Investigation of cerebral perfusion changes following MDMA Ecstasy administration in an animal model using bolus tracking arterial spin labelling MRI

OUscoil Atha Cliath The University of Dublin

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Investigation o f cerebral perfusion changes following

MDMA “Ecstasy” administration in an animal model

using bolus-tracking arterial spin labelling MRI

by

Jennifer Rouine

Thesis subm itted for the degree o f Doctor o f Philosophy at the

University o f Dublin, Trinity College

Subm itted October 2011

2 2 OCT 2 iir /

I Declaration

T h is thesis is su b m itte d by th e u n d e r s ig n e d fo r the d e g re e o f D o c to r o f P h ilo so p h y at the U niversity o f D ublin, T rinity C o lle g e and has not been s u b m itte d as an e x e rc ise for a d eg ree at this o r an y o th e r u n iv e rsity and it is entirely m y o w n w o rk . 1 ag re e to d e p o s it this thesis in the U n iv e r s ity ’s o p en a c c e s s institutional re p o sito ry o r allo w th e library to do so on m y behalf, su b ject to Irish C o p y rig h t L egislation and T rin ity C o lle g e Library c o n d itio n s o f use and a c k n o w le d g e m e n t.

T h e recreation al drug o f ab u se 3 ,4 -m e th y le n e d io x y m e th a m p h e ta m in e (M D M A ; E c sta sy ) carries a risk o f cereb ro v a scu la r a c c id e n ts (C V A ) that m ay relate to the role o f seroton in (5 -H T ) and/or d o p a m in e in the reg u la tio n o f c ereb ro v a scu la r ton e. R e cen t a d v a n c e s in m a g n e tic reso n a n c e im a g in g (M R I) h a v e en a b led m easu rem en t o f cerebral b lo o d p erfu sion u sin g con trast a g en t-free ap p ro a ch es su ch a s b o lu s-tra ck in g arterial spin la b e lin g (b tA S L ). T h is in v estig a tio n a sse sse d c h a n g e s in cerebral p erfu sion f o llo w in g sy ste m ic M D M A ad m in istration to rats u sin g b tA S L M R I. A d u lt m a le W istar rats w e r e a d m in istered M D M A (5 or 2 0 m g/k g; i.p .) or sa lin e , a n a e sth etise d 1, 3 or 2 4 hours later and a h igh reso lu tio n an a to m ica l scan fo llo w e d by a c o n tin u o u s A S L (c A S L ) se q u e n c e w a s c o n d u cted u sin g a 7 T esla M RI scan n er. P e r fu sio n -w e ig h te d im a g e s w ere gen erated by su b traction o f la b elled from con trol im a g e s and ex p e rim e n ta l data w a s fitted to a q u an titative m o d el o f cerebral p erfu sio n to gen erate m ean transit tim e (M T T ), cap illary transit tim e (C T T ) and sign al am p litu d e. M T T and C T T are in v e r se ly proportional to cerebral b lo o d flo w (C B F ) and C B F squared r e sp e c tiv e ly , and sig n a l a m p litu d e is proportional to cerebral b lo o d v o lu m e (C B V ). M D M A in d u ced a red u ction in M T T and C T T and an in crea se in sig n a l a m p litu d e in prim ary m otor, se co n d a ry m otor and so m a to se n so r y co rte x I and 3 hours fo llo w in g ad m in istration . S u ch e ffe c ts w ere n o t o b ta in ed in su b -co rtica l re g io n s. T h e acu te e ffe c ts o f M D M A on cerebral p erfu sion m ay g o so m e w a y to w a rd s p r o v id in g a m e ch a n ism to ex p la in the o c c u r ren ce o f C V A in v u ln era b le recreation al e c sta sy users.

e ffec t o f the d o p a m in e D] re c e p to r a n ta g o n is t S C H 2 3 3 9 0 (1 m g /k g ) w a s also d e te rm in e d . W h ile Di re cep to r a n ta g o n ism p ro v o k e d a d ec rea se in cerebral p erfusion in th e visual and parietal asso c ia tio n cortex, it failed to influence the c h a n g e s in cortical p e rfu sio n o b ta in e d w ith M D M A indicating th a t d o p a m in e Di re cep to rs play a role in re g u la tin g b lo o d flow in s o m e brain regions but no t M D M A - r e la te d p erfu sio n c h a n g e s in the frontal cortex. In c o n c lu sio n a lthough 5 - H T depletio n m a y play a role in m e d ia tin g c h a n g e s in cortical p erfu sio n asso c ia te d w ith M D M A ad m in istratio n , m e c h a n is m s in d e p e n d e n t o f 5 - H T such as d irec t d ru g action on, o r 5 -H T and d o p a m in e Di re cep to r in d e p e n d e n t re g u la tio n o f the c e rebral m ic ro v a sc u la tu re unit sh o u ld also be c o n sid ered .

Finally as re peated M D M A e x p o s u r e leads to lo ng-term 5 -H T depletio n , lo ng-term c h a n g e s in C B F and C B V w ere also a sse sse d 8 w e e k s fo llo w in g a re p eated re g im e o f M D M A (5 and 10 m g/k g ; i.p., tw ic e daily for 4 days). P rior e x p o s u r e to M D M A , h a v in g no effect alo n e , atten u a te d p erfusion c h a n g e s asso c ia te d w ith ac u te M D M A (2 0 m g /k g ) c h a lle n g e . In a d d itio n , prior M D M A e x p o s u r e w a s asso c ia te d with a long-term re d u ctio n in cortical 5- H T co n c en tratio n . T h e results sug g est that a functional deficit d e v e lo p s w ith prio r e x p o s u r e in relation to c e re b r o v a s c u la r to n e an d /o r n e u r o v a s c u la r c o u p lin g in r e s p o n se to acute ch a lle n g e. T h e results h av e im plica tions in relation to long-term d eficits in the re g u la tio n o f cerebral perfusion a sso c ia te d w ith prio r M D M A ex p o su re .

Firstly, 1 wish to thank my supervisor Dr. A ndrew Harkin. Your constant guidance, support

and insight were invaluable and 1 am so grateful for all o f your help over the last four years.

I would also like to say a big thanks to OH and Rustam who helped me so much with

learning about the MRl and how to conduct my scans to the best o f my ability. A big thanks

is also due to Ann in Pharmacy who gave up so many o f her m ornings to help me

photograph my Evans blue infused brains.

A very special thanks goes to Valentina Gigliucci for all o f her dedication to m aking me the

best “master apprentice” in HPLC that she possibly could. I never would have gotten all o f

it done without your help ;-).

In addition a big thank you must go to all o f the m em bers o f the TC/A H lab, both past and

present. In particular I would like to thank Lorna and Natacha for teaching me both lab and

in vivo

techniques that 1 have used over the course o f my research. 1 would also like to say a

big thanks to Aine, Jen, Katie, Eimear, Martina, Barry, Shane, Eadaoin, Raasay and Sinead

for making my years here in TC IN fun and full o f good m emories ©.

IV Table of Contents

I D e clara tio n i

II S u m m a r y ii

III A c k n o w le d g e m e n ts iv

IV T a b le o f C o n te n ts v

V List o f F igures ix

VI List o f T a b le s xi

VII A b b re v ia tio n s xii

1. I n tr o d u c tio n

1.1 3 ,4 - m e th y le n e d io x y m e th a m p h e ta m in e 3

1.1.1 In troduc tion 3

1.1.2 R ecreational use o f M D M A “ E c s ta s y ” 5

1.1.3 M D M A to xicity 7

1.1.4 M D M A n e u ro to x ic ity 9

1.2 M a g n e tic R e s o n a n c e Im ag in g 11

1.3 A rterial Spin L ab e llin g 13

1.4 C ere b ral B lood F lo w 15

1.4.1 In troduc tion 15

1.4.2 N e u r o v a s c u la r C o u p lin g 16

1.4.3 R eg u latio n o f C ere b ral B lo o d F lo w 17

(a) E ndothelial C ells 17

(b) P ericy te s 18

(c) N e u ro tra n s m itte rs 20

1.5 S u m m a r y 27

2.1 M ateria ls 33

2.1.1 A n im a ls 33

2.1.2 E x p erim e n ta l T re a tm e n ts 33 2.1.3 H igh P e rf o rm a n c e Liquid C h ro m a to g ra p h y

R ea g e n ts 33

2.1.4 M R I re a g e n ts 34

2.1.5 E v a n s blue assa y re a g e n ts 34 2.1.6 G eneral L a b o r a to ry C h e m ic a ls 34 2.1.7 G eneral L a b o r a to ry Plastics 34

2.1.8 A n a e sth e tic s 35

2.2 M e th o d s 36

2.2.1 A n im a ls 36

2.2.2 D rug P repa ration and A d m in istra tio n 36 2.2.3 M o n ito rin g B o d y T e m p e r a tu r e 36 2.2.4 A n a e sth e sia an d A n im a l P repa ration 37 2.2.5 M ag n e tic R e s o n a n c e Im ag in g 38 2.2.6 Test for E x tra v a sa tio n o f E v an s B lue 41 2.2.7 High P e rf o rm a n c e Liquid C h ro m a to g ra p h y 42

2.2.8 Statistical A n a ly s is 43

3. R e g io n a l, tim e an d d o se d e p e n d e n t e ffe c ts o f M D M A “ E c s ta s y ” on ce reb ra l p erfu sio n d e te r m in e d by b o lu s-tr a c k in g a r te r ia l sp in la b e llin g (b tA S L ) M R I

3.1 In troduc tion 47

3.2 E x p erim e n ta l P ro c e d u re 54

3.3 R esu lts 55

3.3.1 M D M A p r o v o k e s a tim e d e p e n d e n t d e c re a se in M T T and C T T an d an increase in signal a m p litu d e

M T T a n d C T T w ith a c o r r e s p o n d i n g i n c r e a s e in

s ig n a l a m p l i t u d e in t h e p r i m a r y m o t o r c o r t e x 65 3 .3 .3 C o r tic a l a n d stria ta l 5 - H T a n d 5 - H I A A

c o n c e n t r a t i o n f o l l o w i n g M D M A a d m i n i s t r a t i o n 72

3 .4 D i s c u s s i o n 7 6

4. Investigation o f the role o f 5-H T and d opam ine in m ediating increased cortical

perfusion follow ing M D M A “ E cstasy”

4.1 I n t r o d u c t i o n 85

4 .2 E x p e r i m e n t a l P r o c e d u r e 89 S t u d y I : C a n t h e 5 - H T r e l e a s i n g a g e n t f e n f l u r a m i n e o r t h e 5- H T2 r e c e p t o r a g o n i s t D O l m i m i c c h a n g e s in c o r t i c a l p e r f u s i o n

a s s o c i a t e d w i t h M D M A ? 89 S t u d y 2: C a n c e n tr a l 5 - H T d e p l e t i o n o r 5 - H T r e c e p t o r b l o c k a d e i n f l u e n c e M D M A - i n d u c e d c h a n g e s in c o r t i c a l p e r f u s i o n ? 8 9 S t u d y 3: C a n b l o c k a d e o f t h e 5 - H T t r a n s p o r t e r p r e v e n t M D M A - i n d u c e d c h a n g e s in c o rtic a l p e r f u s i o n ? 9 0 S t u d y 4: C a n p r i o r t r e a t m e n t w ith th e s e l e c t i v e d o p a m i n e

D|/5 r e c e p t o r a n t a g o n i s t , S C H 2 3 3 9 0 , i n f l u e n c e M D M A - i n d u c e d c h a n g e s in c o r tic a l p e r f u s i o n ? 9 0

4 .3 R e s u l t s 91

4.3.1 F e n f l u r a m i n e , b u t n o t D O I , m i m i c s M D M A - i n d u c e d c h a n g e s in c o r tic a l p e r f u s i o n 91 4 . 3 . 2 5 - H T d e p l e t i o n p r o v o k e s an i n c r e a s e in c o r tic a l

p e r f u s i o n a n d p o t e n t i a t e s M D M A r e la te d c h a n g e s 9 9 4 .3 .3 M D M A - i n d u c e d c h a n g e s in c o r t i c a l p e r f u s i o n

4.4.1 Increased cortical perfusion follow ing M D M A

is mimicked by 5-HT depletion

115

4.4.2 Increased cortical perfusion follow ing M D M A

is not mediated by 5-HT depletion

116

4.4.3 A role for dopamine?

117

4.4.4 A role for direct vascular actions o f M D M A ?

118

4.4.5 Concluding remarks

119

5. Investigation of the long-term effects of repeated M D M A “Ectsasy” exposure on

cerebral cortical perfusion with btASL M R I in rats

5.1 Introduction

123

5.2 Experimental Procedure

130

5.3 Results

131

5.3.1 Prior exposure to M D M A has no effect alone

but attenuates increased cerebral cortical perfusion

induced by acute M D M A challenge

131

5.3.2 Cortical 5-HT concentration in response to prior

M D M A exposure and acute M D M A challenge

139

5.4 Discussion

141

6. Discussion

6.1 Discussion

149

6.2 Future Directions

157

References

159

V III

Appendix

x v ii

V List o f Figures

F igure 1.1.1 S tru ctu re o f a m p h e ta m in e an d its d erivatives

F igure 1.2 Protons, o r h y d ro g e n ions, a lig n in g parallel and anti-parallel to a m a g n e tic field F igure 1.3 S c h e m a tic d ep ictin g th e b tA S L M R l te c h n iq u e

F igure 1.4.1 S c h e m a tic o f brain v asc u la tu re

Figure 1.4.3 S chem atic o f the neuro v ascu lar unit and the role it has to play in m odu latin g cerebral blood flow

F igure 3.3.1.1 M D M A p ro v o k e s a tim e d e p e n d e n t d ec rea se in M T T and CTT w ith a c o r re sp o n d in g increase in signal a m p litu d e in the p rim a ry m o to r corte x

F ig u re 3.3.1.2 M R im ag es re p re se n tin g the a p p e a ra n c e and cle a ra n c e o f co n tra st a g e n t o v e r tim e fo llo w in g in trav e n o u s adm in istratio n

F igure 3.3.2 D o s e - d e p e n d e n t d e c re a se s in M T T and C T T and increase in signal a m p litu d e in the prim ary m o to r cortex

F igure 3.3.3 M D M A p ro v o k e s a tim e d e p e n d e n t d e c re a se in 5 -H T and 5 -H I A A co n c en tratio n

F igure 4.3.1.1 F en flu ra m in e , like M D M A , p ro v o k e s a d e c re a se in M T T and C T T w ith a c o rre s p o n d in g increase in signal a m p litu d e in the p rim a ry m o to r cortex

F igure 4.3.1.2 C ortical 5 -H T c o n c e n tra tio n fo llo w in g fe n flu ra m in e a d m in istra tio n and asso c ia te d re p resen ta tiv e blood v o lu m e m ap s

representative blood volume maps

Figure 4.3.3 Cortical 5-HT concentration follow ing citalopram pre-treatment and associated

representative blood volume maps

Figure 5.3.1 Prior exposure to M D M A attenuates increased cerebral cortical perfusion

induced by acute M D M A challenge

VI List o f Tables

Table 3.3.1 M D M A provokes a time dependent decrease in M T T and C T T with a

corresponding increase in signal amplitude in cortex

Table 3.3.2 M D M A provokes a dose-dependent decrease in M TT and C T T with a

corresponding increase in amplitude in cortex

Table 4.3.1 Fenfluramine and M DM A provoke a decrease in M T T and C T T with a

corresponding increase in amplitude in cortex

Table 4.3.2 pCPA potentiates M DM A -induced decreases in M TT and C T T and

corresponding increase in amplitude in cortex

°C d e g r e e s C e lsiu s

5 -H IA A 5 -h y d r o x y in d o le -a c e tic acid

5 -H T ser o to n in /5 -h y d ro x y tr y p ta m in e

5 -H Tia type-1 a sero to n in receptor

5 -H Tid t y p e - 1 D seroton in recep tor 5 -H T 1 Du t y p e - 1 D a sero to n in recep tor 5 -H T 1 DP t y p e - 1 D p seroton in recep tor

5-H T2 ty p e -2 sero to n in receptor

5-H T2A ty p e -2 A seroton in recep tor 5-H T2B ty p e -2 B sero to n in receptor 5-H T2C ty p e -2 C sero to n in receptor

5-H T7 ty p e -7 sero to n in receptor

5 -H T -lR sero to n in im m u n o re a c tiv e

A D P a d e n o sin e d ip h osp h ate

A N O V A a n a ly sis o f varian ce

A S L arterial sp in la b e llin g

A T P a d e n o sin e trip h osp h ate

b tA S L b o lu s-tra c k in g arterial sp in la b e llin g

B B B b lo o d -b ra in barrier

B O L D b lo o d -o x y g e n le v e l d ep en d en t

Ca^"^ ca lc iu m

c r ch lo rid e

C B F cerebral b lo o d f l o w

C B V cerebral bloo d v o l u m e

C N S central n e r v o u s s y s t e m

C T T c apillary transit t im e

C V A cer e b r ov asc u lar a c c id e n t

Di t y p e- 1 d o p a m in e receptor

D2 t y p e- 2 d o p a m in e receptor

D3 ty p e -3 d o p a m in e receptor

D4 t y p e - 4 d o p a m in e receptor

D5 ty p e -5 d o p a m in e receptor

D A d o p a m in e

D A G d ia c y lg ly c e r o l

D A T d o p a m in e transporter

D O ! 1 -( 2 ,5 - d im e t h o x y - 4 - i o d o p h e n y l) - 2 - a m in o p r o p a n e E D T A e th y le n e d ia m in e te tr a -a c e tic acid

e N O S e n d o th e lial nitric o x i d e sy nthase

ET-1 e n d o t h e liu m - d e r iv e d c o n str ic tin g factor fM RI functional m a g n e tic r e so n a n c e im a g in g

g gram

G A B A y - a m in o butyric acid

H P L C high p e r for m an ce liquid c h r om atogr a p h y

H S P 9 0 heat s h o c k protein

L C B F local cerebral blood flow

M C A m id d le cerebral artery

M D A 3 ,4 -m e th y le n e d io x y a m p h e ta m in e

M D M A 3 ,4 -m e th y le n e d io x y m e th a m p h e ta m in e

M R l m a g n e tic re so n a n c e im ag in g

M T T m e a n transit tim e

m R N A m e s s e n g e r R N A

N A n o ra d re n a lin e

N a H 2 P 0 4 so d iu m d ih y d ro g e n p h o sp h a te

NaCI so d iu m chloride

N a O H s o d iu m h y d ro x id e

n g n a n o g ra m

N O nitric o x id e

p C P A /? « r a -c h lo ro p h e n y a la n in e

P K C protein kinase C

r C B F regional cerebral blood flow

rC B V regional cerebral blood v o lu m e

r f radio fr eq u en c y

rpm re v o lu tio n s p er m in u te

R T - P C R re v erse tra n sc rip tio n - p o ly m e r a se chain reaction

sec s e c o n d s

s.c. s u b c u ta n e o u s

S C H 2 3 3 9 0 7 c h lo r o 3 m e th y 11 p h e n y l 1,2,4 ,5 tetrah y d ro 3

S E M standard erro r o f th e m ean

S E R T sero to n in tra n sp o rte r

S S R l selec tive sero to n in re u p ta k e in h ibitor

T C A trich lo ro a cetic acid

V M A T v e sic u la r m o n o a m in e tra n sp o rte r

v/v v o lu m e p er v o lu m e

Chapter 1

Chapter I: Introduction

1.1 3,4-m ethylenedioxym ethamphetamine (MDMA)

I . l . l Introduction

3 ,4 -m e th y le n e d io x y m e th a m p h e ta m in e ( M D M A ) is a re ad ily a v a ila b le illicit p s y c h o a c tiv e drug. M D M A w a s first sy nthesised and p a te n te d by the G e rm a n p h a rm a c e u tic a l c o m p a n y M e r c k in 1914. M D M A is a sy n th etic d ru g an d m e m b e r o f th e a m p h e ta m in e fa m ily o f drugs. It is rin g -su b stitu te d and shares a sim ilar structure to o th e r a m p h e ta m in e d e riv a tiv e s in clu d in g m e th a m p h e ta m in e , 3 ,4 - m e th y le n e d io x y a m p h e ta m in e ( M D A ) an d the h allu c in o g e n m e sc a lin e (F ig u re 1.1.1).

..NH, CH, am pheiam iiic

NHCII

m etham phetam m e

NH,

3.4-m cthy)cnedk)xyam phetanw «e (MDA)

<

1

H

O il,

<:i

N n o i , CH ,

Cl

•NH, CH, 3.4-iiK ih>leiieJ«>xyclham phctam ine 3.4-m cth>leiiedK )xym elham phetam ine p<ifn-chk>roatnplKftaniitur

(VU)HA) (MDMA) (PCA)

F.C NHCH.CH,

fcn tliiram iiie

C H ,0

NH. C H ,0 C H ,0 CM,

NK,

O CH ,

p tiro-nicihoxyam phetam ine 3,4.5-inm elhoxyphcncthyianiin«

CPMA) (m escalincj

Chapter 1: Introduction

am phetam ine, by the presence o f a m eth ylen ed ioxy group attached to p osition s 3 and 4 o f the arom atic ring o f the am phetam ine m olecule. T his group o f com pounds also includes

3,4-m ethylen ed ioxyeth am p h etam in e (M D E A ), and m ethylenedioxyam phetam ine (M D A ) w hich are c lo se ly related to M D M A and share many o f its properties. N either am phetam ine

nor its derivatives are found in nature and are com p letely synthetic substances. T hey structurally resem ble adrenaline and dopam ine, with the substituted am phetam ines also resem bling serotonin (5-H T ), and act to enhance neurotransmitter release into the synaptic cleft. M D M A acts as a m onoam ine releaser, a direct and indirect m onoam inergic agonist

and a m onoam ine re-uptake inhibitor in the brain, it binds to all three o f the m onoam ine pre-synaptic transporters (Green et a l , 2 0 0 3 ), but has the highest affinity for the serotonin (5-H T ) transporter (SE R T ) and acts m ainly on the serotonergic system . It acts to a lesser extent on the dopam inergic and the noradrenergic system s resulting in increased release o f th ese neurotransmitters. Crespi and co llea g u es (1 9 9 7 ) have show n that the release o f 5-H T and dopam ine is both carrier-mediated and calcium -dependent (Ca^^-dependent) with M D M A acting to increase cyto so lic Ca^"^ lev els in neuronal term inals, thereby inducing ex o c y to sis. M D M A also binds to various receptors and its in vitro pharm acological profile

ranks its affin ities at th ese receptors as fo llo w s (B attaglia et a l , 1988; D e S ouza &

Battaglia, 1989): 5-H T uptake > a2 adrenergic = 5-H T i = Mi m uscarinic = a i adrenergic =

1.1.2 Recreational use o f MDMA “E c s ta sy ’

M DM A, when used recreationally, is usually taken orally in a tablet form referred to as

“ecstasy”, with tablets generally containing 50 - 150 mg o f the drug. The relative purity o f

the tablets varies and they have been shown to contain any amount o f extraneous

substances including caffeine, ephedrine, ketamine, paracetamol, LSD and other

amphetamine derivatives (Freese

et a i ,

2002; Parrott, 2004). Patterns o f ecstasy use vary

between countries with a high prevalence for binge use in the United Kingdom, 25% o f

subjects taking 4 or more tablets per session (W instock et a i , 2001).

Onset o f effects are typically observed between 20 and 60 min follow ing ingestion and

peak concentrations are observed at 1.5 - 3 hr with the primary effects o f the drug lasting

between 3 and 5 hr (Green

et al.. 2003). The half life (T

hr. MDMA undergoes metabolism by common metabolic pathways in the liver via several

cytochrome P450 enzym es including CYP2D6 and over a dozen metabolites o f M DM A

have been identified in animals and humans (Green

et a i ,

2003; Kreth

et a i , 2000).

Demethylation o f MDM A which produces reactive catechols is a major degradation step, as

is a parallel side chain pathway initiated by N-dem ethylation to form M DA (Chu

et a i ,

1996). An aromatic hydroxylation pathway also exists, and has been proposed to result in

the

production

o f

trihydroxymethamphetamine

via

6-hydroxymethyienedioxymethamphetamine. The main metabolites o f MDM A and MDA are

4-hydroxy-3-methoxymetamphetamine (HM M A) and 4-hydroxy-3-m ethoxyam phetamine

(HM A) (Green

et a i , 2003). An animal model o f CYP2D6/D1 deficiency, the female Dark

Agouti rat, is w idely used in MDMA research as these rats are poor metabolisers o f

Chapter 1: Introduction

Agouti rats when compared to Sprague Dawley rats. It has been estimated that between 5 -

10% o f Caucasians are deficient in this particular enzym e and are classified as poor

metabolisers (Gonzalez

et a i, 1988). It has been proposed that poor metabolism may

account for some apparently inexplicable or idiosyncratic toxic reactions to the drug

(Tucker

et a l, 1994). The kidneys are the main excretory organs and M DM A has a non­

linear pharmacokinetic profile, probably due to either a saturable or inhibitable metabolic

pathway (de la Torre

et a l, 2004; Farre

et a i, 2004) increasing the chance o f accidental

overdose. There are 2 stereoisomers o f M D M A with S(+) being metabolised faster than R(-

) and demonstrating greater neurotoxicity in the rat (Kalant, 2001).

Increased synaptic 5-HT availability is believed to be responsible for the feelings o f

euphoria and enhanced confidence in addition to increased feelings o f serenity and

calm ness (Liechti

et a i, 2000a,b; Verheyden

et a i, 2003). Many studies have reported a

rapid increase in release o f 5-HT following M D M A administration using

in vivo

m icrodialysis (M eehan

et al., 2002; Shankaran and Gudelsky, 1999) and using

in vitro

approaches (Koch and Galloway, 1997; O ’ Loinsigh

et al., 2001). This is followed by a

pronounced decrease in brain levels o f 5-HT and its primary metabolite,

5-

hydroxyindoleacetic acid (5-H lA A ) and the activity o f the 5-HT synthesising enzym e

tryptophan hydroxylase. Within 24 hr brain 5-HT levels recover to normal baseline values

but 3 days following drug administration a sustained and regionally specific depletion o f 5-

HT and 5-HIAA is seen which has been shown to persist for up to 12 months in the rat

(Battaglia

et a l, 1987; Baumann

et a i, 2007; Harkin

et a l, 2001; M cK enna and Peroutka,

With respect to dopamine (DA) release, there is evidence that MDMA elicits this effect via

5-HT release (Koch and Galloway, 1997) via the

5-HT2A receptor (Nash, 1990) and via a

carrier-mediated mechanism independent o f 5-HT release (Nash and Brodkin, 1991). The

positive effects o f MDMA decrease while the negative effects increase with respondents

reporting an increasing tolerance to the drug with repeated use (Solow ij

et a l ,

1992;

W instock et a l ,

2001).

1.1.3 MDMA toxicity

The toxicity o f MDMA which is exhibited both peripherally and centrally has been

extensively reviewed elsewhere (Green

et a l ,

2003). It has been estimated that ingestion o f

the drug results in the deaths o f 15 persons per year in the UK. Nevertheless at the height o f

its popularity, when approximately 500,000 people consumed the drug in an uncontrolled

manner every week in the UK, it became evident that MDMA is actually not very

dangerous or toxic in the short-term. The major concern relating to MDM A is its putative

long-term neurotoxic effects that may not be apparent for many years after consumption.

Chapter I: Introduction

been reported to cause a marked hyperthermic response (Che

el al., 1995; Dafters, 1994;

O ’Shea

et al., 1998). However, at ambient temperatures below 20 - 22°C, a hypothermic

response has been observed following M DM A administration to rats (Marston

et al., 1999).

Dafters & Lynch (1998) observed that ambient temperatures o f 17°C resulted in a

hypothermic response in rats follow ing administration o f M DM A (10 - 15 mg/kg; s.c.).

These findings indicate that M DM A has a profound effect on thermoregulatory

mechanisms and that the substance is highly sensitive to external temperature changes. It

has been reported that acute 5-HT release is not directly responsible for hyperthermia, but

that 5-HT receptors modulate the response (Docherty & Green, 2010; Green

et a l ,

2004,

for review). In addition dopaminergic Di receptors and a p ,

a

been implicated in the M DM A-induced hyperthermic response (Docherty & Green, 2010).

It is thought that MDMA may compromise thermoregulation or the body’s ability to

maintain a stable core body temperature despite changes in ambient temperature. This is o f

considerable relevance to human M DM A use as the vast majority o f MDMA consumption

occurs at “raves” where a high ambient temperature, overcrowding and excessive dancing

greatly influence the effects o f the drug (Parrott, 2011).

In addition to the thermoregulatory changes observed follow ing MDMA administration

further physiological changes have been reported. In rats “serotonin syndrome” is observed

follow ing

administration

o f

M DM A.

This

behavioural

syndrome

includes

hyperlocomotion, flattened body posture, head weaving, piloerection, hind limb retraction,

Straub tail, sweating and forepaw treading (Colado

et al., 1993; de Souza

et a l ,

1997;

Marston

et a l ,

1999; Shankaran & Gudelsky, 1999). MDMA administration to animals

stimulatory effects resulting in tachycardia and arrhythm ia in rats (Dum ont

et a l, 2009;

Gordon

et a l, 1991; O ’Cain

et a l,

2000; Vanattou-Saifoudine

et al., 2010b) and increased

blood pressure (Broadley, 2010). The num ber o f M D M A associated hospital admissions

presenting with cardiovascular toxicity suggests that M D M A profoundly affects parameters

such as heart rate and arterial pressure (Henry

et al., 1992) however, the cardiovascular

actions o f M D M A have not been well characterised. It is difficult to carry out studies in

hum ans m im icking the uncontrolled conditions the drug is normally taken under, such as

overcrowding, excessive dancing and loud music. From clinical studies that have been

conducted - albeit in a more controlled environm ent than that in which the drug is typically

consumed - M D M A has been shown to produce a m odest tachycardia and hypertension

(Downing, 1986; Mas

et al., 1999; Verheyden

et al., 2003; Vollenweider

et a l, 1998)

although these studies also reported severe responses in certain individuals. When

administered acutely in recreational doses to human volunteers (0.25 - 1.9 mg/kg; p.o.),

M D M A increased cardiovascular activity, which peaked between I and 2 hr following

administration (de la Torre

et a l,

2000a,b; Lester

et a l, 2000; Liechti & Vollenweider,

2000a,b). It could therefore be the case that M D M A exacerbates latent cardiovascular

problems and could pose serious threats in a dance club setting. The physiological changes

alluded to here are the most com m on indicators o f M D M A -induced toxicity.

1.1.4 MDMA neurotoxicity

With respect to long-term effects, 5-H T neurons appear to be alm ost exclusively

susceptible to dam age by M D M A in prim ates and rats (Bankson & Cunningham, 2001;

Colado

et a l, 2004; Green

et a l, 2003; Gudelsky & Yam am oto, 2008; Shankaran &

Chapter 1: Introduction

following M D M A administration. Battaglia and colleagues (1987) and others (M cCann

et

a l, 1998; Reneman

et al., 2001) have reported significant reductions (up to 60 - 70%) in 5-

H T uptake sites following M DM A (20 mg/kg; s.c., twice daily for 4 consecutive days)

administration to rats in comparison to vehicle treated control animals, indicative o f a

reduction in 5-HT nerve terminal integrity. Immunoreactive 5-HT axon density was

quantified by Hatzidimitriou and colleagues (1999) in various brain regions following

M D M A administration (5 mg/kg; s.c., twice daily for 4 consecutive days) to non-human

primates. 83 - 95% reductions in 5-HT immunoreactive (5-HT-lR) axon density were

reported in cerebral cortex two weeks following M D M A administration. Seven years after

treatment with M D M A , reductions in 5-HT-IR were still evident but significant recovery

had occurred in comparison to the two week response. There are two major 5-HT

projections from the raphe nuclei to forebrain areas and immunocytochemistry studies in

animals have shown a differential vulnerability to the neurotoxic effects o f M DM A . Fine 5-

HT axons arising from the dorsal raphe nucleus display an enhanced vulnerability while

beaded 5-HT axons originating from the median raphe nucleus are spared (M am ounas &

Molliver, 1988; Molliver

et al., 1990). Retrograde degeneration does not seem to occur,

leaving cell bodies in the raphe nuclei intact and there is evidence that damaged terminals

can recover (Battaglia

et a l, 1988; M ayerhofer

et a l, 2001). M D M A administration to

mice also results in changes, to a lesser extent, in the concentration o f the catecholamines,

dopam ine (DA) (Bankson & Cunningham, 2001; Colado

et al., 2004; Green

et a l, 2003;

Gudelsky & Y am am oto, 2008; Shankaran & Gudelsky, 1998) and noradrenaline (NA)

(Green et a l, 2003; Rothman

et a l, 2001).

showed a 50 - 80% depletion o f 5-HT and 5-HIAA in the brain o f a chronic M D M A user,

while dopamine concentrations were unaffected. In addition, hum an studies using

neuroimaging techniques have indicated 5-HT neuronal dam age following M D M A

administration (M cC ann

et al, 1998; Obrocki

et al., 2002; Semple

et ai, 1999) but caution

is advised in relation to the interpretation o f these findings (de Win

et al, 2004; Kish, 2002;

Thom asius

et al, 2003). Impairment o f 5-HT function is also supported by blunted

responses to challenge with the 5-HT releasing agent D-fenfluram ine (Gerra

et al, 1998;

2000) and reduced 5-HlAA in the cerebrospinal fluid o f abstinent M D M A users (M cCann

et al, 1994). There has been some speculation that M D M A itself does not mediate the

neurotoxicity (Esteban

et al, 2001; Paris & Cunningham, 1992) and that it may, in fact, be

the products o f metabolism which are taken into the 5-HT neuron which are responsible

(Bai

et al, 2001; Cadet & Brannock, 1998; Capela

et al, 2007; Carvalho

et al, 2004;

Colado et al, 1995; de la Torre

et al, 2004; Jones et al, 2005). In addition oxidative stress

has also been implicated in M DM A -induced neurotxicity (Puerta

et al, 2010; Steinkellner

et al, 2011; Yam am oto & Raudensky, 2008).

1.2 M agnetic Resonance Imaging

Magnetic resonance imaging (MRI) is an imaging technique which m ay be employed in

both clinical and pre-clinical investigations to obtain a high quality image o f the interior o f

the brain.

The use o f MRI, in hum ans and animals, is possible due to the fact that body tissues are

Chapter I: Introduction

large n u m b e rs o f h y d ro g e n a to m s w h ic h c o m p ris e u npaire d protons. T h e se unpaired

pro to n s po ssess a p h e n o m e n o n k n o w n as “s p in ” . T h e spin o f an u n p aire d proton allo w s

pro to n s to line up w ith (parallel fo rm a tio n ) o r ag a in st (anti-parallel fo rm a tio n ) a m ag n etic

field, fo llo w in g ap p lica tio n o f a m a g n e tic field. In M R l, a m ag n etic field is generated b y a

m ag n etic field gra d ie n t coil (F igure 1.2).

Figure 1.2 Protons, or hydrogen ions, aligning parallel and anti-parallel to a magnetic

fie ld

T h e s e p ro to n s po ssess d iffe rent e n e rg y states and a proton has the ability to m o v e from on e

en e rg y state to an o th e r e n e rg y state fo llo w in g the a b so rp tio n o f a p h oton. W h e n the en e rg y

o f th e p h o to n m a tc h e s the en e rg y d iffe re n c e b etw e en the tw o spin states, ab so rp tio n o f

en e rg y occurs. In M R I, the fre q u e n c y o f the p h oton falls w ithin th e radio fr eq u en c y (R F )

range ( w w w .c is .rit.e d u /h tb o o k s /m r i/in s id e .h tm ) and this fre q u e n c y m a y be applied by a

ra d io fre q u e n c y coil. W h e n the RF pulse is tu rn ed off, the h y d ro g e n p ro to n s return to th e ir

natural a lig n m e n t w ith in th e m a g n e tic field and re le ase th e ir e x c e s s stored energy. W h e n

subsequently integrated and converted through the use o f a Fourier transformation into an

M R image (Huettei

et a l, 2008).

1.3 A rterial Spin Labelling

Arterial spin labelling (ASL) is a method used to assess for functionality within an MRl

scan. It acts to assess cerebral blood flow (CBF) or cerebral blood volum e (CBV ) in the

brain without the use o f neuronal activation, it is a technique originally introduced by

Alsop & Detre (1996) and it is the only MRl technique that can directly and absolutely

quantify regional CBF (rC BF) (Beckmann, 2006). An MR image can becom e sensitive to

C B F changes if the m agnetic state o f blood water spins is different to that o f the tissue

water spins (Thom as

et al., 2000). This ASL technique uses magnetically labelled arterial

blood water as an endogenous tracer for the assessment o f perfusion changes (Jahng

et al.,

2007). In this way ASL M Rl is a non-invasive imaging technique that assesses for cerebral

blood perfusion changes. This method is advantageous as it causes minimal disturbance to

the system being imaged (Beckm ann, 2006). T w o separate sets o f M R images are generated

following an A SL scan. The first image contains blood and tissue w ater magnetisations that

are different (the

labelled image) and the second image contains blood and water

magnetisations that are the same (the

control image). Subtraction o f the

labelled from the

control image generates a perfusion weighted image with an intensity that is directly related

to perfusion (Thom as et al., 2000).

Recently, a new quantitative bolus-tracking A SL (btASL) MRl technique was developed

C h apter I: Introdu ction

rodent brain. The technique assesses cerebral perfusion through the calculation o f two

transit times: the mean transit time (M T T) which represents the time taken for labelled

arterial blood water to traverse the vasculature and reach the imaging plane and the

capillary transit time (CTT) which represents the time taken for the arterial blood water to

disperse at the imaging plane. M TT is inversely proportional to CBF, while C T T is

inversely proportional to C B F squared. A third quantifiable output is the btASL signal

amplitude, which is derived from the area under the signal-time ASL curve and has been

interpreted as being proportional to CB V (Figure 1.3).

j-2 r

|CTT

MTT

F igure 1.3 S ch em a tic depictin g th e btA SL M R I techn ique

The schematic depicts ASL as it occurs. Briefly:

1. The inflowing arterial blood water is magnetised.

2. An image o f this magnetised blood is taken at the imaging plane (the

la b elled

image).

3. The inflowing arterial blood water has no magnetic pulse applied to it.

1.4 Cerebral Blood Flow

1.4.1 Introduction

C B F is the blood supply to the brain at any given tim e. The brain is dependent on a

continuous supply o f oxygenated blood and it has the ability to control the blood delivery by

sensing pressure changes in its main arteries and by m onitoring respiratory gas levels. The

m ajor arteries supplying the brain are the internal carotid arteries which divide into the anterior

and middle cerebral arteries. The basilar artery divides into the tw o posterior cerebral arteries at

the upper border o f the pons and the Circle o f Willis links all o f these arteries. The arteries

which arise from this structure branch out into sm aller pial vessels that bring blood to the brain

surface. The pial arteries (Figure 1.4.1) give rise to penetrating arteries and arterioles which

penetrate the substance o f the brain and as the arterioles become progressively smaller with

each branching, by losing their smooth muscle layer, they become cerebral capillaries. These

capillaries are also known as the intracerebral or intraparenchymal micro vessels (Cohen

et a l,

1996) and all the other vessels in the brain including the pial vessels and the major cerebral

arteries are known as the extracerebral vessels (Cohen

et a l,

1996). The endothelial cells o f

these capillaries are not fenestrated, as they are in the periphery, but are instead inter-connected

by focal adhesions (ladecola

et a l,

2004) known as tight junctions which, along with the

astrocytic end-feet, form the blood brain barrier (BBB). The BBB is extrem ely important in the

brain as it m odulates the entry o f m etabolic substances such as glucose, controls the m ovem ent

o f ions, and prevents the access o f toxins and peripheral neurotransm itters to the central

nervous system. The presence o f the BBB is one o f the m ajor differences that exists between

Chapter 1: Introduction

V

P ial a rte ry

G lo l

C en tral p a th w a y s from :

C a p i ll a r y

Figure 1.4.1 Schematic o f brain vasculature (ladecola et al., 2004)

T h e d ia g ra m s h o w s a pial arte ry b ra n c h in g into the intracerebral arte riole and finally the

capillary, in additio n , it s h o w s th e c o n n e c tio n s o f the v esse ls to cells.

1.4.2 Neurovascular Coupling

In the brain en e rg y m u s t be m a d e a v a ila b le w h e re n ee d ed q u ick ly and efficiently to e n su r e

p ro p e r fu n c tio n in g and this m e a n s b lo o d flo w m u st co rrelate clo sely w ith n euronal

ac tivation. A useful c o n c e p tu a l tool in d e s c rib in g this p h e n o m e n o n is th e n e u r o v a sc u la r

unit w h ic h is th e functional unit c o m p r is in g n eu ro n s, blo o d vessels, and glial cells that

w o r k in unison to e n su r e a d e q u a te blo o d flo w is c o u p led to neuronal activation (D ra k e &

ladec ola, 2007). N e urovascula r coupling enables cerebral blood flow to be increased in areas

neurotransm itters such as acetylcholine, glutamate, GA BA , 5-HT, dopam ine and noradrenaline

as well as some non-conventional transm itters such as nitric oxide (NO).

O ur understanding o f the processes involved in the regulation o f CBF is evolving and

subject to debate (Paulson

et a l, 2010). it is not alw ays clear exactly how activation and

blood flow are coupled and this m ay be due to lim itations in the ability to m easure neuronal

activity or C B F and to correlate them accurately (Tan, 2009). The current paradigm is that

rather than being controlled by a negative feedback loop (an energy deficit signalling for

increased blood flow ), feed-forw ard m echanism s (increased neuronal firing am plifying

local and upstream blood flow ) are key in second-to-second changes in C B F (C auli &

H am el, 2010). H ow ever, it is becom ing clear that astrocytes (H aydon & C arm ignoto,

2006), pericytes (K am ouchi

et al., 2 0 1 1), local neurons (D rake & ladecola, 2007), and the

direct and indirect effects o f neurotransm itters (Cauli & H am el, 2010) play a key role in the

process.

/. 4.3 Regulation o f Cerebral Blood Flow

(a) Endothelial Cells

The endothelial cells o f the m icrovessels are regulators o f vascular tone, vasculogenesis,

inflam m ation and throm bosis (A ndresen et a l, 2006). Stimulation o f endothelial cells leads to

Chapter I: Introduction

regulators o f endothelial nitric oxide synthase (eNOS) and so a decrease in the

concentration or inhibition o f the HSP-90 protein can lead to constriction o f cerebral blood

vessels. Three endothelium -derived constricting factor (ET-1) receptors have been found

expressed in the endothelium and sm ooth m uscle (Andresen

et a l,

2006). ET-I is a profound

2+

vasoconstrictor that increases intracellular C a

concentration and m ay also increase release

o f chloride (C1‘) from sm ooth muscle cells (A ndresen

et al,

2006).

E ndothelial-derived vasoactive com pounds are o f im portance because neurotransm itters

such as acetylcholine (H eistad

et a l,

1977), dopam ine (K rim er

et a l,

1998), 5-H T (C ohen

et a l,

1996) and noradrenaline (R aichle

et a l,

1975) have all been show n to have either

vasoconstrictor or vasodilatatory properties and endothelial vasoactive com pounds m ay

play a role in their m echanism s o f action.

(h) Pericytes

In addition, pericytes appear to have a m acrophage-like activity acting as first line defence in

the brain and having the ability to present antigen (Guillem in & Brew, 2004). The pericytes

have processes that surround the capillaries (Peppiatt

et al.^ 2006) and the prim ary processes

extend from the pericyte cell body along the capillary and subsequently branch into secondary

and tertiary processes (Hamilton

et a l ,

2010). Their importance within the nervous system is

further supported by the fact that there are more pericytes per endothelial cell here than in any

other area o f the body (Hamilton

et a l , 2010).

Chapter I: Introduction

fc) N eu ro tra n sm itters

5-HT neurons have been reported to modulate blood flow in the microcirculation (Cohen

et al,

1996). Ultrastructural analysis has revealed an intimate association between serotonergic

neurons and blood vessels in the brain (Cohen

et a l ,

1995; Milner

et al.,

1966). It was the

discovery o f this association that promoted the theory that 5-HT could modulate cerebral blood

flow. The vasoconstrictive actions o f 5-HT have been widely reported in both humans

(Kaumann

et al,

1993; Price

et al,

1997; Ullmer

et al,

1995) and animals (Cao

et al,

1992;

McBean

et al,

1991; Roberts

et al,

1997) however, 5-HT has also been reported to promote

vasodilatation under certain conditions (Cohen

et al,

1996). The predominant vasomotor effect

o f 5-HT on cerebral blood vessels is constriction and it has been suggested that the different

receptor subtypes, and possibly the vessel tone before exposure, mediate the opposing effects o f

5-HT on the microcirculation.

A high number o f perivascular serotonergic neurons synapse on astrocytes, implying an

important role for astrocytes in the regulation o f CBF. Astrocytes have been shown to

express a number o f 5-HT receptor subtypes including 5-HTia,

-HT2a and

-HT2c (Cohen

et a l ,

1996; Osredkar & Krzan, 2009). The 5-H Tia receptors act by inhibiting adenylate

cyclase which decreases cyclic adenosine monophosphate (cA M P) leading to

down-regulation o f particular genes. This can lead to reduced levels o f cyclooxygenase

eicosanoids (Volterra & M eldolesi, 2005) which can result in vessel constriction. The

5-HT2A receptors are associated exclusively with astrocytes in the human brain (Cohen

et a l ,

Seroton ergic Neuron

• = Seroton in

5-H T ,„,

5-H T,d3 h -

-HT7

• 5 - H T 7 « •

Pericyte

t Ca2+ = Vasoconstriction 5 -H T , 5 -H T 5 -H T

5 -H T 5 -H T ,,,

5 -H T

Endothelial Cell

Astrocyte

G-protein

5 -H T 5 -H T

I

t C a2+

t

V asoconstriction 5 -H T

Figure 1.4.3 Schematic o f the neurovascular unit and the rote it has to play in modulating

cerebral blood flo w

Chapter 1: Introduction

oestrogen, adenosine diphosphate (ADP) and adenosine triphosphate (ATP) which activate

G-protein coupled receptors which can stimulate vasodilatation by the production o f nitric

oxide (NO). Endothelium-derived constricting factor (ET-1) receptors have been found

expressed on the endothelium and ET-1 is a profound vasoconstrictor that increases

intracellular Ca^^ concentration.

The catecholamine dopamine has been implicated in the regulation o f CBF. Dopam ine is a

catecholamine and acts as a neurotransmitter in the CNS. It is synthesised from the amino

acid tyrosine which is taken up into the nerve terminal and converted to dopam ine which is

subsequently stored in vesicles by the vesicular m onoam ine transporters (VM ATs). Release

o f dopamine from the nerve terminal leads to the activation o f dopamine receptors. To date

5 different dopamine receptors have been identified (Golan

et al., 2008) which have been

divided into 2 distinct classes D |-like and D

-like. D |-like comprise D| and D

receptors

whereas D

-like comprise D

, D

and D

receptors. Di-like receptors are positively coupled

to G-proteins leading to stimulation o f adenylate cyclase and D

-like receptors are

negatively coupled to G-proteins leading to inhibition o f adenylate cyclase. Dopam ine is

removed from the synaptic cleft via the pre-synaptically located dopam ine transporter

(DAT).

substantia nigra to the limbic system and the neocortex and is implicated in behaviour. The

second is the nigrostriatal pathway which consists o f neurons projecting from the substantia

nigra to the caudate/putamen and is involved in voluntary m ovem ent. The third is the

tuberoinfundibular pathway, which links arcuate nuclei and periventricular neurons to the

hypothalam us and posterior pituitary and is involved in prolactin homeostasis. The fourth is

the medullary-periventricular pathway which consists o f cell projections in the motor

nucleus o f the vagus nerve. The fifth is the incertohypothalamic pathw ay which connects

the medial zona inccrta to the hypothalamus and the am ygdala (Golan

et al.,

2008).

Krimer and colleagues (1998) first speculated that observed changes in vessels innervated

by dopaminergic neurons were due to direct effects o f dopam ine on the vessel rather than

due to neuronal activation. Further studies have been carried out to elucidate a role for

dopamine in mediating CBF changes. Utilising an array o f selective dopam ine agonists,

antagonists, releasers and re-uptake inhibitors, and using both increased relaxation with

iron oxide nanoparticles (IRON) and blood-oxygen level dependent (B O L D ) techniques

investigators exam ined the effects o f dopam ine release on the cerebrovasculature to

elucidate the specific DA receptors underlying the observed changes, investigators reported

a strong correlation between dopamine concentration in the brain, as released by

am phetam ine, and relative cerebral blood flow (rCBF). Administration o f a dopamine

transporter (DAT) blocker produced com parable results (Krim er

et ai,

1998).

A m phetam ine administration alone, and in combination, with a D

/ D

antagonist (SCH

23390) revealed that SCH 23390 produced a small negative rCBF change (approximately

5%) when administered alone, but blocked the rCBF changes normally induced by

am phetam ine and D A T blocker. D

agonists (quinpirole and R(-)-2,IO,

Chapter I: Introduction

the regions where D2 receptors are present. The D3 agonist 7-OHDPAT also produces small

negative rCBF changes, but differed from D

agonists in respect that no CBF changes were

observed in the caudate/putamen. The findings o f this study indicate that increases in CBF

are mediated by agonism o f D

/D

receptors, while decreases in rCBF are mediated by

agonism o f D2

/D

et al,

2006). Ren and colleagues (2009) also reported a

strong relationship between amphetamine dose and dopamine release however, a negative

rCBF change was associated with low dose amphetamine. Microdialysis, performed to

assess dopamine release at low amphetamine concentration, indicates a dose-dependent

release o f dopamine. It was hypothesised that, because D2/D

affinity for dopamine than D

/D

receptors, at low concentrations, they have higher relative

occupancy and thus exert more net effect on the vasculature. As amphetamine, and thus

dopamine, concentration increases, D

studies provide evidence to imply a role for dopamine as a potent modulator o f CBF in the

microcirculation.

not aiD- adrenoceptors, was expressed in the cerebral microvesseis.

iodo-2-[p-(4-

hydroxyphenyl)-ethyl-aminomethyl]tetralone

(['^^1]HEAT)

binding

to

the

cerebral

microvesseis was inhibited by 5-methylurapidil, a selective aiA-adrenergic receptor

antagonist, in a dose-dependent manner.

1.5 Sum m ary

Chapter 1: Introduction

1.6 A im s and Objectives

T h e o v e ra ll aim o f the w ork d escrib e d in th is th e sis w a s to d eterm in e the acu te and lo n g ­

term e ffe c ts o f M D M A on cerebral b lo o d p erfu sio n and to e x p lo r e th e u n d erly in g

m e c h a n ism s in a rodent m o d el o f M D M A a b u se u sin g b tA S L M R I. S p e c ific a lly the

o b je c tiv e s w er e as fo llo w s;

(1 ) T o e m p lo y b tA S L M RI to d e term in e r eg io n a l, tim e and d o se -d e p e n d e n t c h a n g e s to

cerebral p erfu sio n in the rat f o llo w in g a cu te M D M A a d m in istration . It w a s a ls o d eem ed

n e c e ssa r y to cla rify i f an y c h a n g e s o b se r v e d w ere a sso c ia te d w ith B B B disru p tion as

su sta in ed in crea ses in B B B p erm ea b ility h a v e p r e v io u sly been reported fo llo w in g

a d m in istration o f h igh d o s e s o f M D M A to rats.

(2 ) G iv e n the e sta b lish e d role o f 5 -H T and d o p a m in e in the regu lation o f cerebral

p erfu sio n , attem p ts w e r e m ad e to d e term in e the m e c h a n ism s that m ed ia te the a b ility o f

M D M A to in crease cortical p erfu sio n and v o lu m e in rats. First, the a b ility o f M D M A to

g e n e r a lise to fe n flu r a m in e, a sy n th e tic a m p h eta m in e that s e le c t iv e ly in d u ce s th e relea se o f

central 5 -H T w a s d e term in ed , or i f the r e sp o n se to M D M A co u ld be sim u la ted by

a d m in istration o f the n o n -s e le c tiv e 5 -H T 2 recep tor a g o n ist 2 ,5 d im e th o x y -4 -io d o p h e n y

la m in o p ro p la n e h y d r o ch lo rid e (D O l). N e x t, the e ffe c ts o f 5 H T d e p le tio n on M D M A

-in d u ced c h a n g e s -in cortical p erfu sio n w e r e d eterm -in ed . In a d d itio n , -in h ib itio n o f 5-H T

tr a n sm issio n w a s a sse sse d by prior ad m in istration o f the n o n -s e le c tiv e 5 -H T receptor

a n ta g o n ist m e terg o lin e. T h e c o n s e q u e n c e s o f b lo c k in g the 5 -H T transporter and resultant

administration o f tiie selective dopam ine Di receptor antagonist SCH 23390 was assessed

on M D M A -induced changes.

(3) To determ ine if repeated exposure to M D M A with subsequent long-term central 5-HT

loss m ay provoke sustained alterations in cerebral cortical perfusion and blood volum e in

the rodent model established. M oreover it was o f interest to determine if prior exposure to

M D M A and subsequent long-term cortical 5-HT loss would influence the response to acute

Chapter 2