were submitted to a cued

version of the water maze as described previously (Predi-

ger et al. 2006). This consisted of 4 training days (from 1 to4 days pos-injection of METH), four consecutive trials per

day, during which the animals were left in the tank facing

the wall and were then allowed to swim freely to thesubmerged platform placed in the centre of one of the four

imaginary quadrants of the tank. The initial position in

which the animal was left in the tank was one of the fourvertices of the imaginary quadrants of the tank, and this

was varied among trials in a pseudo-random way. Fur-

thermore, the position of the platform was always changedin each trial of the day. If a mouse did not find the platform

during a period of 60 s, it was gently guided to it. After the

animal had escaped to the platform, it was allowed toremain on it for 10 s and was then removed from the tank

for 20 s before being placed in the next random initial

position. The experiments were recorded and the scores forlatency of escape from the starting point to the platform

and swimming speed were later measured through the

ANY-mazeTM video tracking system.

Neurochemistry

Animals were sacrificed by decapitation at 3 or 49 days

post-treatment. Striata and frontal cortices were dissected

on ice and stored at -80 "C until further analyses. Thesebrain regions were chosen because they are dopaminergic

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rich-regions and are well documented to play a major role

in depressive-like conditions (Krishnan and Nestler 2010).Left brain areas were used for the determination of

monoamine (DA, DOPAC, HVA and 5-HT) contents by

high-performance liquid chromatography with electro-chemical detection (HPLC-ED), and right brain areas were

used for the quantification of protein levels by Western

blot.

Monoamine Assessment by HPLC-ED

The left striata and frontal cortices were sonicated in ice-

cold 0.2 M perchloric acid and centrifuged (15.5009g,7 min, 4 "C). Supernatants were filtered (9,0009g, 10 min,

4 "C) using 0.2 lm Nylon microfilters (Spin-X# Centri-

fuge Tube Filter) and stored at -25 "C until further anal-yses. The pellet was resuspended in 1 M NaOH and stored

at -80 "C for total protein quantification by the bicinch-

oninic acid (BCA) protein assay (Thermo Fisher Scientific,MA, USA). A Gilson HPLC system was used to determine

DA, DOPAC, HVA and 5-HT concentrations in the stria-

tum as well as in the frontal cortex as described previously(Pereira et al. 2006, 2012). These compounds were sepa-

rated on a reversed-phase Waters Spherisorb ODS2 column

(24.6 mm 9 250 mm; 5 lm) with a mobile phase(pH = 3.8) consisting of 0.1 M sodium acetate trihydrate,

0.1 M citric acid monohydrate, 0.5 mM sodium octane

sulphonate, 0.15 mM EDTA, 1 mM dibutylamine and10 % methanol (v/v). A flow rate of 1.0 mL/min was

maintained for 60 min, and the detection of the chro-

matographed compounds was achieved using a glassycarbon working electrode set at 0.75 V. Sensitivity was set

at 2 nA/V. Monoamine concentration was determined by

comparison with peak areas of standards, and expressed innanogram per mg of protein.

Western Blot Analysis

For measuring TH and GFAP levels, total extracts were

obtained as previously described (Pereira et al. 2012). Theright striata and frontal cortices were homogenized in lysis

buffer (50 mM Tris–HCl pH 7.4/0.5 % Triton X-100, 4 "C),

supplemented with a protease inhibitor cocktail (1 mMphenylmethylsulfonyl fluoride, 1 mM dithiothreitol, 1 lg/

mL chymostatin, 1 lg/mL leupeptin, 1 lg/mL antipain and

5 lg/mL pepstatin A; Sigma-Aldrich) and centrifuged(15,5009g, 15 min, 4 "C) to discard insoluble material.

Total protein concentration was determined using the BCA

method (Smith et al. 1985) and supernatants were stored at-80 "C until further use. Equal amounts of protein (stria-

tum: 5 lg—TH, 10 lg—GFAP; frontal cortex: 10 lg—TH

and GFAP) were loaded and separated by electrophoresis onsodium dodecyl sulphate polyacrylamide gel electrophoresis

(12 %), transferred to a polyvinylidene difluoride membrane

(Millipore, Madrid, Spain), and blocked with 5 % non-fatdry milk in phosphate buffer saline for 1 h at room tem-

perature. The membranes were probed with mouse anti-TH

(1:5,000; Millipore, MA, USA) and mouse anti-GFAP(1:5,000; Millipore) overnight at 4 "C. Membranes were

then incubated with alkaline phosphatase-conjugated sec-

ondary antibodies (1:10,000 anti-mouse, GE Healthcare,USA). Finally, membranes were visualized on a Storm 860

Gel and Blot Imaging System (GE Healthcare, Bucking-hamshire, UK), using an enhanced chemifluorescence

detection reagent (ECF, GE Healthcare). To confirm equal

protein loading and sample transfer, membranes werereprobed with b-tubulin (1:10,000; Sigma-Aldrich) or

GAPDH (1:5,000; Abcam, Cambridge, UK) antibodies.

Densitometric analyses were performed using the ImageQuant 5.0 software. Results were normalized against

b-tubulin or GAPDH, and then expressed as percentage of

control.

Statistical Analysis

The data are expressed as mean ± SEM. Body temperature

and water maze data were analysed by one-way analysis of

variance (ANOVA) followed by the Newman–Keuls mul-tiple comparison test. Data from the other experiments

were analysed by using unpaired Student’s t test. Signifi-

cant differences were defined at P \ 0.05. All analyseswere performed using GraphPad Prism 5.0 software for

Windows.

Results

Effects of METH on Depressive-Like Behaviour

of Mice

The mice treated with METH were evaluated on the tail

suspension test at 3 and 49 days post-injection. The METH

group showed an increased immobility time on tail sus-pension test at the two evaluated time-points when com-

pared with SAL group (P \ 0.01) (Fig. 1a).

Effects of METH on the Performance of Mice

in Striatal-Dependent Memory Task

As illustrated in Fig. 1b, no significant differences were

observed between METH and SAL groups in the escape

latency to reach the cued platform in the water maze task,indicating absence of procedural learning and memory

impairments. In addition, the swimming speed during the

performance of the water maze was recorded and no signifi-cant differences between groups were observed, indicating

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that METH injected mice exhibited normal motor functioncompared with SAL group (Fig. 1c).

Long-Term Effect of METH on Dopaminergicand Serotonergic Neurotransmission in the Frontal

Cortex and Striatum of Mice

The effects of a single injection of METH (30 mg/kg, i.p.)

on DA and its metabolite content in the frontal cortex are

shown in Fig. 2a–d. METH produced a significant depletionof DA and its metabolites, DOPAC and HVA, in the frontal

cortex at 3 days (DA, 61 %; DOPAC, 29 %; HVA, 37 %;

P \ 0.05) that persisted until 49 days post-injection (DA,62 %; DOPAC, 33 %; P \ 0.05) (Fig. 2a–c). The reduction

in HVA levels seen at 49 days post-METH did not reach

statistical significance. METH also produced a significantreduction in TH levels (28 %; P \ 0.05) at 3 days. Although

TH levels remained reduced at 49 days after treatment, this

reduction did not reach statistical significance, as shown inFig. 2d. The impact of METH on frontal cortical levels of

5-HT was evident by the reduction observed both at 3 (27 %)and 49 days after treatment (25 %) (Fig. 2e) (P \ 0.05).

The effects of a single injection of METH (30 mg/kg,

i.p.) on the levels of DA and its metabolites in the striatumare illustrated in Fig. 3a–d. METH produced a significant

depletion of DA and its metabolites, DOPAC and HVA, in

the striatum at 3 days (DA, 33 %; DOPAC, 20 %; HVA,20 %; P \ 0.05) that persisted until 49 days post-injection

(DA, 25 %; DOPAC, 23 %; HVA, 27 %; P \ 0.05).

METH also produced a marked reduction in TH levels(41 %; P \ 0.001) at 3 days that was also observed at

49 days after treatment (28 %; P \ 0.01), as shown in

Fig. 3d. We also analysed the impact of METH on striatallevels of 5-HT. Notably, METH did not alter 5-HT levels

in striatum neither at 3 nor at 49 days (Fig. 3e) (P [ 0.05).

The 5-HT results obtained at the early-time point areconsistent with our previous data (Pereira et al. 2012).

Long-Term Effects of METH on GFAP Levelsin the Frontal Cortex and Striatum of Mice

METH failed to modify GFAP levels in the frontal cortexat the two investigated time-points (Fig. 4a). On the other

hand, striatal GFAP levels were significantly increased

3 days post-METH, as compared to SAL controls (350 %of SAL; P \ 0.01) (Fig. 4b). However, striatal GFAP

levels were normal at 49 days post-METH.

Discussion

Our results provide the first evidence that a single injection

of a high dose of METH (30 mg/kg, i.p.) triggered a long-lasting depressive-like behaviour in mice, concurrent with

a long-term dopaminergic and serotoninergic disruption in

the frontal cortex and striatum.Clinical studies have documented the relevance of

depressive symptoms in abstinent METH abusers (Glasner-

a

3 days 49 days0

50

100

150

200METHSAL

Imm

obili

ty ti

me

(s)

b

1 2 3 40

20

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METHSAL

training days

Esc

ape

late

ncy

(s)

c

1 2 3 4

0.10

0.15

0.20 SALMETH

training days

Spe

ed (c

m/s

)

Fig. 1 Effects of a single doseof METH (30 mg/kg, i.p.) onthe depressive-like behaviour(tail suspension test) and onstriatal-dependent memory task[water maze (cued version)test]. a The immobility time inthe tail suspension test at 3 and49 days post-injection; b, c thelatency for escape to a cuedplatform (b) and the swimmingspeed (c) on the water maze test1, 2, 3 and 4 days post-injection.The escape latencies (s) and theswimming speed (cm/s) for theindividual trials were averagedeach day. The results areexpressed as mean ± SEM of7–8 animals per group.**P \ 0.01 versus saline(SAL)-treated animals, using anunpaired Student’s t test (a) anda one-way ANOVA withNewman–Keuls post-test (b, c)

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Edwards et al. 2009; Zorick et al. 2010; Panenka et al.2012). Indeed, adolescent consumption of amphetamines

including METH is associated with subsequent depressive

symptoms (Briere et al. 2012). Accordingly, Glasner-Edwards et al. (2009) highlighted the importance of

addressing these negative emotional states in METH users

during substance abuse treatment. Animal studies back thisemergence of negative emotional states upon METH con-

sumption. Thus, a sub-chronic neurotoxic regimen of

another amphetamine-derivative compound (3,4-methyle-nedioxymethamphetamine, MDMA) induced a long-term

depressive-like behaviour in mice evaluated in the forced

swimming test (Renoir et al. 2008). Jang et al. (2013) alsoreported a depressive-like state in rats evaluated in the

forced swimming test during early withdrawal periodsfollowing a chronic METH self-administration. Likewise,

rodents administered with amphetamine (5–10 mg/kg/day

for 7 days using an osmotic pump) exhibited a significantincrease in immobility scores in tail suspension test 24 h

following withdrawal (Cryan et al. 2003). Finally, Iijimaet al. (2013) reported that rats increased the immobility

time during the forced swimming test, thus exhibiting

increased depressive behaviour, at 48 h following with-drawal from sub-chronic treatment with METH (5.0 mg/

kg/day 9 5 days).

Surprisingly, the behavioural profile of laboratoryrodents after an acute single neurotoxic METH dose has

not been documented to date. Herein, we report that mice

injected with METH display an increased immobilityscores at 3 and 49 days post-injection, which is indicative

of a long-lasting depressive-like behaviour. This finding

provides the first demonstration of a long-lasting depres-sive-like phenotype following a single administration of

METH, which is consistent with depression persisting insome METH users for several years after treatment even

where substance use is reduced (Rawson et al. 2002).

However, a major pitfall of this study is that it only used asingle behavioural paradigm to support the sustained

a b

3 days 49 days0

2

4

6

***

DA

(ng/

mg

prot

ein)

3 days 49 days0.0

0.5

1.0

1.5

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******

DO

PA

C (n

g/m

g pr

otei

n)

c d

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2.0

HV

A (n

g/m

g pr

otei

n)

3 days 49 days0

20

40

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120TH

55kDa!-tubulin62kDa

TH (%

SA

L)

e

3 days 49 days0.0

0.2

0.4

0.6

METHSAL

5-H

T (n

g/m

g pr

otei

n)

Frontal cortexFig. 2 Effects of a single doseof METH (30 mg/kg, i.p.) onfrontal cortical monoaminehomeostasis at early and long-term periods. a DA, b DOPACand c HVA cortical tissuecontents (measured by HPLC-ED) were significantlydecreased at 3 and 49 daysfollowing METHadministration. d TH density(measured by western blot) wasalso significantly decreased at3 days but not at 49 days post-treatment. e 5-HT levels(measured by HPLC-ED) weresignificantly decreased at 3 and49 days after METH treatment.The results are expressed asmean or mean percentage ofSAL ± SEM of six animals pergroup. *P \ 0.05, **P \ 0.01,***P \ 0.001, versus SAL-treated animals, using anunpaired Student’s t test

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negative emotional phenotype caused by a single high doseof METH. And, although the tail suspension test is a robust

and validated test to probe negative emotional states

(Cryan et al. 2005) in a variety of mouse models of

depression (Holmes 2003; El Yacoubi and Vaugeois 2007),it will be important to consolidate this finding using other

behavioural tests as well as different endpoints currently

recommended to explore negative emotional disturbances

Striatum

a b

3 days 49 days0

20

40

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DA

(ng/

mg

prot

ein)

3 days 49 days0

5

10

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C (n

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A (n

g/m

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otei

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3 days 49 days0

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120!-tubulin 34kDa

62kDa55kDa

THGAPDH

TH (%

SA

L)

e

3 days 49 days0

1

2

3

4

*

METHSAL

5-H

T (n

g/m

g pr

otei

n)

Fig. 3 Effects of a single doseof METH (30 mg/kg, i.p.) onstriatal monoamine levels atearly and long-term periods.a DA, b DOPAC and c HVAstriatal tissue contents(measured by HPLC-ED) weresignificantly decreased at 3 and49 days following METHadministration. d TH density(measured by western blot) wasalso significantly decreased atthese two time-points. e 5-HTlevels (measured by HPLC-ED)did not differ at both time-points. The results are expressedas mean or mean percentage ofSAL ± SEM of six animals pergroup. *P \ 0.05, **P \ 0.01and ***P \ 0.001, versus SAL-treated animals, using anunpaired Student’s t test

a b

3 days 49 days0

50

100

150GFAPGAPDH 34kDa

50kDa

Frontal cortex

GFA

P (%

SA

L)

3 days 49 days0

100

200

300

400

500 GFAPGAPDH

50kDa34kDa

SALMETH

Striatum

GFA

P (%

SA

L)

Fig. 4 Effects of a single dose of METH (30 mg/kg, i.p.) on frontalcortical (a) and striatal (b) GFAP levels at early and long-termperiods (measured by western blot). METH increased GFAP proteinlevels at 3 days after METH injection in the striatum (a), but not in

the frontal cortex (b). Data are presented as mean percentage ofSAL ± SEM (n = 6 animals per experimental group). **P \ 0.01using an unpaired Student’s t test

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in animal models (Berton et al. 2012; Dzirasa and Cov-

ington 2012). Importantly, the lack of alteration in theswimming speed during the performance of the water maze

test indicates that the METH-exposed mice have a normal

motor function, thus ruling out putative motor deficits asresponsible for the observed increased immobility time in

the tail suspension test. Although motor function was not

directly assessed at 49 days post-METH injection, it islikely that it remained normal at this time-point since

Johanson et al. (2006) reported that either recent or long-term abstinent METH abusers did not display major motor

deficits.

Furthermore, we show that this sustained depressive-likebehaviour is associated with a long-lasting disruption of

DA and 5-HT neurotransmission homeostasis in both the

frontal cortex and striatum. In fact, we provide the firstdemonstration that a single high METH dose caused a

long-term DA and 5-HT cortical depletion in mice. Fur-

thermore, our findings demonstrate that DA/metabolites aswell as TH striatal depletion were already present at 3 days

and remained at 49 days post single-METH injection.

Interestingly, this 5-HT depletion is in line with thereported persistent depletion of cortical 5-HT in rats fol-

lowing a single-day multiple METH-injection regimen

(Friedman et al. 1998). In spite of noradrenaline and 5-HTbeing traditionally envisioned as key players in the aeti-

ology of depressive disorders, there is a growing amount of

data focusing on the implication of the dopaminergic sys-tem (Drevets et al. 1999; Lemke et al. 2004; Nestler and

Carlezon 2006; Nutt et al. 2006; Savitz and Drevets (2012).

In this respect, the observed long-term striatal dopami-nergic disruption is consistent with the prolonged striatal

dopaminergic dysfunction triggered by other neurotoxic

regimen, namely single-day multiple METH-injectionsparadigm, in rodents (Hotchkiss et al. 1979; Friedman et al.

1998; O’Callaghan and Miller 1994). Interestingly, this

striatal dopaminergic disruption was not sufficient toimpair striatal-dependent learning, since METH-treated

mice performed normally in the procedural memory ver-

sion of the water maze. This observation is consistent withprevious findings by Grace et al. (2010) demonstrating a

normal cognitive performance of mice in the cued version

of the water maze following a repeated administration ofhigh METH dose (10 mg/kg s.c. 9 4; 2 h interval). In line

with the ‘‘monoamines hypothesis’’, which suggests that a

deficiency or imbalance of the monoaminergic systemmight be a cause of depression (Lee et al. 2010), the

neurochemical data are consistent with the hypothesis that

disturbances of the dopaminergic and serotonergic neuro-transmission might underlie the depressive-like behaviour

observed following a single high METH administration in

the present study. However, it is important to stress that thepresent study only provides a correlation between the

depletion of frontocortical 5-HT and DA and a depressive-

like phenotype. It will be important to design additionalstudies, namely using anti-depressive drugs targeting the

serotoninergic and the dopaminergic system to consolidate

a possible causal relation between the dysfunctionalfrontocortical monoaminergic systems and the emergence

of a sustained depressive-like phenotype after a single high

METH administration. A word of caution is also requiredto highlight that the changes in DA and 5-HT were only

measured in the frontal cortex and in the striatum becausethese are two brain regions with a rich dopaminergic

innervation and that have been critically associated with

mood disorders (Krishnan and Nestler 2010). This shouldnot underscore the putative importance of other brain

regions in the METH-induced depressive-like behaviour.

For instance, it has been shown that a METH regimen ofadministration similar to that used in the present study

caused a 5-HT depletion (Herring et al. 2010), as well as a

transient astrogliosis and microgliosis (Goncalves et al.2010) in the hippocampus, a brain region also involved in

mood control (Fanselow and Dong 2010). Therefore,

although the focus of this study was directed to thefrontostriatal axis, one cannot rule out the contribution of

other brain regions in the adaptive changes caused by a

high METH dose.Our search for other possible correlates of METH-

induced depressive-like behaviour was also extended to the

possible involvement of astroglial changes. Indeed accu-mulating evidence supports the existence of modifications

of astrocytes in depressive symptoms (reviewed in Pav

et al. 2008; Sanacora and Banasr 2013) in view of theimpact of modified astrocytic function on processes rang-

ing from metabolic support, ion homeostasis, control of

oxidative stress, of synaptic plasticity and global processessuch as sleep or memory (Agostinho et al. 2010; Allaman

et al. 2011; Kimelberg and Nedergaard 2010). This is

emphasized by the demonstration that the pharmacologicalablation of frontal cortical astroglia, confirmed by a

decrease in GFAP density, was sufficient to induce

depressive-like behaviours in rats similar to chronic stress(Banasr and Duman 2008). The evaluation of GFAP den-

sity, a marker of astrocytic dysfunction (Pekny and Nilsson

2005), only revealed changes in the striatum, but not in thecortex, which contrast with the previous report by O’Cal-

laghan and Miller (1994) that detected a small cortical

GFAP increase 3 days post-multiple METH-injection reg-imen using ELISA. This suggests that METH only induced

a transitory change in striatal astrocytes, which might

contribute to the early depressive-like behaviour, but isunlikely to be involved in the long-term depressive-like

behaviour caused by a single METH administration.

In summary, the present results unequivocally highlightthe behavioural consequences, namely the long-lasting

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depressive-like behaviour without clear impairment on

motor function, resulting from an acute high dose ofMETH, which are accompanied by a neurotoxic profile on

frontostriatal dopaminergic and serotonergic system.

Although this model mimics an overdose experienced bynaive METH users, it indeed recapitulated the detrimental

monoaminergic effects seen in abstinent METH abusers

following repeated daily or intermittent drug consumption.Therefore, this experimental model is instrumental to pin-

point the neurobiological substrate underlying depressivebehaviour seen in METH addicts.

Acknowledgments This research was supported by PEst-C/SAU/UI3282/2011 and by FCP011 (Faculty of Medicine, University ofCoimbra, Portugal). SDV is a recipient of a PhD grant from Fundacaopara a Ciencia e a Tecnologia (FCT, Portugal, SFRH/BD/78166/2011). The experiments comply with the current laws of Portugal.

Conflict of interest None.

References

Agostinho P, Cunha RA, Oliveira C (2010) Neuroinflammation,oxidative stress and the pathogenesis of Alzheimer’s disease.Curr Pharm Des 16:2766–2778

Allaman I, Belanger M, Magistretti PJ (2011) Astrocyte–neuronmetabolic relationships: for better and for worse. TrendsNeurosci 34:76–87

Banasr M, Duman RS (2008) Glial loss in the prefrontal cortex issufficient to induce depressive-like behaviors. Biol Psychiatry64:863–870

Berton O, Hahn CG, Thase ME (2012) Are we getting closer to validtranslational models for major depression? Science 338:75–79

Bora E, Harrison BJ, Davey CG, Yucel M, Pantelis C (2012) Meta-analysis of volumetric abnormalities in cortico-striatal-pallidal-thalamic circuits in major depressive disorder. Psychol Med42:671–681

Briere FN, Fallu J-S, Janosz M, Pagani LS (2012) Prospectiveassociations between meth/amphetamine (speed) and MDMA(ecstasy) use and depressive symptoms in secondary schoolstudents. J Epidemiol Community Health 66:990–994

Cappon GD, Pu C, Vorhees CV (2000) Time-course of methamphet-amine-induced neurotoxicity in rat caudate-putamen after single-dose treatment. Brain Res 863:106–111

Carvalho M, Carmo H, Costa VM, Capela JP, Pontes H, Remiao F,Carvalho F, Bastos MDL (2012) Toxicity of amphetamines: anupdate. Arch Toxicol 86:1167–1231

Cryan JF, Hoyer D, Markou A (2003) Withdrawal from chronicamphetamine induces depressive-like behavioral effects inrodents. Biol Psychiatry 54:49–58

Cryan JF, Mombereau C, Vassout A (2005) The tail suspension test asa model for assessing antidepressant activity: review ofpharmacological and genetic studies in mice. Neurosci BiobehavRev 29:571–625

Darke S, Kaye S, McKetin R, Duflou J (2008) Major physical andpsychological harms of methamphetamine use. Drug AlcoholRev 27:253–262

Davidson C, Gow AJ, Lee TH, Ellinwood EH (2001) Methamphet-amine neurotoxicity: necrotic and apoptotic mechanisms andrelevance to human abuse and treatment. Brain Res Rev 36:1–22

Drevets WC, Price JC, Kupfer DJ, Kinahan PE, Lopresti B, Holt D,Mathis C (1999) PET measures of amphetamine-induceddopamine release in ventral versus dorsal striatum. Neuropsy-chopharmacology 21:694–709

Dzirasa K, Covington HE 3rd (2012) Increasing the validity ofexperimental models for depression. Ann N Y Acad Sci1265:36–45

El Yacoubi M, Vaugeois JM (2007) Genetic rodent models ofdepression. Curr Opin Pharmacol 7:3–7

European Monitoring Centre for Drugs and Drug Addiction (2012)Annual report 2012: the state of the drugs problem in Europe.Publications Office of the European Union, Luxembourg

Fanselow MS, Dong HW (2010) Are the dorsal and ventralhippocampus functionally distinct structures? Neuron 65:7–19

Friedman SD, Castaneda E, Hodge GK (1998) Long-term monoaminedepletion, differential recovery, and subtle behavioral impair-ment following methamphetamine-induced neurotoxicity. Phar-macol Biochem Behav 61:35–44

Glasner-Edwards S, Marinelli-Casey P, Hillhouse M, Ang A, MooneyLJ, Rawson R (2009) Depression among methamphetamineusers. J Nerv Ment Dis 197:225–231

Goncalves J, Baptista S, Martins T, Milhazes N, Borges F, RibeiroCF, Malva JO, Silva AP (2010) Methamphetamine-inducedneuroinflammation and neuronal dysfunction in the mice hippo-campus: preventive effect of indomethacin. Eur J Neurosci31:315–326

Gouzoulis-Mayfrank E, Daumann J (2009) Neurotoxicity of drugs ofabuse–the case of methylenedioxyamphetamines (MDMA,ecstasy), and amphetamines. Dialogues Clin Neurosci 11:305–317

Grace CE, Schaefer TL, Herring NR, Graham DL, Matthew R,Gudelsky GA, Williams MT, Vorhees CV (2010) Effect of aneurotoxic dose regimen of (?)-methamphetamine on behavior,plasma corticosterone, and brain monoamines in adult C57BL/6mice. Neurotoxicol Teratol 32:346–355

Herring NR, Gudelsky GA, Vorhees CV, Williams MT (2010) (?)-Methamphetamine-induced monoamine reductions and impairedegocentric learning in adrenalectomized rats is independent ofhyperthermia. Synapse 64:773–785

Holmes PV (2003) Rodent models of depression: reexaminingvalidity without anthropomorphic inference. Crit Rev Neurobiol15:143–174

Homer BD, Solomon TM, Moeller RW, Mascia A, DeRaleau L,Halkitis PN (2008) Methamphetamine abuse and impairment ofsocial functioning: a review of the underlying neurophysiologi-cal causes and behavioral implications. Psychol Bull 134:301–310

Hotchkiss AJ, Morgan ME, Gibb JW (1979) The long-term effects ofmultiple doses of methamphetamine on neostriatal tryptophanhydroxylase, tyrosine hydroxylase, choline acetyltransferase andglutamate decarboxylase activities. Life Sci 25:1373–1378

Iijima M, Koike H, Chaki S (2013) Effect of an mGlu2/3 receptorantagonist on depressive behavior induced by withdrawal fromchronic treatment with methamphetamine. Behav Brain Res246:24–28

Imam SZ, Ali SF (2001) Aging increases the susceptibility tomethamphetamine-induced dopaminergic neurotoxicity in rats:correlation with peroxynitrite production and hyperthermia.J Neurochem 78:952–959

Jang C-G, Whitfield T, Schulteis G, Koob GF, Wee S (2013) Adysphoric-like state during early withdrawal from extendedaccess to methamphetamine self-administration in rats. Psycho-pharmacology 225:753–763

Johanson CE, Frey KA, Lundahl LH, Keenan P, Lockhart N, Roll J,Galloway GP, Koeppe RA, Kilbourn MR, Robbins T, Schuster CR(2006) Cognitive function and nigrostriatal markers in abstinentmethamphetamine abusers. Psychopharmacology 185:327–338

Neurotox Res

123

Author's personal copy

Kimelberg HK, Nedergaard M (2010) Functions of astrocytes and theirpotential as therapeutic targets. Neurotherapeutics 7:338–353

Kish SJ, Fitzmaurice PS, Boileau I, Schmunk GA, Ang L-C,Furukawa Y, Chang L-J, Wickham DJ, Sherwin A, Tong J(2009) Brain serotonin transporter in human methamphetamineusers. Psychopharmacology 202:649–661

Kitamura O, Tokunaga I, Gotohda T, Kubo S (2007) Immunohisto-chemical investigation of dopaminergic terminal markers andcaspase-3 activation in the striatum of human methamphetamineusers. Int J Leg Med 121:163–168

Kitamura O, Takeichi T, Wang EL, Tokunaga I, Ishigami A, Kubo S(2010) Microglial and astrocytic changes in the striatum ofmethamphetamine abusers. Leg Med (Tokyo) 12:57–62

Krasnova IN, Cadet JL (2009) Methamphetamine toxicity andmessengers of death. Brain Res Rev 60:379–407

Krishnan V, Nestler EJ (2010) Linking molecules to mood: newinsight into the biology of depression. Am J Psychiatry 167:1305–1320

Lee S, Jeong J, Kwak Y, Park SK (2010) Depression research: whereare we now? Mol Brain 3:8

Lemke M, Fuchs G, Gemende I, Herting B, Oehlwein C, ReichmannH, Rieke J, Volkmann J (2004) Depression and Parkinson’sdisease. J Neurol 251:24–27

London ED, Simon SL, Berman SM, Mandelkern MA, Lichtman AM,Bramen J, Shinn AK, Miotto K, Learn J, Dong Y, Matochik JA,Kurian V, Newton T, Woods R, Rawson R, Ling W (2004)Mood disturbances and regional cerebral metabolic abnormali-ties in recently abstinent methamphetamine abusers. Arch GenPsychiatry 61:73–84

Miller DB, O’Callaghan JP (2003) Elevated environmental temperatureand methamphetamine neurotoxicity. Environ Res 92:48–53

Moszczynska A, Fitzmaurice P, Ang L, Kalasinsky KS, SchmunkGA, Peretti FJ, Aiken SS, Wickham DJ, Kish SJ (2004) Why isParkinsonism not a feature of human methamphetamine users?Brain 127:363–370

Nestler EJ, Carlezon WA (2006) The mesolimbic dopamine rewardcircuit in depression. Biol Psychiatry 59:1151–1159

Nutt DJ, Baldwin DS, Clayton AH, Elgie R, Lecrubier Y, MontejoAL, Papakostas GI, Souery D, Trivedi MH, Tylee A (2006)Consensus statement and research needs: the role of dopamineand norepinephrine in depression and antidepressant treatment.J Clin Psychiatry 67(Suppl 6):46–49

O’Callaghan J, Miller D (1994) Neurotoxicity profiles of substitutedamphetamines in the C57BL/6J mouse. J Pharmacol Exp Ther270:741–751

O’Callaghan JP, Sriram K, Miller DB (2008) Defining ‘‘neuroinflam-mation’’. Ann N Y Acad Sci 1139:318–330

Panenka WJ, Procyshyn RM, Lecomte T, Macewan GW, Flynn SW,Honer WG, Barr AM (2012) Methamphetamine use: a compre-hensive review of molecular, preclinical and clinical findings.Drug Alcohol Depend 129:1–13

Pav M, Kovaru H, Fiserova A, Havrdova E, Lisa V (2008)Neurobiological aspects of depressive disorder and antidepres-sant treatment: role of glia. Physiol Res 57:151–164

Pekny M, Nilsson M (2005) Astrocyte activation and reactive gliosis.Glia 50:427–434

Pereira FC, Lourenco ES, Borges F, Morgadinho T, Ribeiro CF,Macedo TR, Ali SF (2006) Single or multiple injections ofmethamphetamine increased dopamine turnover but did notdecrease tyrosine hydroxylase levels or cleave caspase-3 incaudate-putamen. Synapse 60:185–193

Pereira FC, Cunha-Oliveira T, Viana SD, Travassos AS, Nunes S,Silva C, Prediger RD, Rego AC, Ali SF, Ribeiro CAF (2012)Disruption of striatal glutamatergic/GABAergic homeostasisfollowing acute methamphetamine in mice. Neurotoxicol Teratol34:522–529

Prediger RDS, Batista LC, Medeiros R, Pandolfo P, Florio JC,Takahashi RN (2006) The risk is in the air: intranasal admin-istration of MPTP to rats reproducing clinical features ofParkinson’s disease. Exp Neurol 202:391–403

Rajkowska G, Stockmeier CA (2013) Astrocyte pathology in majordepressive disorder: insights from human postmortem braintissue. Curr Drug Targets 14:1225–1236

Rawson RA, Huber A, Brethen P, Obert J, Gulati V, Shoptaw S, LingW (2002) Status of methamphetamine users 2–5 years afteroutpatient treatment. J Addict Dis 21:107–119

Renoir T, Paızanis E, El Yacoubi M, Saurini F, Hanoun N, Melfort M,Lesch KP, Hamon M, Lanfumey L (2008) Differential long-termeffects of MDMA on the serotoninergic system and hippocampalcell proliferation in 5-HTT knock-out vs. wild-type mice. Int JNeuropsychopharmacol 11:1149–1162

Rowe DC, Stever C, Gard JM, Cleveland HH, Sanders ML,Abramowitz A, Kozol ST, Mohr JH, Sherman SL, WaldmanID (1998) The relation of the dopamine transporter gene (DAT1)to symptoms of internalizing disorders in children. Behav Gen28:215–225

Sailasuta N, Abulseoud O, Harris KC, Ross BD (2010) Glialdysfunction in abstinent methamphetamine abusers. J CerebBlood Flow Metab 30:950–960

Sanacora G, Banasr M (2013) From pathophysiology to novelantidepressant drugs: glial contributions to the pathology andtreatment of mood disorders. Biol Psychiatry 73:1172–1179

Savitz JB, Drevets WC (2012) Neuroreceptor imaging in depression.Neurobiol Dis 52:49–65

Scott JC, Woods SP, Matt GE, Meyer RA, Heaton RK, Atkinson JH,Grant I (2007) Neurocognitive effects of methamphetamine: acritical review and meta-analysis. Neuropsychol Rev 17:275–297

Segman RH, Shalev AY (2003) Genetics of posttraumatic stressdisorder. CNS Spectr 8:693–698

Sekine Y, Ouchi Y, Takei N, Yoshikawa E, Nakamura K, Futatsub-ashi M, Okada H, Minabe Y, Suzuki K, Iwata Y, Tsuchiya KJ,Tsukada H, Iyo M, Mori N (2006) Brain serotonin transporterdensity and aggression in abstinent methamphetamine abusers.Arch Gen Psychiatry 63:90–100

Semple SJ, Grant I, Patterson TL (2005) Negative self-perceptionsand sexual risk behavior among heterosexual methamphetamineusers. Subst Use Misuse 40:1797–1810

Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH,Provenzano MD, Fujimoto EK, Goeke NM, Olson BJ, Klenk DC(1985) Measurement of protein using bicinchoninic acid. AnalBiochem 150:76–85

Steru L, Chermat R, Thierry B, Simon P (1985) A new method forscreening antidepressants in mice. Psychopharmacology 85:367–370

Tulloch I, Afanador L, Mexhitaj I, Ghazaryan N, Garzagongora AG,Angulo JA (2011) A single high dose of methamphetamineinduces apoptotic and necrotic striatal cell loss lasting up to3 months in mice. Neuroscience 193:162–169

Wilson JM, Kalasinsky KS, Levey AI, Bergeron C, Reiber G,Anthony RM, Schmunk GA, Shannak K, Haycock JW, Kish SJ(1996) Striatal dopamine nerve terminal markers in human,chronic methamphetamine users. Nat Med 2:699–703

Xu W, Zhu JPQ, Angulo JA (2005) Induction of striatal pre- andpostsynaptic damage by methamphetamine requires the dopa-mine receptors. Synapse 58:110–121

Zhu JP, Xu W, Angulo JA (2005) Disparity in the temporalappearance of methamphetamine-induced apoptosis and deple-tion of dopamine terminal markers in the striatum of mice. BrainRes 1049:171–181

Zorick T, Nestor L, Miotto K, Sugar C, Hellemann G, Scanlon G,Rawson R, London ED (2010) Withdrawal symptoms inabstinent methamphetamine-dependent subjects. Addiction 105:1809–1818

Neurotox Res

123

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