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Chronic methamphetamine treatment induces oxytocin receptor up-regulation in the

amygdala and hypothalamus via an adenosine A2A receptor-independent mechanism

Authors: Panos Zanosa, Sherie R. Wright

a, Polymnia Georgiou

a, Ji Hoon Yoo

a, Catherine

Ledentb, Susanna Hourani

a, Ian Kitchen

a, Raphaelle Winsky-Sommerer

a and Alexis Bailey

a

a Sleep, Chronobiology & Addiction group, Department of Biochemistry & Physiology,

Faculty of Health and Medical Sciences, University of Surrey, Guildford, GU2 7XH, Surrey,

U.K.

b Institut de Recherche Interdisciplinaire en Biologie Humaine et Moléculaire, Université

Libre de Bruxelles, B- 1070, Belgium

Correspondence: Alexis Bailey, BS.c, Ph.D

Department of Biochemistry & Physiology

Faculty of Health and Medical Sciences

University of Surrey

Guildford, GU27XH, Surrey, U.K

Tel: +44 (0)1483682564

Fax: +44(0)1483 686401

email: a.bailey@surrey.ac.uk

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Abstract

There is mounting evidence that the neuropeptide oxytocin is a possible candidate for the

treatment of drug addiction. Oxytocin was shown to reduce methamphetamine self-

administration, conditioned place-preference, hyperactivity and reinstatement in rodents,

highlighting its potential for the management of methamphetamine addiction. Thus, we

hypothesised that the central endogenous oxytocinergic system is dysregulated following

chronic methamphetamine administration. We tested this hypothesis by examining the effect

of chronic methamphetamine administration on oxytocin receptor density in mice brains with

the use of quantitative receptor autoradiographic binding. Saline (4 mg/kg/day, i.p.) or

methamphetamine (1 mg/kg/day, i.p.) was administered daily for 10 days to male, CD1 mice.

Quantitative autoradiographic mapping of oxytocin receptors was carried out with the use of

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I-vasotocin in brain sections of these animals. Chronic methamphetamine administration

induced a region specific upregulation of oxytocin receptor density in the amygdala and

hypothalamus, but not in the nucleus accumbens and caudate putamen. As there is evidence

suggesting an involvement of central adenosine A2A receptors on central endogenous

oxytocinergic function, we investigated whether these methamphetamine-induced

oxytocinergic neuroadaptations are mediated via an A2A receptor-dependent mechanism. To

test this hypothesis, autoradiographic oxytocin receptor binding was carried out in brain

sections of male CD1 mice lacking A2A receptors which were chronically treated with

methamphetamine (1 mg/kg/day, i.p. for 10 days) or saline. Similar to wild-type animals,

chronic methamphetamine administration induced a region-specific upregulation of oxytocin

receptor binding in the amygdala and hypothalamus of A2A receptor knockout mice and no

genotype effect was observed. These results indicate that chronic methamphetamine use can

induce profound neuroadaptations of the oxytocinergic receptor system in brain regions

associated with stress, emotionality and social bonding and that these neuroadaptations are

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independent on the presence of A2A receptors. These results may at least partly explain some

of the behavioural consequences of chronic methamphetamine use.

Keywords: methamphetamine, oxytocin, A2A receptor, amygdala, hypothalamus

Abbreviations: Acb, nucleus accumbens; AOL, anterior olfactory nucleus-lateral; AOM,

anterior olfactory nucleus-medial; AOV, anterior olfactory nucleus-ventral; Amy, amygdala;

BNST, bed nucleus of stria terminalis; CA2/CA3, regions of the hippocampus; CgCx,

cingulate cortex; CPu, caudate-putamen; DR, dorsal raphe nucleus; FCx, frontal cortex; Hyp,

hypothalamus; KO, knockout; LS, lateral septum; MAP, methamphetamine; MS, medial

septum; NL, neural lobe of the pituitary; OB, olfactory bulb; OT, oxytocin; OTR, oxytocin

receptor; PAG, Periaqueductal gray; Pir Cx, piriform cortex; PVN, paraventricular

hypothalamic nucleus; Sol, Solitary tract nucleus; SC, spinal cord; SON, supraoptic

hypothalamic nucleus; Th, thalamus; Tu, olfactory tubercle; VDB, ventral limb of diagonal

band of Broca; VMH, ventromedial hypothalamus; WT, wild type

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1. Introduction

Methamphetamine (MAP) is a widely abused drug and its use has become of public health

concern in many parts of the world. The relatively easy clandestine manufacture and

consequently low street cost of MAP means that its use has reached near-epidemic

proportions in the United States (see Gonzales et al. , 2010) and multiple cases of epidemic

MAP use in Japan have also been reported (see Wada, 2011). MAP is a highly addictive

psychostimulant drug (Woolverton et al. , 1984) that acutely stimulates dopamine release

and blocks its re-uptake from the synaptic cleft in the striatum (Krasnova and Cadet, 2009),

thus inducing euphoric and psychostimulant effects. Chronic use of MAP causes neurotoxic

effects and has been shown to induce emotional impairment, including social avoidance and

isolation, aggressiveness, depression, anxiety and can also induce psychotic behaviour in

humans (Grant et al. , 2012, McKetin et al. , 2011) and animal models (Clemens et al. , 2007,

Clemens et al. , 2004). Currently, pharmacotherapy for addiction to psychostimulant drugs

including MAP is limited (Ciccarone, 2011).

Although several mechanisms have been suggested, the molecular mechanism underpinning

the addictive properties of chronic methamphetamine use and its behavioural and emotional

consequences remain unclear. Accumulating evidence showing a strong interaction between

neuronal markers of social behaviour and those of stress and mood regulation (Dabrowska et

al. , 2011, Debiec, 2005, Di Simplicio et al. , 2009, Heinrichs and Gaab, 2007, Windle et al. ,

2004) and of drug reward/withdrawal (Liu et al. , 2011, Young et al. , 2011), suggest that the

“social” neuropeptide oxytocin (OT) and its receptor (OTR) may be implicated in the

modulation of at least some of these methamphetamine-induced effects. OT is primarily

synthesized by the magnocellular neurons of the supraoptic (SON) and paraventricular (PVN)

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nuclei of the hypothalamus projecting to the posterior pituitary gland where it is stored in

vesicles and it is released into the bloodstream to exert its peripheral effects (Fig. 1).

Oxytocinergic neurons also project from the PVN and innervate brain regions associated with

drug-seeking behaviour, stress, mood, fear and emotionality such as the nucleus accumbens,

amygdala, septum and the bed nucleus of stria terminalis, where OTRs are located (Gimpl

and Fahrenholz, 2001) (Fig. 1). OT promotes social and sexual bonding and social memory

(Keverne and Curley, 2004) and has a marked anxiolytic, anti-aggressive and antidepressant

effect in humans when administered as an intranasal spray (Baumgartner et al. , 2008, Di

Simplicio et al., 2009, Kirsch et al. , 2005), or in animal models when administered centrally

or peripherally (Dabrowska et al., 2011, Windle et al., 2004). This has triggered a growing

interest in the involvement of OT in psychiatric conditions characterised by symptoms

associated with increased anxiety and social withdrawal, such as autism and schizophrenia

(Carter, 2007, Heinrichs and Gaab, 2007).

There is emerging evidence supporting the involvement of OT in the effects of a number of

drugs of abuse (Broadbear et al. , 2011, McGregor and Bowen, 2012, McGregor et al. , 2008,

Sarnyai, 2011). With respect to MAP, an inhibitory effect of OT has been demonstrated on

MAP-induced hyperlocomotion and conditioned place preference (CPP) (Qi et al. , 2008, Qi

et al. , 2009) and on dopamine release in the Acb (Qi et al., 2008) demonstrating a key role

for OT in the modulation of behavioural effects of MAP, possibly via a dopaminergic-

dependent mechanism. Moreover, central (i.c.v) OT administration abolished stress-induced

reinstatement of MAP seeking in mice (Qi et al., 2009) while peripheral OT administration

reduced MAP self-administration and MAP-induced reinstatement in rats (Carson et al. ,

2010a) supporting the oxytocinergic system as a novel pharmacotherapeutic target for MAP

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relapse prevention. Interestingly, in rats, OT was also recently shown to reduce MAP-induced

Fos activation in neurons of the basal ganglia such as the subthalamic nucleus (STh) and the

Acb core (AcbC) (Carson et al. , 2010b), providing further mechanistic evidence behind OTs

beneficial effect on attenuating at least some of MAPs effects. Additionally, peripheral

administration or microinjections of OT into the STh and Acb core attenuated the formation

of methamphetamine-induced CPP (Baracz et al. , 2012). These findings provide further

support for a key role of the STh and Acb core in the inhibitory effects of OT on the

rewarding properties of methamphetamine.

Another mechanism underlining the beneficial effect of OT on modulating MAPs

behavioural effects may involve an interaction with adenosine A2A receptors. There is

emerging evidence suggesting an involvement of central adenosine A2A receptors on

endogenous oxytocinergic function in the brain. Indeed, the ubiquitous neuromodulator

adenosine has been shown to play an integral role in the neuroendocrine function of SON

neurons (vasopressin and oxytocin containing), via its action on A1 and A2A receptors. While

A1 activation suppresses neurons of the SON (Ponzio and Hatton, 2005), activation of A2A

receptors located on post-synaptic SON neurons and astrocytes causes depolarisation and

excitation of those neurons (Ponzio et al. , 2006). Although yet unknown, it is likely that A2A

receptors may facilitate the excitation of other oxytocinergic neurons (e.g. PVN projections to

the reward centers of the brain). Similarly to the effect of OT, activation of A2A receptors has

a prominent role in modulating MAP behavioural effects including attenuation of MAP-

induced hyperactivity (Shimazoe et al. , 2000) and stimulus discrimination (Munzar et al. ,

2002), at least partly by inhibiting MAP-induced elevation in dopamine output in the striatum

(Golembiowska and Zylewska, 1998, Yoshimatsu et al. , 2001). As a result, it is likely that

OT (endogenous and exogenous) might exert its modulatory effect on MAP-induced

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behaviours via an A2A dependent mechanism. At the receptor level, while structural

interactions between OTR and A2A have yet to be identified, A2A receptors are well known to

be co-localised and form opposing functional interactions with dopamine D2 receptors in the

striatum (Ferre et al. , 2007, Ferre et al. , 2008). As OTR and D2 receptors have recently been

shown to co-localise and facilitate allosteric functional interactions in the nucleus accumbens

(McKetin et al., 2011), it is tempting to hypothesise that OTR (endogenous and exogenous)

may also interact with A2A in the striatum and this interaction might be involved in the

modulatory effect of OT on MAP-induced behavioural effects.

The outcomes of these studies have led us to hypothesise that the central endogenous

oxytocinergic system may be dysregulated following chronic methamphetamine use. We

tested this hypothesis by examining the effect of chronic methamphetamine administration on

oxytocin receptor density in mice brains with the use of quantitative receptor

autoradiographic binding. As there is evidence for interaction between A2A receptors and the

endogenous oxytocinergic system in the brain, we investigated whether possible

methamphetamine-induced oxytocinergic neuroadaptations are mediated via an A2A receptor-

dependent mechanism, with the use of A2A receptor knockout mice.

2. Methods and Materials

2.1 Animals

All animal care and experimental procedures complied with protocols approved by the UK

Home Office, Animals (Scientific Procedures) Act, 1986. All studies are reported in

accordance with the ARRIVE guidelines (McGrath et al. , 2010), and were designed to

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minimise suffering and to reduce the number of animals used. 8-12 weeks old, male CD-1

mice were used in all experiments (total number: 24). The animals were individually housed

in a temperature-controlled environment, under a 12 h light/dark cycle (lights on: 7.00 am).

Food and water were available ad libitum. Wild type and adenosine A2A receptor knockout

(KO) mice, originally generated by Ledent et al., (1997) were obtained from the breeding

colony at the University of Surrey, as previously detailed (Bailey et al. , 2004a, Bailey et al. ,

2002a). Briefly, mutant mice were obtained from heterozygote breeding pairs, which

originated from the individual crossing of a number of heterozygote males from the existing

colony, each with a WT female of the CD-1 strain, purchased from an external supplier

(Charles River, UK). All mice were genotyped at weaning using a polymerase chain reaction

(PCR) based method. Tail tip samples were collected at 3 weeks of age, and DNA was

extracted using the DNeasy tissue kit, according to the manufacturer’s instructions

(QIAGEN, Germany).

2.2 Drug treatment

Mice were randomly divided into four groups; chronic saline-treated WT and KO and chronic

MAP-treated WT and KO animals. Chronic MAP-treated animals were injected

intraperitoneally (i.p.),once per day (11:00am), for 10 days with MAP (1mg/kg) (Sigma-

Aldrich, Poole, UK) in accordance with studies carried out by Liang et al., (2006), with minor

modifications. The chronic saline-treated group were administered with saline (4ml/kg body

weight, i.p.), , once per per day (11:00) for 10 days.

2.3 Oxytocin receptor autoradiography

Animals were killed via cervical dislocation 3 h following the last treatment injection (saline

or MAP) on Day 10 of the steady-dose MAP administration paradigm. Brains were

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immediately removed and frozen in -25oC isopentane solution for 30 seconds and stored at

-80oC until use. Adjacent 20μm coronal brain sections were cut at 300μm distances at -21

°C

using a cryostat apparatus (Zeiss Microm 505E, Hertfordshire, U.K.) and thaw-mounted onto

gelatine-coated ice-cold microscope slides to define receptor binding levels from fore- to

hind-brain regions. Brain slides were stored at -20oC until use. OTR binding was carried out

on sections from chronic saline- and MAP-treated WT and A2A KO mice in accordance with

previously described methods (Jarrett et al. , 2006) with minor modifications. Sections were

rinsed twice for 10 min in a buffer solution (50mM Tris-HCl, pH 7.4, room temperature) to

remove endogenous oxytocin. Total binding was determined by incubating sections with

50pM 125

I-vasotocin (OVTA) (Perkin Elmer, 81.4 TBq/mmol) in an incubation buffer

medium (50mM Tris-HCl, 10mM MgCl2, 1mM EDTA, 0.1 % w/v bovine serum albumin,

0.05 % w/v bacitracin; Sigma-Aldrich, Poole, UK, pH 7.4 at room temperature). Adjacent

sections were incubated with [125

I]-OVTA (50 pM) in the presence of 50μM unlabelled (Thr4

,Gly7)-oxytocin (Bachem, Germany), to determine non-specific binding (NSB). Following a

radioligand binding period of 60 min, slides were rinsed for 3 x 5 min in ice cold rinse buffer

(50 mM Tris-HCl, 10 mM MgCl2, pH 7.4) followed by a 30 min wash in the ice cold rinse

buffer. That was followed by a 2 sec wash in ice cold distilled water. Slides were then dried

under a stream of cool air for 2 hours and stored in sealed containers with anhydrous calcium

sulphate (Drierite-BDH Chemicals, Dorset, U.K.) for 2 days. Slides were then apposed to

Kodak MR-1 films (Sigma-Aldrich, UK) in Hypercassettes with autoradiographic [14

C]

microscales of known radioactive concentration (GE Healthcare Life Sciences, Amersham,

U.K.) for 3 days and developed using 50% Kodak D19 developer (Sigma-Aldrich, Poole,

UK). Quantitative autoradiographic analysis of all structures were carried out by reference to

the mouse brain atlas of Frankin and Paxinos (1997) and binding was analysed as previously

described (Kitchen et al. , 1997), using MCID image analyser (Image Research, Ontario,

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Canada). Briefly, optical density values, which were quantified from autoradiographic [ 14

C]

microscales of known radioactive concentration (GE Healthcare, UK), were entered with

their corresponding radioactivity values into a calibration table, and the relationship between

radioactivity and optical density was subsequently determined using the MCID software.

Sections for all treatment groups were processed in parallel and apposed to the same film at

the same time.

2.4 Confirmation of genotype with autoradiographic binding

To confirm the genotype of the animals used in the present studies, brain sections from the

aforementioned treated WT and KO mice were processed for A2A receptor binding according

to Bailey et al., (2004b), (2002b), (2003). Briefly, sections were pre-incubated for 30 min in

170 mM Tris-HCl buffer, containing 1 mM EDTA (pH 7.4, room temperature). Sections

were then incubated for 2 h at room temperature, in 170 mM Tris-HCl buffer, containing 10

nM [3H]CGS21680 (PerkinElmer; specific activity 30

Ci/mmol), 10 nM MgCl2, and 2 U/ml

adenosine deaminase (pH 7.4). Incubations were terminated by 3 x 5 min washes into ice

cold 50 mM Tris buffer (pH 7.4), followed by a rapid rinse in ice cold distilled water.

Radioligand bound sections were apposed to film for a period of 3 weeks and developed as

previously described above.

2.5 Statistical analysis

Three-way ANOVA for the factors treatment, genotype and brain region were used to

compare receptor binding levels in saline- and MAP- treated, WT and adenosine A2A receptor

KO animals. Moreover, two-way ANOVA was performed in each individual brain region for

factors ‘treatment” and ‘genotype’. Where ANOVA yielded significant main effects, post

hoc Bonferroni analysis was used to determine differences in radioligand binding between

treatment groups.

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Statistical significance was set at level of <0.05. All data were analysed using the

Statistica V10 software (Statsoft Inc., France), and are presented as mean ± SEM of 5-6

animals/group.

3. Results

[3H]-CGS 21680 autoradiography showed restricted localisation of A2A receptors in the

striatum of WT brains (CPu, NAcb, olfactory tubercle) and a complete absence of A2A

receptor binding sites in the brains of homozygous A2A receptor knockout mice, confirming

the genotype of the animals used in this study (Fig. 2). The levels of A2A receptor binding in

the thalamus, hippocampus, septum, amygdala, hypothalamus and cortex was at the level of

background (see Fig. 2)

High levels of OTR binding (1.17-1.46fmol/mg) were observed within the olfactory nuclei,

moderate levels (0.33-0.85 fmol/mg) in the piriform cortex, septum, amygdala and

hypothalamus, and low levels (0.03-0.24fmol/mg) in the cingulate cortex, thalamus, Acb,

olfactory tubercle, caudate-putamen in all treatment groups (Fig. 3, Table 1). Threeway

ANOVA showed significant treatment effect (F[1,273]=13.22, p<0.001) and region effect

(F[14,273]=4.27, p<0.001). No genotype ( p>0.05) or genotype x treatment interaction effects

were observed (p>0.05). Two-way ANOVA for factors ‘treatment’ and ‘genotype’ in each

individual brain region revealed a significant treatment effect in the amygdala (F[1,19]=73.21,

p<0.001) and the hypothalamus (F[1,19]=28.08, p<0.001) but no effect of genotype was

observed in any of these brain regions (p>0.05). Chronic MAP treatment induced similar

degree of OTR upregulation in both WT (78%) and KO mice (54%) in the amygdala and

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52% (WT) and 55% (KO) in the hypothalamus (Table 1). Chronic MAP administration

significantly increased OTR binding in both the amygdala (p<0.001) and the hypothalamus

(p<0.001) of WT and A2A KO mice compared with their respective saline controls

(Bonferroni post-hoc analysis). No genotype or other effect was observed in any other brain

regions analysed (p>0.05).

4. Discussion

This is the first study to demonstrate profound region-specific alterations of the OTR system

in the brain following chronic MAP administration, which may be involved in the modulation

of long-term behavioural and neurochemical adaptations taking place during and following

chronic drug use. We show that in both WT and A2A receptor KO mice, chronic MAP

induces a near 50-80 % increase of OTR binding in the hypothalamus and amygdala,

demonstrating that this dysregulation of the OTR system is not dependent on an A2A receptor

mediated mechanism.

The qualitative binding profile of the OTR in the brain of the CD-1 strain of mice used in the

present study is in accordance with Campbell et al. (2009) and Curley at al., (2009) who also

showed high levels of binding in the olfactory nuclei, septum, amygdala and endopiriform

and lower binding in the striatum, thalamus and hippocampus of mice. This distribution of

OTR is in sharp contrast to the monogamus prairie vole (Lim et al. , 2004) which shows very

high levels of OTR binding in striatal regions including the Acb, the caudate putamen and the

olfactory tubercle clearly indicating a striking species difference with respect to OTR

localization and density. It is generally accepted that these differences may underline species

differences in the ability to form pair bonds (Aragona et al. , 2003, Wang et al. , 1997).

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Although dysregulations of the endogenous oxytocin system have been reported following

acute or repeated alcohol (Kovacs et al. , 1998), cocaine (Sarnyai, 1998, Sarnyai et al. ,

1992), GHB (van Nieuwenhuijzen et al. , 2010) , 3,4-methylenedioxymethamphetamine

(MDMA) (Thompson et al. , 2007) and morphine (Kovacs et al. , 1987) administration in

different brain regions of rodents, this is the first study to report any oxytocinergic

neuroadaptations following chronic MAP administration. The upregulation of the OTR

following chronic MAP treatment was localised in the amygadala and the hypothalamus, two

regions involved in stress regulation. This is in agreement with van Nieuwenhuijzen et al.,

(2009), (2010) who also showed an increase in hypothalamic OTR and OT mRNA levels

following repeated administration of other drugs of abuse (GBH and MDMA respectively) in

the rat; and an increase of OTR binding in the amygdala following treatment with another

psychostimulant (cocaine) in rat dams (Johns et al. , 2004). In contrast to our results, Jarrett

et al., (2006) observed a reduction in OTR binding density in the ventromedial hypothalamus

and failed to demonstrate an increase of OTR binding in the central nucleus of the amygdala

of postpartum rat dams repeatedly treated with cocaine. This discrepancy could reflect

differences in cocaine vs MAP effects, species, methodology and/or simply gender.

Nonetheless, the MAP induced upregulation observed in our study was profound (50-80 %)

and was replicated in genetically modified A2A receptor KO mice.

The significance of this MAP induced upregulation of OTRs is yet to be determined.

However, given the ability of OT to modulate MAP induced hyperactivity (Carson et al.,

2010a, Qi et al., 2008) and to attenuate MAP induced CPP (Qi et al., 2009) and stress- and

priming induced reinstatement of MAP self-administration (Carson et al., 2010a) or CPP (Qi

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et al., 2009), it is highly likely that this oxytocinergic dysfunction may be underlining some

behavioural consequences of chronic MAP use. Indeed, it has been suggested that based on

the ability of OT to supress Fos activity in brain regions associated with motivation and

reward such as subthalamic nucleus (STh) and the Acb (Carson et al. , 2010c), and the ability

of OT to reduce MAP induced dopamine activity in Acb (Qi et al., 2008), OT may be

involved in the modulation of the incentive motivational properties of MAP. Based on these

observations, alterations of OTR would be been expected in the STh and the Acb following

chronic MAP treatment. The reason why these alterations were not observed in these regions

may be due to the relatively low expression of OTR in these regions in the CD1 mouse used

in the present study (see Fig. 3, Table 1). This does not, however, preclude the possibility that

profound changes in OT release occur in these brain regions following chronic MAP

administration, which may indeed lead to long-lasting morphological, functional and

behavioural changes.

Although alterations of OTR binding were not observed in brain regions traditionally

associated with drug dependence, it is important to highlight the possibility of OTR

dysregulation taking place in the amygdala in the present study, to influence dopaminergic

neurotransmission in the Acb and thus motivation. A clear connection between the Acb and

the amygdala has been proposed to control limbic function and motivation. For instance, it

has been suggested that the central amygdala affects tonic dopaminergic tone in the ventral

tegmental area, thereby regulating dopaminergic tone in the Acb and modulating the

incentive value of environmental stimuli (Phillips et al. , 2003). As a result the profound

upregulation of OTR system following chronic MAP administration in the amygdala may

affect dopamine levels in the Acb in order to modulate its addictive-related behavioural

effects.

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The amygdala is a limbic structure which is involved in a number of facets of emotional

analysis (Phelps and LeDoux, 2005) and is a key component of social cognitive circuitry in

both animals and humans (Rosenfeld et al. , 2011). There is considerable evidence

demonstrating abnormalities in the amygdala following chronic MAP use (Kim et al. , 2011,

London et al. , 2004) which is likely to be at least partly responsible for the development of

anxiety, stress, depression (Dawe et al. , 2009, Goeldner et al. , 2011, Morley et al. , 2001,

Thompson et al. , 2004) and social cognitive deficits (Dawe et al., 2009, Volkow et al. ,

2011). Given that OT is known to stimulate oxytocinergic neurons in the amygdala (Carson

et al., 2010c) and that the amygdala is partly responsible for the prosocial, antidepressant and

anxiolytic properties of OT (Baumgartner et al., 2008, Debiec, 2005, Domes et al. , 2007,

Febo et al. , 2005, Febo et al. , 2009), it would be intriguing to speculate that the upregulation

of OTRs in this regions, following chronic MAP administration, may be involved in the

modulation of the behavioural consequences of chronic MAP use including depression,

anxiety and social deficits. Interestingly, Thompson et al., (2007) strongly implicated the

oxytocinergic system of the central medial amygdala in the prosocial effect of the drug

MDMA (“ecstasy”).

MAP induced upregulation of OTR observed in the present study is likely to be the result of

an endogenous central oxytocinergic deficiency which may represent a compensatory

homeostatic mechanism to modulate the anxiety, aggressiveness, depression and social deficit

and drug seeking motivation evident in MAP dependent individuals (Grant et al., 2012,

McKetin et al., 2011) and in animal models (Clemens et al., 2007, Clemens et al., 2004).

Thus, this OTR upregulation may play a protective role in modulating MAP dependence and

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the consequences of chronic MAP use, which is in line with the efficacy of OT in decreasing

MAP self-administration (Carson et al., 2010a).

Interestingly, chronic MAP also induced an upregulation of OTR in the hypothalalamus,

another brain region associated with stress regulation. Given that OT is known to stimulate

oxytocinergic neurons in the hypothalamus (Carson et al., 2010c) and that the hypothalamus

is partly responsible for the antidepressant and anxiolytic properties of OT (Baumgartner et

al., 2008, Dabrowska et al., 2011, Di Simplicio et al., 2009, Kirsch et al., 2005, Windle et al.,

2004), it would be intriguing to speculate, as in the case of the amygdala, that the

upregulation of OTRs in this regions, following chronic MAP administration, may be

involved in the modulation of the behavioural consequences of chronic MAP use including

depression and anxiety. Further studies are needed to test the validity of this speculation.

Based on the evidence suggesting an involvement of central adenosine A2A receptors on the

endogenous oxytocinergic function in the brain (Ponzio et al., 2006), we hypothesised that

the neuroadaptations of the OTR system following chronic MAP administration may be

mediated by an A2A receptor dependent mechanism. However, as in WT mice, the profound

upregulation of OTR following chronic MAP administration was also observed in the

hypothalamus and amygdala of MAP-treated A2A KO mice clearly demonstrating a lack of

A2A involvement in this region-specific upregulation. The reason that we did not observe any

modulation of this MAP-induced upregulation in A2A KO mice might be due to the low

expression of A2A in these brain regions. Although, there is some evidence of A2A expression

in the amygdala (Hume et al. , 1996, Mak et al. , 2012) and hypothalamus (Kokoshka et al. ,

1998, Volkow et al. , 2009), our autoradiographic binding revealed that A2A receptors are

17

almost entirely restricted to the striatum (see Fig. 2). As a result, the lack of A2A receptors in

in those low A2A expressing regions is unlikely to influence OTR binding. This of course

does not preclude the possibility of A2A receptors affecting oxytocinergic function by

modulating endogenous OT release. Indeed, A2A have been shown to be localised

postsynaptically on SON neurons and that activation of those receptors on those neurons and

astrocytes causes depolarisation and excitation of oxytocin and vasopressin containing

neurons (Ponzio et al., 2006). It would be worth investigating if oxytocin release is affected

by A2A deletion in MAP-treated mice and whether A2A receptors are involved in the

modulatory properties of OT on MAP-induced behaviours, by modulating its release.

Interestingly, although A2A receptors functionally interact with D2 receptors restrictedly in the

striatum (Acb, CPu, see Fig. 2), a region where functional interactions between OTR and D2

receptors have been demonstrated (McKetin et al., 2011), no alteration of OTRs were

observed in the striatum of A2AKO mice suggesting a lack on A2A-OTR interaction in the

striatum. This may be due to very low levels of OTR in the caudate putamen and Acb of CD1

mice (Fig. 3). This of course does not exclude the possibility that striatal A2A receptors may

be affecting oxytocinergic function by modulating endogenous OT release in the nucleus

accumbens. Given the presence of strong interaction between D2 and OTR receptors in the

striatum (Romero-Fernandez et al. , 2012), it would be indeed worth investigating the role of

striatal A2A receptors on OT release in the Acb and whether this is involved in the modulatory

properties of OT on MAP-induced behaviours.

It is important to point out here that, despite its significant contribution towards

understanding the mechanisms underlyining the effects of drugs of abuse, full developmental

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KO mouse technology does not come without limitations (see review by Yoo et al., (2012)).

One of the problems arising from studies in KO mice is the emergence of compensatory

adaptations of various systems in the mouse triggered by the gene deletion. For instance, an

upregulation of opioid receptors has been observed in preproenkephalin and preprodynorphin

KO mice (Clarke et al. , 2003). In the case of A2A KO, we showed no compensatory changes

in D2R, MOPr, DOPr, KOPr, 42 and 7 nAChR binding (Bailey et al., 2004a, Bailey et

al., 2002a, Campbell et al., 2009). No change in OTR was also observed in saline-treated A2A

KO vs WT in the present study. As a result, we can conclude that it is unlikely that the

upregulation of OTR in chronically MAP treated WT and A2A KO mice are caused by

compensatory neuroadaptations induced by A2A gene deletion, but rather to the effect of the

psychostimulant itself.

This is the first study to show profound alterations in the endogenous oxytocinergic system

following chronic MAP administration in specific brain regions. We showed that this MAP-

induced upregulation of OTR in the hypothalamus and amygdala does not depend on the

presence of A2A receptors. This may have implications for the delineation of the role of

endogenous OT system in modulating a number of MAP effects including social behaviour,

anxiety, depression and dependence.

5. Acknowledgments

Supported by the European Commission EC (LSHM-CT-2004-005166), FHMS studentship,

The Royal Society research grant (RG120556) and Spyroula and Soteris Zanos. The

sponsors had no involvement in the study design of the study and in the collection, analysis

19

and interpretation of the data nor in the writing of the report and the decision to submit this

article for publication. The authors report no conflict of interest.

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Figure legends

Figure 1. Prominent oxytocinergic projections in the brain of rodents. Oxytocin is primarily

synthesized in the magnocellular neurosecretory cells in the supraoptic (SON) and

paraventricular (PVN) nuclei; Oxytocin is then transported to the posterior pituitary gland

where it is stored in vesicles and is released into the bloodstream to exert its peripheral

effects. Moreover, OT is also synthesised in the parvocellular neurons in the PVN which

29

innervate several extrahypothalamic regions in the CNS including the olfactory nuclei, the

nucleus accumbens, the lateral septum, the amygdala, bed nucleus of the stria termnalis and

also to the brainstem and spinal cord to exert its local effects.

( Magnocellular neurons; Parvocellular neurons)

Abbreviations: Acb, nucleus accumbens; Amy, amygdala; BNST, bed nucleus of stria

terminalis; CgCx, cingulate cortex; CPu, caudate-putamen; DR, dorsal raphe nucleus; FCx,

frontal cortex; LS, lateral septum; NL, neural lobe of the pituitary; OB, olfactory bulb; PAG,

Periaqueductal gray; PVN, paraventricular hypothalamic nucleus; Sol, Solitary tract nucleus;

SC, spinal cord; SON, supraoptic hypothalamic nucleus; Th, thalamus; Tu, olfactory tubercle;

VDB, ventral limb of diagonal band of Broca. Figure founded on reviews and research

publications by Baskerville and Douglas, (2010) , Fuxe et al., (2012), Meyer-Lindenberg et

al., (2011).

Figure 2. Representative computer-enhanced pseudocolour autoradiograms of adjacent

coronal brain sections labelled with [3H]-CGS 21680 from saline and MAP treated wild-type

(WT) and adenosine A2A receptor knockout (KO) mice. Bain sections were incubated for 120

min in [3H]-CGS 21680 (10mM) to label A2A receptors. Sections presented are taken from

the level of the striatum (Bregmas 1.18mm, 0.62mm for first and second row of images

respectively), at the level of the globus pallidus (Bregma -1.22 mm, third row of images) and

at the levels of the thalamus (Bregmas - 1.94 mm, fourth row of images).

Figure 3. Region-specific upregulation of oxytocin receptor binding following chronic

methamphetamine administration in wild-type and A2A receptor knockout mice. Male wild-

type (WT) and A2A knockout (KO) mice were treated with either saline or MAP (1 x 1mg/kg,

i.p. per day) for 10-days. Computer-enhanced representative autoradiograms of adjacent

30

coronal brain sections from chronically saline and MAP treated WT and A2A KO mice

labelled with [125

I]-OVTA (50pM) . The represented images are at the level of the olfactory

nuclei (Bregma 2.46 mm; first row), the caudate (Bregma 0.86mm; second row), the septum

(Bregma 0.14 mm; third row) and the thalamus (Bregma -2.06 mm; fourth row). The colour

bar illustrates a pseudo-colour interpretation of black and white film images in fmol/mg

tissue equivalent. Representative images for the non-specific binding (NSB) (50pM [123

I]-

OVTA in the presence of 50μM unlabelled oxytocin; fifth row) are shown for all the bregma

coordinates.