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Correlation of in vivo positron emission tomography and in vitro autoradiography measurements using radiotracers… Palmer, Kasandra Lynne 2007

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C O R R E L A T I O N O F IN V I V O P O S I T R O N E M I S S I O N T O M O G R A P H Y A N D IN V I T R O A U T O R A D I O G R A P H Y M E A S U R E M E N T S U S I N G R A D I O T R A C E R S F O R T H E D O P A M I N E S Y S T E M by K A S A N D R A L Y N N E P A L M E R B . S c , The University of British Co lumb ia , 2005 A T H E S I S S U B M I T T E D IN P A R T I A L F U L F I L L M E N T O F T H E R E Q U I R E M E N T S F O R T H E D E G R E E O F M A S T E R O F S C I E N C E in T H E F A C U L T Y O F G R A D U A T E S T U D I E S (Experimental Medic ine) T H E U N I V E R S I T Y O F BRIT ISH C O L U M B I A June, 2007 © Kasand ra Lynne Palmer , 2007 Abstract Positron emiss ion tomography (PET) is a noninvasive imaging technique that al lows visualization of physiological function in living, consc ious beings. In the field of Park inson 's d i sease (PD), P E T is used in both bas ic and cl inical research to provide insights into P D pathophysiology. Most importantly, it a l lows longitudinal analysis of d i sease progression and exploration of the compensatory neurochemical changes assoc ia ted with long-term treatment or surgical intervention. Al though the popularity of P E T is increasing, the val idation of many of the tracers in human use is lacking. F e w studies have attempted to correlate the in vivo P E T data with actual physiological p rocesses . E lements of physiological p rocesses are often measured in vitro by autoradiography (ARG) to el iminate confounding factors such as competit ion between the tracer and endogenous l igand. This study examines the relationship between P E T and A R G measurements obtained with the s a m e tracers, which target components of the dopamine sys tem, in an M P T P non-human primate model of P D . Both presynapt ic and postsynapt ic tracers were examined in the s a m e individuals representing a wide range of M P T P - i n d u c e d clinical severity. P E T and A R G measurements were also compared to behavioural assessmen t scores rating the severity of PD- l ike motor symptoms. P E T binding potentials and A R G binding va lues obtained using the presynaptic tracers [ 1 1 C ] D T B Z , [ 1 1 C ] M P , and [ 3H]WIN 35,428 correlated significantly with each other and with behavioural assessmen ts . In contrast, P E T and A R G using the postsynaptic tracers raclopride and S C H - 2 3 3 9 0 labeled with either [ 1 1 C] or [ 3H] correlated weakly or not at all with each other and with behavioural scores . The results of this study suggest that both P E T and A R G using presynaptic tracers provide comparab le insights into d isease pathophysiology and can evaluate clinical severity over a wide range of P D severity. They a lso raise awareness of the high individual variability in D1 and D2 dopamine receptor binding with progression of P D . T h e s e f indings support the utility of P E T using presynapt ic tracers as a valuable brain imaging technique. ii Table of Contents Abstract : ii List of Tab les iv List of F igures v List of Abbreviat ions ix Acknowledgements x Chapter 1 1 Introduction 1 Hypotheses 1 Background 2 Posi t ron Emiss ion Tomography 2 Autoradiography 7 Compar i son of in vivo P E T and in vitro A R G 11 The Dopamine Sys tem 14 The Neurotransmitter Dopamine 14 Neuroanatomy of the Dopamine Sys tem 17 The Direct and Indirect Pa thways 20 Park inson 's D i sease 21 Radiot racers for the Dopamine Sys tem 23 Binding Stud ies and Park inson 's D isease 31 Cl in ical A s s e s s m e n t of Park inson 's D i sease 35 A n An ima l Mode l of Park inson 's D isease 36 Hypotheses revisited 39 Signi f icance 40 Chapter 2 42 Methods 42 Subjects 42 M P T P Les ions 43 Behavioura l A s s e s s m e n t 43 P E T Exper imental Procedure 44 P E T Data Ana lys is 47 A R G Exper imental Procedure 48 A R G Data Ana lys is 53 Statistical Ana lys is 57 Chapter 3 59 .Resu l ts 59 Cl in ical Severi ty and P E T measurements 59 A R G measurements 60 Correlat ions Be tween in vivo P E T and in vitro A R G Measu remen ts 61 Presynapt ic Tracers 61 Postsynapt ic Tracers 71 Chapter 4 80 Discuss ion and Conc lus ions 80 Sou rces of Error 80 Hindsight is 20 /20 87 D iscuss ion 88 Conc lus ions 97 Re fe rences 99 iii List of Tables Table 1.1: Radiot racers deve loped for studying the function of the striatal D A system in vivo with P E T 24 Tab le 2.1. Summary of subjects investigated in this study 42 Table 2.2. Behavioura l assessmen t sca le used to obtain score for degree of severity of parkinsonian syndrome 44 Table 3.1. Behavioura l scores and binding potentials (BP) of radiotracers in striatium used for compar isons with in vitro data. Behavioural scores were measured using rating sca le of cl inical severity of P D symptoms; B P w a s measured in vivo by P E T 59 Table 3.2. Resul ts of radiotracer binding in striatum, obtained in vitro by autoradiography. Binding va lues are reported in units of pmol /cc 61 iv List of Figures Fig. 1.1. Phys i cs of P E T . Top : Unstable radioisotope decays by emitting positrons, which combine with electrons and annihilate. Bottom: Anti-paral lel g a m m a rays from annihilation are detected by tomograph and used to create image of tracer distribution (from http://www.petnm.unimelb.edu.au/pet/) 4 Fig. 1.2. Pathway for dopamine synthesis in the central nervous sys tem. T H , tyrosine hydroxylase; A A D C , aromatic amino acid decarboxy lase; L - D O P A , dihydroxyphenylalanine (adapted from Vo lkow et a l . , 1996b) 15 Fig. 1.3. Pathway for dopamine metabol ism in the central nervous system. M A O , monoamine ox idase; A D , a ldehyde dehydrogenase; C O M T , catechol -O-methyltransferase; D O P A C , dihydroxyphenylacet ic ac id ; H V A , homovanil l ic ac id , (adapted from Vo lkow et a l . , 1996b) 16 Fig. 1.4. Diagram of dopamine nerve terminal, including P E T tracers that target terminal components (adapted from diagram by Sarah Lidstone) 17 Fig. 1.5. Anatomica l posit ions of the basa l gangl ia and tha lamus in the human brain (from cti. i tc.virgina.edu/pscy220/) 18 Fig. 1.6. Pathological changes in neurodegenerat ive d i seases . (A) Midbrain from a patient with Park inson 's d i sease shown on left demonstrates loss of D A neurons in substant ia nigra (pigmented area, arrows) relative to normal subject shown on right. (B) The s ize of the striatum is dramatical ly reduced in patients with Huntington's d isease (from Bradley e t a l . , 1991) 19 Fig. 1.7. The direct pathway. Activity in this pathway ultimately facil itates movement (from Purves et a l . , 2001) 20 Fig. 1.8. The indirect pathway (shaded). Activity in this pathway ultimately inhibits movement (from Purves et a l . , 2001) 21 Fig. 1.9. Funct ional ou tcomes of direct and indirect pathways in Park inson 's d isease . Thinner arrows represent reduced neuronal activity and thicker arrows represent increased activity (from Purves et al . , 2001) 23 Fig. 2 .1. Stereotaxic f rame used to align all monkeys in P E T scanner in s a m e plane 46 Fig. 2.2. Posi t ioning of ROIs on P E T images in four sequent ia l s l ices through the striatum. The ROIs encompass ing total striatum, as shown here in left hemisphere in each image, were used to obtain the binding potential va lues for each subject. Images obtained from an exper iment using [ 1 1 C]raclopr ide (courtesy of Doris Doudet) 48 Fig. 2.3. [ 1 1 C] standard d isp layed relative to activity in the 6 t h point (left), 2 n d point (middle), and with ROIs drawn on (right). Standards were made by pipetting 5 uL drops of a set of eight serial dilutions onto a strip of T L C plate 54 v Fig. 2.4. Image from a [ 3H]WIN 35,428 binding experiment, demonstrat ing a [ 3 H]microscale (Amersham) on the left, which was used to cal ibrate optical density of images to quantitative measu res of activity 55 Fig. 2.5. ROI p lacement method used to calculate binding measurements from A R G exper iments <. 57 Fig. 3.1. P E T images produced by reconstruction of data col lected by P E T tomograph during scann ing. Images show single coronal p lane at s a m e striatal level in s a m e subject from separate exper iments using the four radiotracers investigated in this study. The white outline indicates the edges of the brain (courtesy of Doris Doudet) 60 Fig. 3.2. Images of [ 1 1 C ] D T B Z binding obtained by in vitro autoradiography in hemi-M P T P animal (unlesioned hemisphere; s l ides 1-3) and bilateral M P T P animal (slides 4-6). Total binding was measured by incubating t issue sect ions in buffer containing [ 1 1 C](+)DTBZ for 30 minutes (sl ides 1,2,4,5); nonspeci f ic binding was measured by addition of 5 u M (±)TBZ to incubation solution (sl ides 3,6). Nonspeci f ic binding represented only 1-2% of total binding on average for [ 1 1 C ] D T B Z , thus these sect ions are not visible when images are displayed relative to much higher activity in sect ions exposed to radioactive tracer only for measurement of total binding 62 Fig. 3.3. Correlat ion of [ 1 1 C](±)DTBZ B P measurements from in vivo P E T and [ 1 1 C](+)DTBZ binding measurements from in vitro A R G 63 Fig. 3.4. Correlat ion of behavioural scores with [ 1 1 C] (± )DTBZ B P from P E T exper iments 64 Fig. 3.5. Correlat ion of behavioural scores with [ 1 1 C](+)DTBZ binding measurements from A R G exper iments 64 Fig. 3.6. Images of [ 1 1 C ] M P binding obtained by in vitro autoradiography in a hemi-M P T P monkey (unlesioned hemisphere; s l ides 1-3) and a bilateral M P T P monkey (slides 4-6). Total binding was measured by incubating s l ides for 40 minutes in buffer containing 15 nM [ 1 1 C ] M P (slides 1, 2, 4, 5); nonspeci f ic binding was measured by addition of 10uM nomifensine (slides 3,6) 66 Fig. 3.7. Correlat ion of [ 1 1 C ] M P B P measured in vivo using P E T with [ 1 1 C ] M P binding measurements obtained in vitro using A R G 67 Fig. 3.8. Correlat ion of behavioural scores with [ 1 1 C ] M P B P measurements using in vivo P E T 67 Fig. 3.9. Correlat ion of behavioural scores with [ 1 1 C ] M P binding measurements from in vitro A R G 68 vi Fig. 3.10. Images of [ 3 H]WIN 35,428 binding obtained by in vitro autoradiography in a h e m i - M P T P animal . S l ides 1-3 from unlesioned hemisphere and sl ides 4-6 from lesioned hemisphere. Total binding was measured by incubation in buffer containing 15nM [ 3 H]WIN 35,428 for 40 minutes (sl ides 1,2,4,5); nonspeci f ic binding was measured by addit ion of 10uM nomifensine to incubation buffer (sl ides 3,6) 69 Fig. 3.11. Correlat ion of [ 1 1 C ] M P B P measurements from in vivo P E T and [ 3H]WIN 35,428 binding measurements from in vitro A R G 70 Fig. 3.12. Correlat ion of behavioural scores with [ 3 H]WIN 35,428 binding measurements from in vitro A R G 70 Fig. 3.13. Correlat ion of [ 1 1 C ] M P and [ 3H]WIN 35,428 binding va lues from in vitro A R G 71 Fig. 3.14. [ 3 H]SCH-23390 binding image obtained by in vitro A R G in unlesioned hemisphere of h e m i - M P T P monkey. Total binding measured by incubating sl ides in buffer containing 2 n M [ 3 H]SCH-23390 (4 dark sect ions); nonspeci f ic binding measured by addition of 10uM (+)butaclamol to incubation solution (2 sect ions at right) 72 Fig. 3.15. Correlat ion of [ 1 1 C ] S C H - 2 3 3 9 0 B P measurements taken in vivo using P E T and [ 3 H]SCH-23390 binding measurements taken in vitro using A R G 73 Fig. 3.16. Correlat ion of behavioural scores with in vivo P E T [ 1 1 C ] S C H - 2 3 3 9 0 B P measurements 73 Fig. 3.17. Correlat ion of behavioural scores with in vitro A R G [ 3 H]SCH-23390 binding measurements . Sol id line represents correlation including all data points; dotted line represents nonsignif icant correlation after exc lus ion of outlier (highest behavioural score) 74 Fig. 3.18. Images of in vitro A R G using [11C]raclopride (sl ides 1-3) and [3H]raclopride (slides 4-6; darker staining around sect ions is nai lpol ish, used to keep tracer on sect ions throughout incubation) in s a m e h e m i - M P T P animal . Binding with both radiotracers fol lowed s a m e protocol, including incubation in buffer containing 3 n M radiolabeled raclopride, with addit ion of 10pm (+)butaclamol for nonspeci f ic binding (slides 3 and 6; barely visible for [11C]raclopride binding at top). Note higher proportion of nonspeci f ic binding in s l ides incubated with [3H]raclopride (darker appearance) than in s l ides incubated with [11C]raclopride 75 Fig. 3.19. Relat ionship between P E T B P and A R G binding using [ 1 1 C]raclopr ide 76 Fig. 3.20. Correlat ion of behavioural scores and in vivo P E T B P va lues obtained using [ 1 1 C]raclopr ide 76 Fig. 3.21. Correlat ion of behavioural scores and in vitro A R G binding va lues obtained using [ 1 1 C]raclopr ide 77 vii Fig. 3.22. Correlat ion of [ 1 1 C]raclopr ide B P values obtained in vivo using P E T and [ 3H]raclopride binding va lues obtained in vitro using A R G 78 Fig. 3.23. Correlat ion between behavioural scores and in vitro A R G [ 3H]raclopride binding 78 Fig. 3.24. Correlat ion of in vitro A R G binding values obtained using [ 1 1 C]raclopr ide and [ 3H]raclopride 79 viii List of Abbreviations 6 - O H D A - 6-hydroxydopamine A A D C - aromatic amino acid decarboxy lase A D - A lzhe imer 's d i sease A D H D - attention deficit hyperactivity disorder A R G - autoradiography Bmax- receptor/transporter density B G - basa l gangl ia B P - binding potential C O M T - catechol-O-methyl t ransferase DA- dopamine D A T - dopamine transporter DLU-d ig i ta l light unit D O P A - d ihydroxyphenylalanine D T B Z - dihydrotetrabenazine dpi- dots per inch F D O P A - 6-f luoro-L-dopa G P i - external g lobus pal l idus G P e - internal g lobus pall idus H D - Huntington's d i sease K d - receptor/transporter apparent affinity K O - knock-out L - D O P A - levodopa; dihydroxyphenylalanine M A O - B - monoamine ox idase B M L C R A - multiple l igand concentrat ion receptor a s s a y M P - methylphenidate M P T P - 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine O D - optical density P D - Park inson 's d i s e a s e P E T - positron emiss ion tomography ROI - region of interest S A - speci f ic activity S N c - substant ia nigra pars compac ta SNr - substant ia nigra pars reticulata S T N - subthalamic nucleus ty2- half-life T H - tyrosine hydroxylase U P D R S - Unif ied Park inson 's D i sease Rating S c a l e V M A T 2 - vesicular monoamine transporter type 2 ix Acknowledgements I thank Matthew Harr iman for his continual love and support throughout the course of this project. I a lso thank my parents, Lynne and Booth Pa lmer , for their uncondit ional love and encouragement . I would like to thank my supervisor, Dr. Doris Doudet, for her mentorship and for helping me learn to think critically. I would a lso like to thank the members of my committee for their t ime and participation in the complet ion of this project. Many thanks are due to the individuals who col lected the positron emiss ion tomography data and behavioural a s s e s s m e n t s of the subjects, as well as those who cared for the animals over the course of their l ives. Whi le working on this project, the author was supported by a Canad ian Institutes of Health R e s e a r c h C a n a d a Graduate Scho larsh ips Master 's Award , and by a Graduate Entrance Scho larsh ip awarded by the U B C Faculty of Medic ine, Department of Medic ine. x Chapter 1 Introduction The research presented here examines the relationship between in vivo positron emiss ion tomography (PET) data and in vitro autoradiography ( A R G ) data using various radiotracers for the dopamine (DA) system in a non-human primate model of Park inson 's d i sease (PD). The present study invest igates the correlation between binding potentials (BP) of select tracers routinely used in P E T with binding values obtained in vitro, in the s a m e individuals, with the s a m e tracers. It a lso investigates the correlation between behavioural assessmen ts of cl inical severity and both in vivo B P values and in vitro binding va lues for six radioactive tracers. Th is is the first study to investigate the relationship between in vivo P E T and in vitro binding using the s a m e tracers and behavioural measurements in the s a m e individual rhesus monkeys representing a wide range of lesion severity induced by the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine ( M P T P ) . This study will examine six tracers of the dopamine sys tem using one or both of in vivo P E T and in vitro A R G in the s a m e subjects. Presynapt ic radiotracers will be investigated as fol lows: [ 1 1 C]dihydrotetrabenazine (DTBZ) , which labels vesicular monoamine transporter type 2 (VMAT2) , by both P E T and A R G ; [11C]d-tf7reo-methylphenidate (MP) , which labels dopamine transporter (DAT), by P E T and A R G ; and [ 3 H]WIN 35,248, which also labels D A T , by A R G only. Postsynapt ic radiotracers will be investigated as follows: [ 1 1 C ] S C H - 2 3 3 9 0 , which labels dopamine D1 receptor, by P E T only and [ 3 H]SCH-23390 by A R G only; [ 1 1 C]raclopr ide, which labels dopamine D2 receptor, by P E T and A R G , and [ 3H]raclopride by A R G only. Both in vivo and in vitro measurements will be compared to behavioural scores representing severity of cl inical parkinsonian symptoms for each subject. Hypotheses 1) In vivo P E T data and in vitro A R G data for all t racers investigated will be significantly correlated with one another. 1 2) There will be a signif icant correlation between behavioural scores representing cl inical severity and P E T and A R G data obtained with presynapt ic tracers ( [ 1 1 C]DTBZ, [ 1 1 C ] M P , [ 3H]WIN 35,428), but little or no correlation between clinical severity and postsynapt ic tracer ( [ 1 1 C ] S C H - 2 3 3 9 0 , [ 3 H]SCH-23390 , [ 1 1 C]raclopr ide, [ 3H]raclopride) P E T and A R G measurements . Background Positron Emission Tomography Positron emiss ion tomography (PET) is a noninvasive imaging technique that al lows in vivo v isual izat ion of physiological function in var ious t issues throughout the brain and body. P E T images can be used to local ize and quantify aspec ts of physiological , metabol ic and molecular p rocesses in normal and pathophysiological condit ions. Cl inical use of P E T is well establ ished in the field of oncology, where the technique is used for d iagnos is and staging of malignant d i sease , as well as for monitoring therapy (Blodgett et a l . , 2007; ls rae l and Kuten, 2007 ;Schoder and G o n e n , 2007 ;Seve et a l . , 2007;Hicks et al . , 2007;Juweid et a l . , 2007). P E T is a lso used clinically in cardiology patients and select neurodegenerat ive d i sease patients (Townsend et al . , 2004 ;Koeppe et al . , 2005;Mil ler, 2006;Di Carl i and Dorbala, 2006). Severa l potential cl inical appl icat ions in above fields as well as sch izophrenia , attention deficit hyperactivity disorder (ADHD) , depress ion , substance abuse , musculoske le ta l d i seases , pain treatment, p lacebo effect, and infectious d i seases are currently under investigation and the field of P E T imaging is rapidly advancing (Farde et al . , 1987;Ernst et al . , 1996;Antonini et a l . , 1996;Volkow et al . , 1997b;Andrews et a l . , 1999;Volkow et a l . , 2003 ;He iss and Hilker, 2004 ;Townsend et al . , 2004;Fur tado et a l . , 2005 ;Koeppe et a l . , 2005;Mil ler, 2006 ;Ed ison et a l . , 2007;Duet et al . , 2007;Blodget t et a l . , 2007). In the field of neurodegenerat ive d iseases , P E T plays an instrumental role in both basic and clinical research. S ince P E T is noninvasive and is performed in living subjects, it al lows longitudinal analys is of d i sease progression and explorat ion of the compensatory neurochemica l changes assoc ia ted with long-term treatment or surgical intervention. It cont inues to provide insights into the pathophysiology and underlying neurochemical changes of severa l neurological d i seases (e.g. Park inson 's d i sease (PD), A lzhe imer 's d i sease (AD), Huntington's d isease (HD)), improve differential d iagnoses, and 2 contribute to the understanding of compl icat ions of treatments and mechan isms of drug action. Al though differentiation of frontotemporal dement ia from A D is the only application in cl inical use at present, the potential cl inical appl icat ions of P E T under study in this field are vast (Antonini et al . , 1996;Andrews et a l . , 1999;Heiss and Hilker, 2004;Kung et a l . , 2004 ;Koeppe et al . , 2005;Mil ler, 2006). In P E T , a drug or analog compound select ive for a speci f ic biological p rocess is labeled with a relatively short half-life radionucl ide and injected intravenously at a very smal l dose that does not induce pharmacological effects. A P E T tomograph is used to detect both speci f ic and non-speci f ic uptake of the radioactive tracer by measur ing its distribution over time. The tomograph, or scanner , col lects data that can be used to generate an image of this distribution. Calibrat ion of P E T images yields quantitative est imates of the radioactivity in a speci f ic region of interest. Carbon-11 ([ 1 1C]) and Fluor ine-18 ([ 1 8F]), with half-l ives of 20.4 minutes and 120 minutes respectively, are radionucl ides commonly used to synthesize P E T tracers. T h e s e proton-rich radionucl ides decay by emitting posit ive electrons (positrons) from their nuclei. Emitted positrons travel a short d istance from the nucleus before they combine with nearby electrons and annihilate (Fig. 1.1). Upon annihilation, the m a s s e s of both particles are converted to electromagnet ic radiation in the form of two g a m m a rays of equal energy (511 keV), which are emitted at 180° to e a c h other. This radiation is detected by the P E T scanner and used to determine the location and quantity of the positron emitter. 3 Fig. 1.1. Phys ics of P E T . Top : Unstable radioisotope decays by emitting positrons, which combine with electrons and annihilate. Bottom: Anti-paral lel g a m m a rays from annihilation are detected by tomograph and used to create image of tracer distribution (from http://www.petnm.unimelb.edu.au/pet/). E a c h emitted photon is referred to as a "single". S ince the two singles from an annihilation are emitted s imultaneously and in opposi te directions, they should theoretically be detected at oppositely-posit ioned detectors (coincidence detectors) at the s a m e time. The scanner generates an output only when two 511 keV photons trigger two co inc idence detectors within a short t ime, def ined as the "coincidence timing window". The duration of the co inc idence timing window varies by detector model , but is in the range of 6-12 ns and shorter windows lead to a better signal (Zanzonico, 2004). W h e n these condit ions are met, the event is cal led a "true co inc idence event" or "true". The collection of co inc idence annihilation events detected over the course of the scan is 4 reconstructed using mathemat ical algorithms to relate the col lected data to the distribution of activity within the subject. Unfortunately other events detected by the scanner meet these requirements and are recorded as true events, degrading the quantitative accuracy and image quality of P E T . For example, random events occur when two g a m m a rays from separate annihilation events are recorded at two different detectors within the co inc idence timing window, increasing the number of recorded events. G a m m a rays can a lso be redirected or scattered, which leads to misposi t ioned events. Even though scatter causes photons to lose energy, the low energy resolution of the scintil lation detectors used in P E T tomographs al lows detect ion of photons with 2 5 0 k e V to 6 5 0 k e V (Humm et al . , 2003). Extensive data correct ions are avai lable to minimize the impact of these unwanted events, but the correct ions increase noise in the data, which indirectly leads to further reductions in resolut ion. In addition to the data correct ions required to address physica l scanner limitations, corrections must be made to account for photon interactions with t issue that prevent them from being detected (attenuation correction), subject movement during the s c a n , as well as partial vo lume effect, which occurs when radioactivity in high receptor density regions spil ls over into neighbouring regions (Hoffman et a l . , 1979). Al l of these factors must be taken into account when obtaining quantitative data from P E T , as they may distort images. However, scanner development is progressing rapidly and improving P E T resolution. Newer tomographs have shorter co inc idence timing windows and energy windows that reduce detection of randoms and scattered events as well as other improvements that increase P E T accuracy (Zanzonico, 2004). The most common end measurement in P E T studies is the binding potential (BP) of the tracer (Holden et a l . , 2002). B P is a measurement est imate of the receptor availability and combines the density of the receptor ( B m a x ) with its apparent affinity ( K d ) ( B P = B m a x / K d ) . Therefore, finding a change in B P does not clarify whether the density or affinity of the receptors or both are changing, and finding no change in B P does not rule out the possibil ity that both B m a x and K d changed proportionally to one another. S o m e P E T studies go further than B P and determine the density and apparent affinity of 5 receptors, which provides a more comprehens ive idea of speci f ic changes at the synapse with d i sease progression and in response to therapeut ic treatments. There are multiple ways to del ineate B m a x and K d . In our lab, multiple l igand concentrat ion receptor a s s a y s ( M L C R A s ) were performed, in which subjects were scanned three or four t imes using radiotracers at decreas ing speci f ic activity (SA) (Holden et al . , 2002). S A is a measure of the radioactivity of the ligand relative to its weight. The P E T data obtained from each scan were cal ibrated and reduced to produce a Logan plot from which B P was determined. Resul ts from all s c a n s were plotted on a single graph and B m a x , apparent K d , and B P were easi ly determined (from the x-intercept, inverse of the s lope, and y-intercept, respectively). B P is much eas ie r to measure than B m a x and rvj because determining B P requires only a single P E T scan using a radiotracer at high speci f ic activitiy. B P measurements are also more reproducible and less variable than B m a x and K d measurements , so they are more comparab le ac ross sites and between exper imental groups in P E T studies (Gatley et al . , 1995;Hietala et a l . , 1999). B P measurement is, however, dependent on the properties of the tracer and on measurement and analys is of the raw P E T data. Tracer kinetics and central and peripheral tracer metabol ism differ among tracers and different sites use different analys is techniques to obtain quantitative B P values from data col lected by P E T tomographs (Phelps et al . , 1986). Multiple mode ls exist for analyzing raw P E T data and e a c h involves different assumpt ions, constraints, and some degree of subjectivity on the part of the researcher, from the cho ice of analys is technique to the p lacement of regions of interest (ROIs) (Phelps et a l . , 1986;Suzuk i et al . , 2005). Al though the popularity of P E T is increasing, the val idation of many of the tracers in human use is lacking. F e w studies have attempted to correlate in vivo P E T data with actual physiological p rocesses . P E T provides a valuable tool to investigate actual neural p rocesses in living, consc ious human beings, but the technique has limitations. The advantages of using a living system are counteracted by the inability to control all var iables and know definitively all aspects of the physiological situation. For example, in vivo binding of the radiotracer [ 1 1 C]raclopr ide to D2 receptor is affected by extracellular 6 D A c o n c e n t r a t i o n s , w h i c h c a n v a r y t h r o u g h o u t t he s c a n a n d a f fec t e n d m e a s u r e m e n t s of b i n d i n g po ten t i a l ( S a s a k i , 2 0 0 2 ; S e e m a n et a l . , 1 9 8 9 ) . P E T is a l s o a v e r y e x p e n s i v e t e c h n i q u e a n d r e q u i r e s a c c e s s to a n e a r b y c y c l o t r o n fo r s y n t h e s i s of sho r t ha l f - l i fe r a d i o t r a c e r s . T h e l o w s p a t i a l r e s o l u t i o n o f P E T i m a g e s cu r ren t l y l imi ts its util ity to l a rge n e u r o n a l s t r u c t u r e s , s u c h a s t he s t r i a tum, but t he o n g o i n g d e v e l o p m e n t o f P E T s c a n n e r s s h o w s a p r o m i s i n g fu tu re for i n c r e a s e d reso lu t i on . T h e n e w e s t h i g h - r e s o l u t i o n r e s e a r c h t o m o g r a p h s h a v e r e s o l u t i o n s of 2 . 3 m m to 3 . 2 m m (full w id th at ha l f m a x i m u m ) a c r o s s the f ie ld o f v i e w , w h i c h is m o r e t h a n tw i ce the r e s o l u t i o n o f t he P E T s c a n n e r s m a d e t e n y e a r s a g o (de J o n g et a l . , 2 0 0 7 ) . I n c r e a s e d r e s o l u t i o n wi l l i m p r o v e p r e c i s i o n of m e a s u r e m e n t s in l a rge n e u r a l s t r uc tu res a n d po ten t ia l l y e n a b l e t h e i nves t i ga t i on of s m a l l e r n e u r a l s t r u c t u r e s that w e r e p r e v i o u s l y too s m a l l to b e s t u d i e d u s i n g P E T . Autoradiography A u t o r a d i o g r a p h y ( A R G ) is a n in vitro t e c h n i q u e u s e d to e x p l o r e t he m e c h a n i s m s by w h i c h r e c e p t o r s a n d t r a n s p o r t e r s m e d i a t e p h y s i o l o g i c a l e f f e c t s in c o m p l e x o r g a n i s m s . A R G u s e s r a d i o t r a c e r s to v i s u a l i z e a n d quan t i f y b i n d i n g s i t e s s i m i l a r to t h o s e e v a l u a t e d by P E T , but p o s t m o r t e m , in t i s s u e s e c t i o n s . In e a r l y s t u d i e s i nves t i ga t i ng b ind ing s i t e s , m e m b r a n e h o m o g e n a t e s w e r e u s e d a s s a m p l e s fo r r a d i o t r a c e r b i nd i ng a n d w e r e o b t a i n e d by h o m o g e n i z i n g t i s s u e f r o m a s p e c i f i c r e g i o n a n d i so la t i ng t he p ro te in fo r i n c u b a t i o n w i th r a d i o l i g a n d ( P h e l p s et a l . , 1 9 8 6 ) . W i t h t h e e m e r g e n c e of a u t o r a d i o g r a p h i c f i lm, it b e c a m e m o r e c o m m o n to p e r f o r m A R G o n s l i d e - m o u n t e d t i s s u e s e c t i o n s , w h i c h a l l o w e d v i s u a l i z a t i o n of s p e c i f i c a n a t o m i c a l t r a c e r d is t r ibu t ion . T h e h igh reso lu t i on of A R G ( ~ 2 5 u m ) a l s o pe rm i t t ed quan t i t a t i ve m e a s u r e m e n t o f t r a c e r b ind ing in s m a l l n e u r a l s t r u c t u r e s ( L i b e r a t o r e et a l . , 1 9 9 9 ; P a v e y et a l . , 2 0 0 2 ) . D e v e l o p m e n t of p h o s p h o r i m a g i n g A R G t e c h n o l o g y u s i n g h igh ly sens i t i v i t e p h o s p h o r s c r e e n s r e d u c e d e x p o s u r e t i m e s to o n e ten th of x - r ay f i lm e x p o s u r e t i m e s , e f f ec t i ve l y r e p l a c i n g f i lm A R G . T h e h igh sens i t i v i t y of p h o s p h o r s c r e e n s a l s o a l l o w s t he u s e o f t r a c e r s wi th shor t hal f -l i ves , s u c h a s t h o s e u s e d in P E T ( S i h v e r et a l . , 1 9 9 9 ; B e r g s t r o m et a l . , 2 0 0 3 ; S t r o m e et a l . , 2 0 0 5 ) . 7 In storage phosphor A R G , sl ide-mounted frozen t issue sect ions are pre-washed to remove endogenous l igand, then incubated with a radioactive l igand. Unlike P E T , A R G can be performed with radiol igands incorporating short or long half-life (ty2) isotopes. Therefore, the range of radioisotopes useful for A R G is much larger than for P E T , including 1 1 C (t y=20.4 minutes), 1 8 F (tj_=120 minutes), 3 2 P (t y =14 days), 1 2 5 l ( t y=60 days), 3 H (ty2=12.3 years), 1 4 C (ty2=5730 years), etc. Nonspec i f ic binding is determined by incubating adjacent sect ions with the radioligand and a high concentrat ion of speci f ic receptor agonist or antagonist. Under these condit ions, speci f ic binding sites are saturated with cold l igand and radioisotope is able to bind only nonspeci f ic binding sites, which result from trapping, ionic interaction, or van der W a a l s forces between the ligand and different chemica l components of the t issue (Phelps et a l . , 1986). Sect ions are apposed to phosphor sc reens for a t ime period dependent on the half-life of the radioisotope. Phosphor sc reens are photost imulable imaging plates that trap energy from the decay ing radioisotopes in their crystal layer. Th is energy is later re leased by scann ing sc reens in a phosphor imager, which st imulates the crystals with laser light and re leases trapped electrons, in turn releasing photons that are detected by a photomultiplier tube. The imager thereby produces a high-resolution latent image of radioisotope distribution in t issue sect ions (L 'Annunziata, 1998). The location of binding is usual ly easy to detect by v isual examinat ion of the A R G images; darker areas of the image represent greater binding than light areas. The amount of radioactivity in the t issue sect ions can be quantif ied too. Cal ibrated radioactivity s tandards are used to relate the optical densi ty in an image to the activity contained in the s tandard ized sample that produced the image. This process creates a standard curve that can be used to determine the amount of activity in certain t issue regions of interest from the optical density of the images they produce (Pollak and Whar ton, 1993). A R G is commonly used for evaluating e lements of physiological p rocesses , similar to P E T , but without the confounding factors present in vivo, such as competit ion between the tracer and endogenous ligand (Phelps et a l . , 1986;Laruel le , 2000). It is widely employed to determine the characterist ics and distribution of neurotransmitter receptors 8 in the brain. For example , A R G with D2-speci f ic radioactive l igands such as [ 3H]raclopride have been used extensively in P D research in both humans and animal models to determine the fate of D2 receptors during d i sease progression (Ungerstedt, 1968;Hal l et a l . , 1988;Hal l et al . , 1994;Minuzzi et a l . , 2006). Resu l ts of these studies have improved understanding about the neurochemical changes assoc ia ted with P D and contributed to identifying the D2 receptor as a target for P D treatments. A R G plays a fundamenta l role in P E T tracer d iscovery (Myers et a l . , 1996;Bergstrom et al . , 2003;Pate l et a l . , 2003). Cons iderab le time, effort, and cost are assoc ia ted with validating a P E T tracer for human use, so it is helpful to have a screening technique for candidate tracers that will identify those with substantial potential for s u c c e s s and those not worth pursuing. A R G provides a quick, cost-eff icient method of assess ing radiotracer distribution and binding parameters of potential P E T tracers. A R G experiments are much less costly than in vivo P E T exper iments, s ince they are quick, work with a variety of radiotracers, and do not require numerous personnel or elaborate equipment. The use of A R G in identification of [ 1 1 C](+)DTBZ as the best tetrahydrabenazine derivative (over (±)DTBZ and (±)MTBZ) for in vivo P E T studies demonstrates the utility of A R G (Myers et al . , 1996;Ki lbourn et a l . , 1997). Al though other information must be taken into account in the ultimate select ion of new radiotracers, such as radiotracer metabol ism, toxicity, and serum protein binding, in vitro binding data plays a significant role in precl inical evaluation of potential P E T tracers. B m a x and K d can be measured in vitro using Scatchard studies that are much more cost-efficient than in vivo M L C R A s . Binding is performed on either t issue homogenates or serial t issue sect ions using severa l different concentrat ions of radiol igand. S ince different sect ions are used for each concentrat ion, all sect ions can be incubated simultaneously. Th is greatly reduces assay time relative to in vivo M L C R A s , which require a separate scan for each individual l igand concentrat ion, as well as sufficient t ime between s c a n s to maximize radioisotope decay and t issue c learance. Furthermore, use of t issue instead of living organisms permits use of more l igand concentrat ions (~5-12), which improves the accuracy of est imates for B m a x and K d . Saturat ion curves are drawn using data from analys is of A R G images and provide quantitative est imates of B m a x and K d (Geary and Woo ten , 1983;Revi l la et al . , 2000). 9 Whi le B m a x and K d va lues obtained in vitro are more comparab le to in vivo P E T measures than s imple binding va lues, major di f ferences between the two methods still generate different va lues for B m a x and K d . For example , in vivo P E T measurement of raclopride K d in one study was 10-fold smal ler than in vitro homogenate binding measurement of the s a m e parameter obtained in another study (Hall et al . , 1988;Farde et al. , 1989). The limited amount of t issue avai lable for the present study did not permit saturation studies, so A R G binding exper iments were performed at a single ligand concentrat ion for e a c h tracer. The role of A R G with [ 1 1 C]- labeled l igands in P E T tracer d iscovery is much more establ ished than its role in quantif ication of binding si tes. F e w studies have attempted to use in vitro phosphor imaging with P E T tracers to obtain absolute quantitative data (Gatley et al . , 1998;St rome et al . , 2005;St rome et al . , 2006). Furthermore, there is a great deal of variation among the A R G studies that have reported quantitative est imates of tracer binding character ist ics. The variation likely results from inter-site differences in t issue process ing, exper imental protocol, and subject group. Binding exper iments using t issue homogenates have found vastly different results from exper iments using t issue sect ions, as have exper iments using X-ray film and those using phosphor sc reens (Dohanich et al . , 1986 ;Pavey et al . , 2002). Exper imental binding protocols a lso vary by dif ferences in buffer composi t ion, p H , and temperature, radiotracer incubation concentrat ion and duration, number and duration of p re -washes and post-washes, and exposure, imaging, and analys is methods (Phelps et a l . , 1986;Darchen et a l . , 1989 ;Faedda et a l . , 1989;Schwer i , 1990 ;Masuo et a l . , 1990;van K a m p e n and Stoess l , 2003;Minuzz i e t a l . , 2006). Binding condit ions must be opt imized to attain max imum binding eff iciency, but optimal condit ions are determined individually by site, which results in di f ferences among sites. Furthermore, optimal condit ions differ by tracer, subject spec ies and t issue type (Phelps et al . , 1986;Ros tene et a l . , 1992;Pavey et al . , 2002). Buffer composi t ion, temperature, and incubation t ime are the major factors that variably affect in vitro binding. The concentrat ion of cat ions affects the total to nonspeci f ic binding ratio, which ultimately affects speci f ic binding va lues. For example , addit ion of 1 5 0 m M sodium to the 10 incubation buffer increased B m a x of [ 3 H]SCH-23390 by 1 9 % and dec reased K d by 2 9 % (Phelps et a l . , 1986). Temperature affects the time to reach equil ibrium. For example, speci f ic binding with [ 3 H]SCH-23390 reached a max imum within 20-30 minutes at 30 and 37°C, but required 180 minutes at 4°C (Faedda et a l . , 1989). On the contrary, [ 3H]WIN 35,428 binding to D A T is more effective at 4°C (Pavey et a l . , 2002;Marazzi t i et al . , 2006 ;Chen et a l . , 2006). Buffer pH has a lso been shown to differentially affect in vitro binding character ist ics of multiple tracers (Phelps et a l . , 1986). Investigation of a speci f ic molecule using different radiotracers a lso produces different quantitative results for B m a x (Hall et a l . , 1990;Pavey et a l . , 2002). Measured density of a molecule should be the s a m e no matter which tracer is used , but di f ferences exist and likely result from properties of individual radiotracers, such as affinity for the molecule of interest, binding site, or sensitivity to endogenous l igand. Whi le most studies obtain comparab le data, the absolute quantitative va lues of binding character ist ics obtained at different si tes are not in agreement. The variation in exper imental methods and radiotracer propert ies makes it difficult to reconci le f indings among sites in order to obtain a comprehens ive understanding about the molecu le of interest. For this reason, changes in binding va lues (often % change or directional change, i.e. increase or decrease) between normal controls and experimental groups are often reported in the literature instead of absolute binding values. Comparison of in vivo PET and in vitro ARG Although P E T and A R G share the s a m e goal of elucidating biological p rocesses under speci f ic condit ions, there are important distinctions between the two methods. T h e s e dif ferences must be taken into considerat ion when assess ing results obtained by either method. Tracer binding characterist ics reported in the literature vary between the two techniques, for reasons that will be d iscussed below. To the best of my knowledge, no single study has compared in vivo and in vitro binding methods with the s a m e radiotracers to obtain quantitative est imates of B m a x and K d . However, a few studies have been performed in the s a m e individuals with the s a m e l igands labeled with [ 1 1 C] for P E T and with [3H] for A R G to measure B m a x a n d K d (Suzuki et al . , 2001;Minuzz i et a l . , 2006). S ince the advent of phosphor imaging, which al lowed 11 use of [ 1 1 C]- and [ 1 8 F]- labeled l igands for A R G , more recent studies have used the s a m e radiol igands both in vivo and in vitro to a s s e s s B P by P E T and binding by A R G , although none have del ineated B m a x and K d (Nikolaus et a l . , 2003;lnaj i et al . , 2005;St rome et a l . , 2006). They have reported significant correlat ions between P E T B P and A R G binding measurements , but these correlat ions are not perfect (correlation coefficient r<1.0). Severa l explanat ions have been proposed for observed d iscrepancies among B m a x and K d va lues obtained by P E T and A R G in the literature (Nader et al . , 1999;Holden et a l . , 2002;Niko laus et al . , 2003 ;Rosa -Ne to et al . , 2004;lnaj i et al . , 2005;Kung and Kung , 2005). In vivo P E T studies are performed in living beings and thus measure dynamic p rocesses in functioning living sys tems. This quality provides the advantage of studying real p rocesses as they occur in their true physiological environment. P E T also avoids confounding inf luences of the sample preparation required for in vitro A R G . Homogeniz ing t issue and purifying cell membrane proteins or sl icing t issue into very thin sect ions (usually 15-20um) may damage or alter the receptor of interest (Pollak and Whar ton, 1993;Holden et a l . , 2002). Both homogenizat ion and sect ioning can also expose internalized receptors that may not be readily avai lable to bind radiol igands in vivo. Moreover, methods of t issue isolation and preservat ion may also differentially affect binding character ist ics. This point is of particular importance in c a s e s where there is a significant amount of t ime between subject death and t issue preservation, as is often the c a s e in human A R G studies (Hall et al . , 1994). A primary caveat of P E T is that the speci f ic binding environment is not entirely def ined and may vary during the s c a n . Endogenous ligand may compete with the radioligand for receptor binding si tes and affect the binding of certain radiol igands (Mintun et al . , 1984). Raclopr ide binding, for example, is known to be sensi t ive to endogenous D A levels, whereas D T B Z binding is thought to be unaffected by changes in synaptic D A levels (Fuxe et a l . , 1992;Vander Borght et al . , 1995a;Wi lson and K ish , 1996;Suzuki et al . , 2001). Sensit ivity to D A levels may be the result of dynamic changes in affinitiy of the receptors as a function of neurotransmitter re lease, especia l ly in the case of the low-affinity l igand raclopride (Fuxe et al . , 1992;Suzuk i et a l . , 2001). In support of this hypothesis, compar ison of raclopride binding in vivo and in vitro has found different 12 apparent affinity and density measurements in response to drug treatments that vary extracellular D A concentrat ion (Logan et al . , 1991;Young et a l . , 1991;Minuzzi et al . , 2006;Chefer et a l . , 2007). For in vitro A R G , on the other hand, endogenous ligand is washed from the t issue before incubation with the tracer and thus does not affect l igand-receptor interactions. The sensitivity of [ 1 1 C]raclopr ide to synapt ic D A concentrat ions is not always a d isadvantage. Th is property is being exploited in s o m e areas of P E T research, such as sch izophrenia and addict ion to v isual ize and quantify changes in extracellular D A concentrat ion (Laruel le, 2000). However, it is problematic in other areas such as P D research, where it interferes with measurements of D2 receptor changes (Volkow et a l . , 1994;Laruel le, 2000). Another factor assoc ia ted with the inability to control the binding environment in vivo is the inability to attain a true equil ibrium condit ion. Most binding data analys is techniques assume that the sys tem is at equil ibrium (Phelps et a l . , 1986;Pol lak and Whar ton, 1993;Logan et a l . , 1996 ;Lammer tsma and Hume, 1996). In the in vivo situation, receptors are embedded in t issue that is surrounded by interstitial fluid exposed to variable concentrat ions of endogenous ligand that change cont inuously to adapt to the environment. There is a lso a limited amount of s p a c e in the synapse for tracer diffusion and only a smal l amount of radiotracer is injected so as to prevent the ligand from inducing a physiological effect. T h e s e condit ions make it rare that P E T measurements be obtained at a true equil ibrium, which inhibits accurate a s s e s s m e n t of nonspecif ic binding (Phe lps et a l . , 1986;Doudet et a l . , 2002a). In Logan analys is of P E T data, as in many other methods, nonspeci f ic binding is determined using the measured radioactivity in a brain region void of speci f ic binding sites (reference region, such as the cerebel lum in most studies investigating the striatal D A system) (Logan et al . , 1996). Activity in the reference region is a reflection of both nonspeci f ical ly bound radioligand and unbound free radiol igand (that which is avai lable for binding) and is only useful for measur ing nonspeci f ic binding when the system is at equil ibrium. The bolus plus constant infusion method of tracer injection improves the l ikelihood of obtaining a true equil ibrium, but does not ensure it (Carson, 2000). In contrast, in in vitro A R G studies, t issue sect ions are incubated with a speci f ic concentrat ion of radiol igand that remains 13 constant throughout the incubation and the incubation concentrat ion is essential ly equivalent to the free l igand concentrat ion (Phelps et a l . , 1986). Nonspeci f ic binding is a lso eas ier to measure in vitro by addition of cold l igand to the incubation solution. In animal studies using P E T , anesthet ics provide an addit ional source of disparity between in vivo and in vitro techniques. An imals must remain mot ionless throughout the duration of the scan and few sites have been able to perform P E T studies in animals without the use of anesthet ics. Most anesthet ics have been shown to affect binding of P E T tracers, which would lead to dif ferences between P E T measurements obtained in vivo and A R G measurements obtained in the absence of anesthet ics (rodent studies) or after different anesthet ic treatment (for non-human primates) (F ink-Jensen et al . , 1994;Tsukada et a l . , 1999 ;Tsukada et al . , 2000 ;Tsukada et a l . , 2001;Votaw et al . , 2003). Mechan i sms of action of most anesthet ics are largely unknown, but their effects appear to vary by anesthet ic, dosage , animal spec ies , and by individual within a spec ies . The Dopamine System The Neurotransmitter Dopamine Dopamine (DA) plays an important role in severa l neural sys tems, regulating such diverse funct ions as movement, motivation and cognit ion (Le Moa l and S imon , 1991,Volkow et a l . , 1996b;Hyman et a l . , 2006). It has a lso been assoc ia ted with reward, reinforcement and addict ion. The wide range of problems assoc ia ted with abnormali t ies in brain dopamine signal ing demonstrates its importance and widespread role in overall brain function (Volkow et a l . , 1996b). Neurodegenerat ive d isorders (e.g. P D , A D , HD) as well as psychiatr ic i l lnesses such as subs tance abuse , sch izophren ia , and depress ion have been linked to alterations in D A signaling (Bernheimer et al . , 1973;Calne et a l . , 1992;Volkow et a l . , 1997a;Volkow et al . , 1999 ;Werkman et a l . , 2006;Malh i and Berk, 2007). D A is the most abundant catecholamine in the brain (Val lone et al . , 2000). Dopaminergic cel ls reside primarily in the midbrain in the substant ia nigra (SN), the ventral tegmental a rea (VTA) , and the retrobulbar a rea (Volkow et a l . , 1996b). Those originating in the S N project predominantly to the dorsal striatum (caudate nucleus and 14 putamen) and are involved mainly with movement initiation and execut ion (Alexander and Crutcher, 1990;Mink, 2003). V T A neurons project predominant ly to limbic and l imbic-associated regions including nucleus accumbens , orbital and cingulate cort ices, amygdala , and h ippocampus (Succu et al . , 2006). They are involved in reinforcement, motivation, mood, and thought-organization (Yun et a l . , 2004;Nest le r and Car lezon, Jr., 2006;Margol is et a l . , 2006). D A neurons in the retrobulbar area project to the hypothalamus and regulate hormone secret ion from the pituitary (de Haan et al . , 2004). The movement -assoc ia ted nigrostriatal dopamine sys tem is the main system affected in early P D . DA is synthes ized inside D A neurons. Figure 1.2 shows the pathway for D A synthesis in the central nervous sys tem. In the rate-limiting step, tyrosine is converted to dihydroxyphenylalanine ( L - D O P A ) by tyrosine hydroxylase (Nagatsu et al . , 1964). D O P A decarboxy lase then converts this intermediate to DA , which is loaded into synapt ic ves ic les by the neuronal vesicular monoamine transporter (VMAT2) (Liu and Edwards, 1997). T h e vesicu lar membrane protects D A from oxidation by monoamine ox idase A (MAO-A) , a degrading enzyme present in D A neurons. M A O - B and catechol -O-methyl t ransferase ( C O M T ) can also oxidize DA. D A degradat ion in all c a s e s results in the formation of homovani l l ic acid (Fig. 1.3). DOPAMINE SYNTHESIS OH fit If l l AAOC Tyrosine L-DOPA Dopamine Fig. 1.2. Pathway for dopamine synthesis in the central nervous sys tem. T H , tyrosine hydroxylase; A A D C , aromatic amino acid decarboxy lase ; L - D O P A , dihydroxyphenylalanine (adapted from Vo lkow et a l . , 1996b). 15 Fig. 1.3. Pathway for dopamine metabol ism in the central nervous system. M A O , monoamine ox idase; A D , a ldehyde dehydrogenase; C O M T , catechol -O-methyltransferase; D O P A C , dihydroxyphenylacet ic ac id ; H V A , homovanil l ic ac id , (adapted from Vo lkow et a l . , 1996b). Figure 1.4 il lustrates the locations of D A system components at the D A neuron terminal, as well as s o m e of the P E T radiotracers that bind these components . A n action potential in the D A neuron triggers fusion of DA-contain ing synapt ic ves ic les with the p lasma membrane to re lease the neurotransmitter into s y n a p s e s where it interacts with postsynapt ic D A receptors. Synapt ic D A concentrat ions are maintained at low (nanomolar) levels by the action of the D A transporter (DAT) as well as M A O and C O M T . D A T plays the most significant role, with reuptake of D A into the presynaptic neuron, while M A O and C O M T in surrounding neurons and glia degrade DA by oxidation. D A re lease in the striatum is jointly regulated by multiple presynaptic and postsynapt ic mechan i sms including D A D2 autoreceptor activity, V M A T 2 activity, dopamine D2 receptor activity and dopaminergic neuron interactions with other neurotransmitters (Volkow et al . , 1996b;Pothos, 2002). 16 Fig. 1.4. Diagram of dopamine nerve terminal, including P E T tracers that target terminal components (adapted from diagram by Sarah Lidstone). Neuroanatomy of the D o p a m i n e S y s t e m DA functions largely in neuronal circuits involving the basa l gangl ia (BG). B G are large subcort ical structures that participate in parallel circuits integrating cerebral regions and thalamus in five def ined cort ico-BG-thalamo-cort ical loops: 1) motor, 2) oculomotor, 3) associat ive, 4) l imbic, and 5) orbitofrontal (Alexander et a l . , 1986;Mink, 2003). S ince the present study focuses on the D A system in relation to Park inson 's d isease and motor behaviour, the following d iscuss ion will focus on the motor B G circuit. The B G nuclei important in movement control include dorsal striatum, subthalamic nucleus (STN) , g lobus pall idus (internal segment [GPi] and external segment [GPe]), and substant ia nigra (pars compac ta [SNc] and pars reticulata [SNr]). Figure 1.5 shows the anatomical posit ions of basa l gangl ia in the human brain. Located in the forebrain, the dorsal striatum is a large structure compr ised of the caudate nucleus and putamen, which are separated by white matter tracts of the internal capsu le . The striatum is one of the primary afferent nuclei of the B G , receiving input mainly from the cortex. The 17 primary B G output structures are the G P i and S N r (Albin et al . , 1989;Obeso et al . , 2002;Mink, 2003). S ince these nuclei contain cytologically similar neurons that form parts of a single neuronal sys tem, they are often lumped together and referred to as G P i / S N r in d iscuss ions of basa l gangl ia circuits (Albin et al . , 1989). Fig. 1.5. Anatomica l posit ions of the basal gangl ia and tha lamus in the human brain (from cti. i tc.virgina.edu/pscy220/). Understanding the role and function of the B G is a long-standing chal lenge that remains unresolved to this day. Much of our understanding has c o m e from the study of disorders involving damage to B G nuclei , such as P D and HD (Fig. 1.6). T h e s e chronic d i seases are character ized by progressive degenerat ion of B G structures accompan ied by motor, cognitive, and emot ional symptoms (Bradley et a l . , 1991). In P D , motor symptoms normally appear much earl ier than cognitive deficits (Calne et al . , 1992). Models attempting to explain B G organizat ion and the causa l relationship between D A def ic iency and motor d is turbances have been proposed and refined s ince the late 1980's (Albin et a l . , 1989 ;Obeso et al . , 2000). It is now accepted that the B G motor Substantia nigra Subthalamic 18 circuit is involved in both planning and production of movement (Alexander and Crutcher, 1990;Mink, 2003). (A) Parkinson's disease (B) Huntington's disease Fig. 1.6. Pathological changes in neurodegenerat ive d i seases . (A) Midbrain from a patient with Park inson 's d i sease shown on left demonstrates loss of DA neurons in substant ia nigra (pigmented area, arrows) relative to normal subject shown on right. (B) The s ize of the striatum is dramatical ly reduced in patients with Huntington's d isease (from Bradley e t a l . , 1991). Information for movement control originating in motor-associated cortical regions is processed in the B G and returns, via the thalamus, to movement-assoc ia ted areas in the frontal lobes and brain stem (Albin et al . , 1989;Alexander and Crutcher, 1990). The c lass ica l model of B G motor circuitry offers an idea of B G functional anatomy and has provided targets for effective treatment of several movement disorders, including P D . In the c lass ica l model , the striatum receives direct excitatory input from the cortex, which is modified by dopaminerg ic input from S N c to modulate G P i / S N r output via the "direct" and "indirect" pathways (Obeso et al . , 2002;Mink, 2003). S N c dopaminergic neurons synapse on striatal medium spiny neurons of morphological ly indistinguishable but functionally distinct identities. O n e subset of striatal neurons contains D1 receptors and sends inhibitory projections directly to the G P i / S N r , representing the "direct pathway". The other main subset of striatal neurons contains D2 receptors and excites the G P i / S N r through a sequence of connect ions involving G P e and S T N in the "indirect pathway". T h e s e pathways have opposing effects on G P i / S N r and thus oppositely affect the amount of inhibition that the output nuclei exert on their thalamic targets (Alexander 19 and Crutcher, 1990;Obeso et al., 2000). In essence, the direct pathway is thought to facilitate desired movements while the indirect pathway suppresses undesired movement (Mink, 2003). The Direct and Indirect Pathways The direct pathway (Fig. 1.7) consists of a monosynaptic, inhibitory connection from the putamen to GPi/SNr (Purves et al., 2001). Since D1 receptors are G protein-coupled receptors that stimulate adenylate cyclase activity upon activation by DA binding, these neurons are thought to potentiate the effects of cortico-striatal activity (Mink, 2003). These neurons release GABA, which inhibits GABA-releasing GPi/SNr neurons that project to the thalamus. The GPi/SNr neurons normally exhibit a high spontaneous firing rate that tonically inhibits the thalamus, whereas the striatal medium spiny neurons are quiescent and transiently fire action potentials only when stimulated. Therefore, inhibiting the GPi/SNr output neurons with activity in the direct pathway disinhibits the thalamus and effectively increases movement. Premotor cortex Q ( t r a n s i e n t ) pars' J^1 Caudate/putamen / • compacts / WKBmBBBBmm (transient) O i l (transient) ML (transient) Globus pall idum internal segment Fig. 1.7. The direct pathway. Activity in this pathway ultimately facilitates movement (from Purves etal., 2001). In contrast, activity in the indirect pathway (Fig. 1.8) ultimately decreases activation of the motor cortex and decreases movement. Striatal neurons in this pathway contain D2 receptors, which are indirectly coupled to adenylate cyclase. DA binding to D2 receptors 20 l e a d s to inh ib i t ion of a d e n y l a t e c y c l a s e act iv i ty , t h e r e b y d e c r e a s i n g e f fec ts of co r t i co -st r ia ta l act iv i ty (M ink , 2 0 0 3 ) . T h e s e n e u r o n s s e n d G A B A e r g i c , inh ib i tory p ro jec t i ons to t he G P e a n d s y n a p s e wi th inh ib i tory n e u r o n s that p ro jec t to t h e S T N . T h e S T N c o n t a i n s g l u t a m a t e r g i c n e u r o n s that e x c i t e t he G P i / S N r s u c h that ac t iv i ty in t he ind i rect p a t h w a y i n c r e a s e s t h a l a m i c inh ib i t ion a n d d e c r e a s e s t h a l a m o c o r t i c a l act iv i ty , t h e r e b y d e c r e a s i n g m o t o r act iv i ty . Indirect F i g . 1.8. T h e ind i rec t p a t h w a y ( s h a d e d ) . Ac t i v i t y in th is p a t h w a y u l t imate ly inh ib i ts m o v e m e n t ( f rom P u r v e s et a l . , 2 0 0 1 ) . Parkinson's Disease P a r k i n s o n ' s D i s e a s e ( P D ) is a p r o g r e s s i v e n e u r o d e g e n e r a t i v e d i s e a s e that is e s t i m a t e d to a f fec t a p p r o x i m a t e l y 1 3 0 p e o p l e p e r 1 0 0 , 0 0 0 p o p u l a t i o n in Br i t i sh C o l u m b i a a n d a l m o s t 4 , 000 ,000 p e o p l e w o r l d w i d e ( W o r l d H e a l t h O r g a n i z a t i o n , 1 9 9 7 ; L a i et a l . , 2 0 0 3 ) . P D is c h a r a c t e r i z e d by d e g e n e r a t i o n of D A n e u r o n s in t he S N c a n d is a s s o c i a t e d wi th f ou r c a r d i n a l c l i n i ca l s y m p t o m s : res t ing t remor , r igidity, b r a d y k i n e s i a ( s l o w n e s s o f m o v e m e n t ) , a n d p o s t u r a l instab i l i ty ( B e r n h e i m e r et a l . , 1 9 7 3 ; C a l n e e t a l . , 1 9 9 2 ; S h i n o t o h e t a l . , 2 0 0 0 ) . 21 In humans, cl inical symptoms of P D appear only when striatal D A concentrat ion is reduced by 80%, or when dopaminergic neuronal death reaches a certain threshold: approximately 8 0 % of striatal nerve terminals or 50 -60% of S N c cell bodies (Bernheimer et al . , 1973;Riederer and Wuket ich , 1976 ;Seeman and Niznik, 1990;Koller, 1992). Symptoms of striatal dysfunct ion are observed in animal mode ls of P D after the s a m e severe D A def ic iency of 8 0 % or more (Poirier et a l . , 1966;Burns et a l . , 1983;Donnan et al . , 1987). Degradat ion of the D A system in idiopathic P D occurs in a non-uniform manner. D A loss is much more severe in the putamen than in the caudate nucleus and there is differential loss within these regions as well . In the putamen, D A loss follows a rostrocaudal gradient, with more severe deplet ion in the cauda l region. In the caudate, D A loss fol lows a rostrocaudal gradient of similar strength, but in the opposite direction with greatest D A deplet ion in the rostral region. Both structures show a similar dorsoventral gradient of D A loss along the entire rostrocaudal extent of the striatum (Kish et a l . , 1988;Leher icy et al . , 1994;Wi lson and K ish , 1996;Lee et al . , 2004). The more prominent D A loss in the putamen fits with the finding that the putamen is the striatal region primarily concerned with movement control in basa l gangl ia circuits. Neuroanatomical and neurophysiological studies have found dense reciprocal connect ions between the putamen and the motor, premotor, and supplementary motor cort ices (Kish et a l . , 1988). The caudate is involved in these motor circuits, but is a lso interconnected with the dorsolateral prefrontal cortex through which it plays a role in working memory and planning (Alexander et al . , 1986;Leher icy et al . , 1994;Fuente-Fernandez and S toess l , 2002;Haber , 2003). Accord ing to the c lass ica l model of basa l gangl ia circuitry, degenerat ion of S N c D A neurons in P D ultimately c a u s e s G P i / S N r activity to increase, leading to excess ive thalamocort ical inhibition (Albin et al . , 1989 ;Obeso et a l . , 2 0 0 0 ; O b e s o et al . , 2002). The resulting reduction in activity of cortical and brain s tem motor a reas partially accounts for motor deficits assoc ia ted with P D . Abnorma l activity in addit ional circuits that contribute to motor symptoms have been identified more recently, but the speci f ic mechan isms of P D symptoms are unclear (Schnitzler et a l . , 2006). Figure 1.9 shows schemat ical ly how loss of S N c D A neurons affects the direct and indirect pathways. D A def ic iency reduces excitation of striatal G A B A e r g i c neurons in the direct pathway and reduces inhibition of striatal G A B A e r g i c neurons in the indirect pathway. Decreased 22 activity in the direct pathway reduces inhibition of the G P i / S N r . Decreased inhibition of indirect pathway striatal neurons leads sequential ly to overinhibition of G P e , decreased inhibition of S T N and increased excitation of the G P i / S N r . In this manner, both pathways contribute to excess ive thalamic inhibition exerted by the G P i / S N r and decreased motor activity. This model partially accounts for the bradykinesia and eventual ak ines ia (loss of movement) observed in P D patients. Fig. 1.9. Funct ional ou tcomes of direct and indirect pathways in Park inson 's d isease . Thinner arrows represent reduced neuronal activity and thicker arrows represent increased activity (from Purves et al . , 2001). Al though the c lass ica l model of B G circuitry has contributed to development of several effective P D treatments such as L - D O P A therapy and pall idotomy, it is a greatly simplif ied model of the true circuitry and does not explain a number of anatomical , physiological, exper imental and clinical f indings (Obeso et a l . , 2000 ;Obeso et al. , 2002). More complex models have been proposed to integrate new findings, but B G circuitry is still not fully understood (Obeso et a l . , 2002;Mink, 2003). Radiotracers for the Dopamine System Severa l radiotracers have been developed to investigate var ious components of the DA system using P E T . S ince D A does not c ross the blood brain barrier, P E T tracers must target assoc ia ted molecu les such as D A receptors, D A transporters, D A precursors, and 23 enzymes that degrade DA. Radioact ive isotopes have been attached to many such molecules for use in P E T studies. Severa l P E T tracers are used to target the s a m e molecule or p rocess at different research sites or under different study condit ions. In most c a s e s , these tracers have different properties that vary in importance depending on the research quest ion, but the combinat ion of advantages and d isadvantages of each determines its functionality for a select purpose. Tracers for P E T examinat ion of the D A sys tem can be categor ized into 2 main groups: those that target presynaptic function and those that target postsynaptic function. Tab le 2.2 lists radiotracers developed for P E T investigation of the striatal D A system, although it is not al l- inclusive. Most of these compounds have also been labeled with [ 3H] for use in in vitro binding studies. Tab le 1.1. Radiot racers deve loped for studying the function of the striatal D A system in vivo with P E T . Radiotracer Target Reference Presynaptic 6-[18F]Fluoro-L-dopa (FDOPA) DA synthesis Garnettefa/. (1983) [11C]dihydrotetrabenazine (DTBZ) VMAT2 Ding etal. (1994) [1 1C]MTBZ VMAT2 Kilbourn etal. (1997) [11C]FP-(3-CIT DAT Mulleref a/. (1993) [11C]d-f/?reo-methylphenidate (MP) DAT Volkow etal. (1995) [11C]WIN 35,428 (CFT) DAT Frost etal. (1993) [11C]nomifensine DAT Aquilonius etal. (1987) [11C]RTI-32 DAT Guttman etal. (1997) [11C]PE2I DAT Halldin etal. (2003) Postsynaptic [11C]SCH-23390 D1 receptor Billard etal. (1984) [ 1 1C]SKF 82957 D1 receptor Neumeyeretal. (1991) [ 1 1C]NNC756 D1 receptor Andersen etal. (1992) [1 1C]NNC 112 D1 receptor Andersen etal. (1992) [11C]raclopride D2 receptor Antonini etal. (1995) [11C]apomorphine (APO) D2 receptor Zijlstra ef al. (1993) [11C]propylnorapomorphine (NPA) D2 receptor Seeman etal. (1984) [18F]fallypride D2 receptor Mukherjee etal. (1995) [18F](3-N-methyl)benperidol D2 receptor Nikolaus etal. (2003) [18F]N-methylspiroperidol D2 receptor Volkow ef al. (1996) VMAT2: Vesicular Monoamine Transporter Type 2; DAT: Dopamine Transporter 6- [ 1 8 F]f luoro-L-dopa ( [ 1 8 F ]DOPA) is one of the best-character ized radiotracers for investigation of the presynapt ic D A sys tem. In the striatum, [ 1 8 F ] D O P A is 24 decarboxylated by aromat ic acid decarboxy lase and converted to [ 1 8 F]DA, which accumulates in synapt ic ves ic les in D A nerve terminals (Doudet et al . , 1997). S ince [ 1 8 F]DA gets t rapped in ves ic les and is only mildly metabol ized, the activity detected by the scanner in [ 1 8 F ] D O P A P E T represents mainly D A synthesis and storage in the striatum (Buu, 1989;Lee et a l . , 2000). [ 1 8 F ] D O P A uptake thus expresses D O P A decarboxy lase activity, as well as D A storage capaci ty (Antonini and DeNotar is, 2004). The finding that [ 1 8 F ] D O P A uptake correlated with nigral cell counts in both humans and MPTP- t rea ted monkeys instigated the use of [ 1 8 F ] D O P A P E T as a method of investigating D A nerve terminal integrity (Snow et a l . , 1993;Pate et a l . , 1993). S ince its first use for P E T in humans in 1983, it has been instrumental in providing insight into various physiological condit ions ranging from cancer to coronary arteriosclerosis to neurodegenerat ive d i seases (Brooks et a l . , 1990;Di Car l i and Dorbala , 2006; lsrael and Kuten, 2007). Al though [ 1 8 F ] D O P A has a high degree of within-subject reproducibility, its utility is compromised b e c a u s e it measures a combinat ion of p rocesses that cannot be isolated (Vingerhoets et a l . , 1996). The striatal activity detected by the P E T scanner is the end result of uptake of [ 1 8 F ] D O P A across the blood brain barrier, uptake into the DA neuron (where [ 1 8 F ] D O P A must compete with large neutral amino acids) , decarboxylat ion to [ 1 8 F]DA inside the terminal, and trapping of [ 1 8 F]DA in synapt ic ves ic les (Stoessl and Ruth, 1999). Therefore, [ 1 8 F ] D O P A P E T is an accurate way to measure D A synthesis and storage as an entire process , but is not as useful for evaluat ing speci f ic molecular changes to the D A sys tem. Dihydrotetrabenazine (DTBZ) binds to V M A T 2 , the central vesicular transporter local ized to monoaminerg ic terminals (Scherman et a l . , 1988). It is highly l iposoluble and thus penetrates through membranes to interact with V M A T 2 throughout the t issue (Scherman et a l . , 1988). Al though it is a chiral molecule, only the (+) enant iomer is capab le of speci f ic binding with V M A T 2 (Kilbourn et a l . , 1997). S ince D T B Z is not select ive for dopamine neurons, it labels serotonergic and noradrenergic neurons as well (Scherman et a l . , 1986). However, several studies confirm that striatal monoamine activity is primarily dopaminergic . The large majority of ca techo lamine transporter sites in rodent and baboon striatum are dopamine transporters (Donnan et al . , 1989;Ding et 25 al. , 1995). D A accounts for approximately 9 0 % of monoamines in the dorsal striatum (Scatton et al . , 1983) and only about 5 % of striatal neurons are serotonergic (Scherman et al . , 1986). Therefore, D T B Z binding is widely accepted as a marker of dopamine neurons in this region (Stoess l and Ruth, 1999). D T B Z binding is largely regarded as the "gold s tandard" for measur ing the integrity of the D A system in vivo. V M A T 2 appears to be unaffected by pharmacological modulat ion, which makes it an ideal target for estimating D A neuron density (Kilbourn et al . , 1996;Stoess l and Ruth , 1999). At tempts to induce regulation of V M A T 2 binding si tes with either acute or chronic drug treatments, in severa l different spec ies , have been unsuccessfu l both in vivo (Kilbourn et al . , 1996) and in vitro (Naudon et al . , 1994;Vander Borght et a l . , 1995a ;Vander Borght et al . , 1995b;Wi lson and K ish , 1996;Kemmerer et al . , 2003). This characterist ic makes V M A T 2 especia l ly useful for measur ing the integrity of the striatal D A system in patients who are currently undergoing treatment by allowing them to cont inue treatment while undergoing diagnost ic testing for treatment eff icacy or d i sease severity. D T B Z binding dec reases with age in rodents, non-human primates, and humans (Masuo et al . , 1990;Frey et a l . , 1996;Doudet et.al . , 2006). D e c r e a s e s in M T B Z binding are proportional to dec reases in tyrosine hydroxylase activity (DA neuron marker) measured by immunohis tochemica l techniques in rats, support ing its use as a marker of D A neuron density (Calne et a l . , 1985;Feuerste in et a l . , 1989;Vander Borght et al . , 1995b). D T B Z binding measured in vitro by A R G has a lso been shown to correlate with u P E T D T B Z B P va lues and with behavioural measurements in the 6 - O H D A rat model of P D , support ing the belief that D T B Z binding cor responds to D A sys tem integrity (Strome et a l . , 2006). Methylphenidate (MP) binds p lasma membrane catecho lamine transporters. It recognizes both dopamine transporters (DAT) and noradrenal ine transporters (NAT), but has low affinity for serotonin transporters ( S E R T ) (Ding et a l . , 1995;Gat ley et al . , 1996). S ince the great majority of catecholamine transporter si tes in rodent and non-human primate striatum are assoc ia ted with D A transporters, M P is a good marker of D A T s in this region (Donnan et al . , 1989;Ding et a l . , 1995). In the cortex and 26 diencephalon, however, N A neurons predominate and M P binding cannot be used to investigate D A T binding sites. Like D T B Z , M P is a chiral molecu le and only one of its isomers, d-threo-MP, b inds D A T with high affinity (Schweri et a l . , 1985;Kimko et a l . , 1999;Ding et a l . , 2004). M P is more select ive for D A T relative to N A T and S E R T and has faster kinetics than other tropane analogs used for P E T tracer synthesis (Stoessl and Ruth, 1999 ;Chan et a l . , 1999;Lee et al . , 2000). D A T is found exclusively in D A neurons, local ized to axons and dendrites in peri-synapt ic a reas (Nirenberg et al . , 1996;Hersch et a l . , 1997;Torres et al . , 2003). D A re leased at the synapse must therefore diffuse out of the cleft to be transported back into the terminal. D A T is driven by the ion concentrat ion gradient generated by the p lasma membrane Na+/K+ A T P a s e (Giros et a l . , 1994). D A T plays an important role in maintaining presynapt ic homeostas is , as has been shown by studies involving mice lacking D A T ( D A T - K O ) . D A T - K O mice exhibit a 300-fold increase in striatal extracellular D A and dec reased levels of both D1 and D2 receptors (Giros et a l . , 1996). Furthermore, chronic increased extracel lular D A concentrat ions are assoc ia ted with increased M P binding and chronic dec reased D A concentrat ion are assoc ia ted with decreased M P binding (Lee et a l . , 2000 ;Bezard et a l . , 2001). C h a n g e s in M P binding to D A T may result from altered trafficking of D A T between external membrane and internalized locations, changes in D A T density in the p lasma membrane, and changes in affinity of D A T for DA. Both high and low affinity D A T binding sites have been identif ied, but P E T studies cannot dist inguish between them (Madras et a l . , 1989;Morr is et a l . , 1996). P E T studies have demonstrated that D A T levels naturally dec rease with age (Volkow et al . , 1996a) and are a lso affected in severa l brain disorders including P D and addict ion (Kim et a l . , 1997;Volkow et a l . , 1997a). M P binding to D A T is not regulated by acute changes in synapt ic D A levels (Gat ley et al . , 1995). The coca ine analog 2B-carbomethoxy-3(3-(4-f luoro-phenyl)tropane (commonly known as WIN 35,428 or C F T ) is another ligand used in P E T studies to investigate D A T binding. WIN 35, 428 is a potent inhibitor of D A transport and is select ive for D A T in severa l spec ies including humans and non-human primates (Madras et a l . , 1989;Canf ie ld et a l . , 1990;Kaufman and Madras , 1992b). Its utility as a l igand for both in vivo and in vitro binding studies has been well-val idated (Hantraye et a l . , 1992;Kaufman and Madras , 27 1992b;Wong et a l . , 1993;Frost et a l . , 1993). It shows high levels of speci f ic binding in vivo and in vitro and provides replicable quantitative binding data (Morris et a l . , 1996). Studies investigating D A T often use [ 1 1 C ] M P for in vivo P E T and [ 3H]WIN 35,428 for in vitro A R G and compare results (Madras et a l . , 1989;Kaufman and Madras , 1992a;Gat ley et a l . , 1995;Ding et al . , 1995). A l though these studies make the assumpt ion that [ 1 1 C ] M P and [ 3H]WIN 35,428 are assess ing the s a m e binding site, this assumpt ion has never been val idated. The fact that both tracers bind D A T is well establ ished, but it is poss ib le that they do not bind in the s a m e manner. Al though M P and WIN binding have never been compared directly in vitro using t issue sect ions, they have been compared in a mouse membrane homogenate binding study (Gatley et a l . , 1995). Methodology w a s quite different in this study, s ince mice were injected with tracer and then sacr i f iced, so tracer distribution occurred in vivo before purification of membrane proteins. Gat ley et al. (1995) detected the s a m e regional distribution and obtained an excel lent correlation coefficient (0.96) between striatal membrane binding with [ 1 1 C ] M P and [ 3 H]WIN 35,428. They also found that speci f ic W I N 35,428 binding was approximately twice as high as speci f ic M P binding in vitro, showing that M P binds more reversibly to D A T than W I N . They suggested that M P has a lower affinity than WIN for D A T . D1 and D2 receptors are the major D A receptor subtypes and their expression varies throughout the brain. D1 receptors are the most w idespread and most highly expressed D A receptors in the brain, residing mainly in the striatum, nuc leus accumbens , olfactory tubercle, cerebral cortex, amygda la , is lands of Cal le ja, and subthalamic nucleus (Jaber et al . , 1996;Val lone et al . , 2000 ;Radad et al . , 2005). D2 receptors are also mainly expressed in the striatum, nucleus accumbens , and olfactory tubercle, but are a lso found in the substant ia nigra pars compacta and ventral tegmental area, where they are bel ieved to function as autoreceptors. Severa l studies support this autoreceptor role of presynapt ic D2 receptors. Firstly, D1 receptors are primarily located postsynaptical ly, whereas D2 receptors are located in both presynapt ic and postsynapt ic neurons (Jaber et a l . , 1996 ;Radad e t a l . , 2005). D1 and D2 receptors a lso play functionally opposing roles in the nigrostriatal pathways of 28 the basal gangl ia, which modify inhibition of the excitatory thalamic input to the cerebral cortex and ultimately regulate movement. D1 receptors are located primarily on medium spiny striatal neurons that project to the G P i in the direct pathway (Gerfen et a l . , 1990;Harr ison et a l . , 1990;Val lone et al . , 2000). D2 receptors are expressed by G P e -projecting striatal neurons in the indirect pathway, and a lso by striatal chol inergic interneurons and nigrostriatal D A neurons in which they appear to play an autoregulatory role (Morelli et a l . , 1988 ;Radad et a l . , 2005). T h e s e presynaptic D2 receptors are bel ieved to modulate the synthesis, re lease, and re-uptake of D A (Tsukada et a l . , 1999). W h e r e a s the density of postsynapt ic D2 receptors is highly sensit ive to regulation by synapt ic D A levels, D2 autoreceptor density in the S N is not affected by drug- induced changes in synapt ic D A concentrat ion (Moore et al . , 1998). Raclopr ide is a l igand used to investigate the D2 dopamine receptor and is one of the most commonly used P E T l igands (Hall et al . , 1988). Rac lopr ide is a D2/D3 receptor antagonist and binds both of these receptor subtypes with equa l affinity (Sokoloff et a l . , 1990;Hal l et a l . , 1994). However, D3 is predominantly exp ressed in the ventral striatum, so raclopride binding in the caudate and putamen represents primarily D2 receptor. Raclopr ide a lso indiscriminately labels both presynapt ic and postsynapt ic D2 receptors, so autoreceptors and postsynapt ic receptors are indist inguishable by P E T (Doudet et al . , 2000). Despi te its high specificity for D2/D3 receptors, raclopride has a relatively low affinity and is therefore extremely suscept ib le to competi t ion with endogenous dopamine (Seeman et al . , 1989;Dewey et a l . , 1993;Vo lkow et a l . , 1994). This characterist ic makes it useful for measur ing not only the density and affinity of the D2 receptor, but a lso acute fluctuations in synapt ic D A concentrat ions. M L C R A s with [ 1 1 C]raclopr ide are useful for determining D2 receptor B m a x and K d under certain condit ions, such as during progression of neurodegenerat ive d i seases . Competi t ion studies with [ 1 1 C]raclopr ide are useful for dynamic measurement of D A neurotransmission. In competi t ion studies, di f ferences in [ 1 1 C]raclopr ide binding before and after a DA-modi fy ing intervention are used to measure changes in synaptic D A concentrat ion (Laruel le, 2000). S u c h competit ion studies can provide insight into neuropsychiatr ic condit ions such as schizophrenia, depress ion , and addict ion. Whi le the ability for raclopride to compete with endogenous D A is obviously advantageous in some c a s e s , it is important to take this property into considerat ion when interpreting 29 P E T B P va lues. C h a n g e s in B P may be caused by changes in the density and/or affinity of the receptor, changes in synapt ic D A levels, or both. S C H - 2 3 3 9 0 is a select ive D1 receptor antagonist, but a lso binds D5 receptors (Billard et al . , 1984;Sunahara et al . , 1991 ;Gagnon et a l . , 1995). S o m e studies suggest that it may bind 5 -HT 2 receptors as well , but this binding is insignificant in striatal t issue where 5-H T 2 receptors are spa rse (Faedda et al . , 1989;Hal l et a l . , 1994). S ince D5 receptor binding is local ized not to striatum, but to h ippocampus, mammil lary bodies and thalamus, S C H - 2 3 3 9 0 binding in the striatum represents primarily D1 receptor binding (Tiberi et al . , 1991 ;Hall et a l . , 1994). The sensitivity of D1 receptor binding to physiologic changes in synapt ic D A concentrat ions is uncertain. Many in vivo studies have suggested that D1 receptor is insensit ive to acute changes in synapt ic DA levels (Thibaut et a l . , 1996;Ab i -Dargham et a l . , 1999;Laruel le, 2000). In contrast, other studies have detected changes in D1 receptor binding under certain condit ions, such as severe D A loss in the striatum, or D A hyperactivity (Marshal l et a l . , 1989; lwata et al . , 1996). Immunohistochemical investigation of D A T - K O mice with chronic hyperdopaminergia revealed low D1 receptor densit ies at the p lasma membrane and high intracellular D1 receptor densi t ies in rough endop lasmic reticulum and Golg i apparatus (Dumartin et a l . , 2000). D1 receptor w a s a lso internalized when st imulated directly with D A agonists or upon increased endogenous D A re lease in response to amphetamines or L - D O P A treatment (Dumartin et a l . , 1998;Muriel et a l . , 1999). The mechan i sms of D1 receptor regulation are unclear. Far fewer studies have been performed with S C H - 2 3 3 9 0 than with raclopride. Both D A receptor l igands have been labeled with tritium for in vitro investigation of D1 and D2 binding as well (Kohier and Radesater , 1986;Hal l et a l . , 1994). Al though both l igands exhibit similar regional distribution in vitro as their [ 1 1 C]- labeled counterparts in vivo, quantitative binding measurements reveal more var iable results, especial ly for raclopride binding (Kohier and Radesater , 1986;Hal l et a l . , 1988;Hal l et al . , 1990;Hal l et al . , 1994;Minuzz i et a l . , 2006). 30 Binding Studies and Parkinson's Disease In vivo P E T and in vitro binding studies in human P D patients and animal models of P D have contributed immensely to our understanding of the d i sease . They have provided insights into the mechan i sms underlying d i sease progression and compl icat ions of therapy (Stoessl and Ruth, 1999). Binding studies have led to discovery of compensatory changes that occur in response to synapt ic D A loss. A R G is fundamental in the search for new P E T tracers and future appl icat ions for P E T include diagnostic purposes and cl inical assessmen t of both d isease progression and effect of treatment interventions. [ 1 8 F ] D O P A was the first P E T tracer used to investigate the human presynaptic D A system in P D (Garnett et al . , 1983). S ince [ 1 8 F ] D O P A uptake was shown to correlate with nigral cell counts in human P D patients and non-human primates treated with M P T P , it has been used to a s s e s s the degree of degenerat ion of the D A system (Snow et al . , 1993;Pate et a l . , 1993). In idiopathic P D patients, [ 1 8 F ] D O P A uptake is reduced in an asymmetr ic fashion similar to post-mortem neurochemica l observat ions, with the putamen more severe ly affected than the caudate (Kish et a l . , 1988;Brooks et al . , 1990). Whi le initial observat ions suggested that the differential degenerat ion of D A neurons between the putamen and caudate was speci f ic for idiopathic P D , studies have s ince detected similar patterns in patients with other forms of park insonism (Brooks et a l . , 1990;Stoess l and Ruth, 1999). Reduct ions in [ 1 8 F ] D O P A uptake have also been detected in patients with familial P D , park insonian syndromes (progressive supranuclear palsy, multiple sys tems atrophy) and acute park insonism following viral encephal i t is (Antonini et a l . , 1997;Stoess l and Ruth, 1999). The high within-subject reproducibility exhibited by [ 1 8 F ] D O P A P E T makes it a useful technique for monitoring effects of P D treatment interventions (Vingerhoets et al . , 1996). For example , fetal nigral transplants have been proposed as a P D treatment to replace D A neurons. [ 1 8 F ] D O P A P E T after transplant shows a susta ined increase in [ 1 8 F ] D O P A uptake in the transplanted striatum relative to the untransplanted striatum, suggest ing success fu l reinnervation of D A neurons (Wenning et a l . , 1997). Immunocytochemical studies support the idea that increased [ 1 8 F ] D O P A uptake is correlated to transplant-derived reinnervation (Kordower et al . , 1995). However, [ 1 8 F ] D O P A uptake does not 31 correlate completely with cl inical severity of P D symptoms or with s ide effects assoc ia ted with transplant, which raises quest ions as to the accuracy of [ 1 8 F ] D O P A uptake rates a lone for evaluating D A system integrity in P D patients (Movement Disorder Society T a s k Fo rce on Rat ing S c a l e s for Park inson 's D i s e a s e , 2003). A more recently proposed method of assess ing the integrity of the D A system is [ 1 1 C ] D T B Z P E T . S ince D T B Z binds V M A T 2 specif ical ly, it targets a single process rather than a combinat ion of p rocesses like F D O P A . The insensitivity of D T B Z binding to pharmacological regulation a lso gives it an advantage over M P and other DAT-spec i f ic radiol igands, lending to its potential to become the "gold s tandard" for estimating D A terminal density (Vander Borght et al . , 1995a;Wi lson and K ish , 1996). It has been wel l -establ ished that D T B Z binding is dec reased in P D (Frey et a l . , 1996;Lee et al . , 2000 ;Au et al . , 2005 ;Bohnen et a l . , 2006). In early P D patients, P E T with [ 1 1 C ] D T B Z shows a significant anteroposterior gradient of D T B Z binding ac ross putamen (greater loss posteriorly) in agreement with previous h istochemical and A R G studies (Lehericy et a l . , 1994;Lee e t a l . , 2004). A P E T study compar ing early P D patients reported reduced [ 1 1 C ] D T B Z B P accompan ied by lesser reduct ions in [ 1 8 F ] D O P A uptake rates and greater reductions in [ 1 1 C ] M P B P (Lee et a l . , 2000). Th is study suggests that D O P A decarboxy lase activity may be upregulated in P D and D A T may be downregulated, both as compensatory mechan isms to account for dec reased synapt ic D A levels. Increased D A turnover and decreased reuptake of D A would both lead to increased synapt ic D A concentrat ions and would counteract the dec reased D A levels caused by degenerat ion of D A neurons. Similar results were obtained in MPTP- t rea ted monkeys using the s a m e radiol igands for P E T (Doudet et al . , 2006). In a 6 - O H D A rat study, [ 3 H]MTBZ binding correlated with D A neuron density in the S N c (Vander Borght et al . , 1995b). T h e s e studies and others suggest that D T B Z is the most accurate radiotracer avai lable to a s s e s s the integrity of the D A system (Kilbourn et a l . , 1996;Bohnen et al . , 2006). There is a great dea l of support for downregulat ion of D A T in P D . Both in vivo P E T studies and in vitro A R G studies have reported larger d e c r e a s e s in radiotracer binding to D A T than in radiotracer binding to V M A T 2 . Normal ized [ 1 1 C ] M P B P values are 32 reduced to a greater degree than normal ized [ 1 1 C ] D T B Z B P va lues in P D striatum (Lee et al . , 2000). Moreover , the ratios of normal ized [ 1 1 C ] M P / [ 1 1 C ] D T B Z are smal ler in the caudal P D striatum, where D A neuron degenerat ion and reduct ions in D A concentration are most severe . A R G studies have found corresponding ev idence of more severe reductions in [ 3 H]WIN 35,428 binding than in [ 3 H]DTBZ binding in P D striatum (Wilson et al . , 1996). For example , [ 3H]WIN 35,428 binding in human post-mortem P D brains demonstrated losses of D A T comparable to losses of D A (Kaufman and Madras , 1992b). Binding studies using radiotracers speci f ic for postsynapt ic D A components have been less helpful in providing insight into compl icat ions of P D therapy. Unl ike presynaptic D A nerve terminals that degenerate with progression of P D , postsynapt ic terminals are relatively spared in both idiopathic P D and in M P T P - i n d u c e d park insonism (Bernheimer et al . , 1973 ;German et a l . , 1996). D A D1 and D2 receptors are the most common postsynapt ic targets for radiol igands, but binding studies targeting these receptors have not elucidated reasons for di f ferences between dyskinet ic and non-dyskinet ic P D patients (Turjanski et a l . , 1997;Kishore et al . , 1998). D1 receptor binding in P D is not well understood in humans or animal models. P E T studies investigating D1 receptor in P D patients have shown unchanged or dec reased binding (Rinne et a l . , 1990;Shinotoh et al . , 1993;Turjanski et a l . , 1997). In these studies, data is normally col lected when the patient is off medicat ion, but the long-term effects of P D treatments and interventions cannot be ignored. Postmor tem studies, on the other hand, show unchanged or increased D1 binding (Ra isman et a l . , 1985;Pimoule et al . , 1985 ;Seeman et a l . , 1987;Pierot et al . , 1988;Hurley et a l . , 2001). Studies in M P T P -treated nonhuman primates and rodent P D models have found similar variability in results, detecting dec reased , unchanged, and increased D1 receptor binding (Graham et al . , 1990 ;Gagnon et al . , 1990;Alexander et a l . , 1991;Gnana l ingham et a l . , 1993 ;A lexandere t a l . , 1993 ;Graham e t a l . , 1993a;Doudet et a l . , 2002b). Studies in non-human primates with severe , short-term lesions or with recent exposure to therapeutic drugs support the postmortem findings, with unchanged or slightly increased D1 binding (Graham et a l . , 1990;Gnana l ingham et al . , 1993 ;Graham et a l . , 1993b ;Gagnon et a l . , 1995;Rioux et a l . , 1997). P E T studies in non-human pr imates with more mild or long-33 term lesions and free of ant iparkinsonian treatments, however, have found mildly dec reased striatal D1 binding (Doudet et al . , 2002b). Roden ts les ioned with 6 - O H D A support the latter f inding of dec reased D1 binding (Marshal l et a l . , 1989;Joyce, 1993). Evidently, the fate of D1 receptors in P D is unclear. It is possib le that D1 receptor regulation is more compl icated than a linear model . D1 regulation may vary depending on the stage of d i sease progression or the severity of the lesion. A study by Iwata al. (1996) using 6 -OHDA- l es i oned rats supports this explanat ion with the finding that D1 binding is increased in severe ly lesioned rats with less than 5 % of normal D A levels, but dec reased in rats with 5 -25% of control D A levels. Another explanat ion for var iable results is short-term compensat ion . In the studies performed in an imals with short-term lesions, observed elevat ions in D1 binding may be the result of D1 upregulat ion. However, the system may have a maximum threshold above which it cannot compensa te for further degenerat ion or for a prolonged period. D2 receptor changes assoc ia ted with P D have been studied more extensively, but are still somewhat unclear. Ear ly P D is assoc ia ted with normal or increased D2 receptor as measured using [ 1 1 C]raclopr ide P E T in human P D patients (Rinne et a l . , 1990;Brooks et al . , 1992;Antonini et a l . , 1995a). Advanced P D patients, however, exhibit normal or dec reased D2 receptor number compared to normal controls (Rinne et a l . , 1981;Guttman et a l . , 1986;Pierot et al . , 1988;Antonini et a l . , 1995a). Loss of D2 supersensit ivi ty in advanced P D patients has been partly attributed to antiparkinsonian medicat ions such as L-dopa and D A agonists, but this hypothesis does not fully explain other changes accompany ing late-stage P D (Antonini et a l . , 1994;Tedroff et al. , 1996). Simi lar increases in D2 binding have been observed postmortem in humans and in animal models of P D including MPTP- t rea ted monkeys and 6-OHDA-t reated rodents (Guerin et a l . , 1985;Joyce et al . , 1986;Perlmutter et a l . , 1987;Falardeau et a l . , 1988 ;Graham et a l . , 1990). The use of animal mode ls avo ids confounding effects of ant iparkinsonian medicat ions, which alter the D A sys tem and radiotracer binding in clinical P E T studies. [ 1 1 C]raclopr ide P E T and [ 3H]raclopride A R G studies in rhesus monkeys with stable M P T P lesions suggest that D2 upregulat ion is susta ined long-term in the absence of ant iparkinsonian treatment (Doudet et a l . , 2000). [ 1 1 C]raclopr ide B P measurements were correlated with in vitro [ 3H]raclopride A R G binding measurements , 34 but the sample s ize was smal l . D2 upregulation a lso appears to be restricted to animals displaying P D symptoms, whereas asymptomat ic an imals exhibited a smal l but significant dec rease in D2 binding (Falardeau et a l . , 1988;Doudet et al . , 2000). Th is decrease may have resulted from loss of D2 autoreceptors in mildly- lesioned animals that was not severe enough to trigger compensatory upregulat ion of postsynaptic D2 receptors. In summary , the literature suggests that D2 receptor binding is increased in untreated P D . Clinical Assessment of Parkinson's Disease Since no objective imaging methods have been val idated for P D , the d isease is evaluated clinically by physic ians by use of physical examinat ions that a s s e s s several aspects of motor, cognit ive, and emotional qualtities. Multiple cl inical rating sca les are in use for assessmen t of P D severity, but the Unified Park inson 's D i sease Rating Sca le ( U P D R S ) is the most c o m m o n (Movement Disorder Soc ie ty T a s k Force on Rating S c a l e s for Park inson 's D isease , 2003;Bur ini et al . , 2006). It provides a comprehens ive evaluation of P D symptoms, especia l ly motor symptoms, and demonstrates high reliability and validity, but a lso p o s s e s s e s w e a k n e s s e s in a s s e s s m e n t of non-motor aspects of P D , ambiguit ies in written text, and inadequate instructions for raters ((Martinez-Martin et a l . , 1994;2003c). Pat ients must be off medicat ion for at least twelve hours prior to assessmen t , which presents a difficulty for cl inical evaluat ion of advanced P D patients (Shinotoh et a l . , 2000). A n objective measure of d i sease severity that avoids the need to withdraw patients from medical treatment in order to a s s e s s d isease progression and evaluate possib le treatments would be benef ic ia l . G iven that cl inical rating sca les are the best tools avai lable for assess ing P D , patient scores on these e x a m s are used as primary outcome measu res to evaluate response to treatment, ef fect iveness of new potential treatments, eff iciency of potential diagnost ic techniques, etc. (Kelly and Gi l l ingham, 1980;Lozano et a l . , 1995;Benabid et a l . , 1996;2003b). S i nce clinical rating sca les are used for such diverse purposes, it is worthwhile to investigate the relationship between cl inical scores and actual physiological p rocesses . 35 A n A n i m a l M o d e l of P a r k i n s o n ' s D i s e a s e Severa l animal mode ls of P D have been deve loped in multiple spec ies by numerous mechan isms, including administration of neurotoxins, deplet ion of neurotransmitters, and physical destruction of certain brain regions. A c o m m o n model of P D is the 6-hydroxydopamine (6 -OHDA) model . Intracerebral injection of 6 - O H D A (often unilateral) causes degenerat ion of catecholaminergic neurons in severa l spec ies , but is most often used in rats to minimize costs (Tolwani et a l . , 1999). A l though this model has contributed to ascertaining the eff icacy and mechan ism of action of select P D treatments, it has limitations (Dunnett et al . , 1981;Schwart ing and Huston, 1996). 6-O H D A administrat ion induces an acute lesion that affects only the neurons that come into contact with the toxin and does not mimic the slow, progressive degenerat ion of D A neurons that is characterist ic of P D . Furthermore, neuroanatomy in rats is distinct from that of pr imates and behavioura l 'e f fects are a lso difficult to translate to the human condit ion. Non-human primates resemble humans more closely than any other animal , especial ly with respect to behaviour, and thus provide invaluable animal mode ls for investigating human condit ions. P D is one condit ion that has benefited immense ly from an excel lent non-human primate model . In the late 1970's and early 1980's , severa l young adults in Cal i fornia deve loped acute park insonism following the use of synthetic heroin. The contaminant that c a u s e d the outbreak was identified as 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine ( M P T P ) (Langston et al . , 1983;Bal lard et a l . , 1985). M P T P causes a persistent park insonian syndrome that resembles idiopathic P D in several animal spec ies including mice, cats, dogs, sheep , pigs, and non-human primates (Langston et al . , 1983;Burns et a l . , 1983;Langston et al . , 1984;Bal lard et a l . , 1985;Tolwani et a l . , 1999). This neurotoxin induces clinical symptoms as well as pharmacologic, neuropathologic and neurochemica l changes similar to those observed in idiopathic P D (Burns et al . , 1983;Schne ider et al . , 1987;Doudet et a l . , 1993;Tolwani et al . , 1999). S ince non-human pr imates exposed to M P T P develop similar cl inical symptoms as P D patients, cl inical rating sca les such as the U P D R S have been adapted to a s s e s s lesion severity in non-human primates for use in studies pursuing comprehens ion of d i sease progression and development of potential treatments (Langston et al . , 2000;Bezard et a l . ,2001) . 36 Debate as to the mechan ism of action of M P T P is ongoing, but it ultimately leads to striatal D A deplet ion and degenerat ion of D A neurons in the S N c (Burns et al . , 1983;German et a l . , 1988;Doudet et al . , 1993). M P T P is uncharged and thus c rosses the blood-brain barrier. In the brain, it is metabol ized by the enzyme M A O - B to 1-methyl-4-phenylpridinium ion (MPP+), which is taken up into D A neurons by D A T (Javitch and Snyder , 1984;Gainetd inov et al . , 1997;Tolwani et al . , 1999). This select ive uptake by D A T leads to accumulat ion of the toxin and the observed specificity for D A neurons. S o m e studies suggest that M P P + acts on mitochondria, thereby inhibiting A T P production and stimulating free radicals, which trigger downst ream effectors of apoptosis and inf lammation (Przedborski and V i la , 2003). Susceptibi l i ty to M P T P differs between and within spec ies and even among individuals. For example , rats are almost resistant to the toxin, whereas mice are somewhat sensit ive. Different strains of mice are more suscept ib le than others and the C 5 7 B L / 6 mouse exhibits the highest sensitivity (German et a l . , 1996;Tolwani et a l . , 1999). The rhesus monkey (Macaca mulatta) is an Old Wor ld non-human primate and is more sensit ive to M P T P than the N e w Wor ld squirrel monkey (Saimiri sciureus) (Tolwani et al . , 1999). S o m e individual subjects in this study required significantly higher doses of M P T P than others to produce similar clinical effects. Two M P T P models have dominated P D research in non-human primates; the bilateral parkinsonian model and the hemi-parkinsonian model . Sys temic M P T P injections cause bilateral les ions of the nigrostriatal sys tem, while M P T P injections into one internal carotid artery are intended to produce unilateral lesions of the injected hemisphere only. In addition to the route of administration, the number of d o s e s , amount per dose, and timing of doses can be varied to create lesions of varying severity. The validity of the M P T P non-human primate model has been supported by several studies demonstrat ing commonal i t ies with P D . M P T P administrat ion produces clinical symptoms of bradykinesia, rigidity, freezing (inability to initiate movement) , ba lance impairment, and tremor (postural and/or resting) (Doudet et a l . , 1985;Schneider et a l . , 1987). It c a u s e s similar chemica l and pathological changes as those observed in 37 idiopathic P D , such as dec reased [ 1 8 F ] D O P A uptake and cerebral blood flow in the striatum, select destruction of nigrostriatal D A neurons, and dec reased striatal D A levels (Burns et al . , 1983;Stern, 1990;lrwin et al . , 1990;Doudet et a l . , 1993;Tolwani et al . , 1999). The symptoms induced by M P T P administration can be reversed by the most effective drug used to treat P D patients, L - D O P A , and susta ined use of this drug produces the s a m e dysk ines ias observed in P D patients after prolonged L - D O P A treatment (Peppe et a l . , 1993;Tolwani et a l . , 1999). In rhesus monkeys who receive systemic M P T P injections, D2 receptor densit ies in the posterior striatum are increased, while those in the anterior striatum are not affected (Bedard et al . , 1986;Kish et a l . , 1988 ;Gagnon et a l . , 1995). This phenomenon is similar to that observed in the putamen of idiopathic P D patients, where D A loss follows a rostrocaudal gradient and the caudal region shows greatest lost (Kish et al . , 1988). Al though the M P T P non-human primate model repl icates many cl inical, chemica l , and pathological effects observed in idiopathic P D , it does not completely replicate the latter situation. In idiopathic P D , the putamen is more severe ly affected than the caudate (Kish et a l . , 1988). Sys temic M P T P treatment successfu l ly mimics this effect, but unilateral M P T P injection appears to produce equal changes to caudate and putamen, as measured by striatal D A levels, [ 1 8 F ] D O P A uptake, and cerebral blood flow (Gibb and Lees , 1991;Doudet et a l . , 1993 ;Gagnon et al . , 1995). Differential sensitivity of nigral neurons to M P T P neurotoxicity is a likely cause for this di f ference (Doudet et al . , 1993). The putamen receives projections from the more ventral and posterior nigral neurons, which are more sensi t ive to M P T P than the caudate-project ing neurons located more dorsally in the S N c (Schneider et al . , 1987 ;German et a l . , 1988). Whi le the caudate-projecting neurons are resistant to the smal l M P T P d o s e s assoc ia ted with systemic administration, intra-arterial infusion during unilateral lesioning brings them into contact with high toxin concentrat ions, leading to equal effects in both putamen and caudate (Doudet e t a l . , 1993). Another distinction is that acute M P T P administration general ly does not appear to produce the progressive neuronal loss characterist ic of idiopathic P D , but some studies have indicated that repeated low doses of M P T P can replicate idiopathic P D progression (Russ et a l . , 1991;Bezard et a l . , 1997). M P T P a lso fails to affect D A 38 terminals in the nuc leus a c c u m b e n s and olfactory tubercle, which originate in the ventral midbrain (Burns et al . , 1983), but does affect serotonin and norepinephrine neurons that are not affected in P D patients (Burns et al . , 1983;Doudet et a l . , 2006). Unilaterally MPTP- t rea ted animals are often used to represent both a normal situation (untreated side) and a parkinsonian situation (MPTP- in fused side). However, the assumpt ion that the untreated s ide is not affected by M P T P has been cal led into quest ion. P E T and A R G exper iments in rhesus monkeys have shown that the untreated s ide of uni lateral ly- lesioned animals is significantly more affected by M P T P than controls, as measured by [ 1 8 F ] D O P A uptake and D2 receptor binding (Doudet et al . , 1993). M P T P may reach the contralateral striatum if it is not completely taken up during its first pass and not fully metabol ized by the liver before being recirculated. C r o s s perfusion between the left and right arterial sys tems may a lso occur (Doudet et al . , 1993). Th is point is not of major concern in this study, b e c a u s e behavioural scores, P E T data, and A R G data were calculated for each individual hemisphere and were not compared between normal and treated individuals. T h e a im of this study was to determine the correlation between these parameters within subjects, not between subjects, so the method of lesioning was not important. H y p o t h e s e s revisi ted Few studies have attempted to compare in vivo P E T B P va lues with in vitro A R G binding va lues in the s a m e individuals (Gatley et al . , 1998;St rome et a l . , 2005;Strome et al . , 2006). Wh i le severa l studies involving solely in vivo P E T or in vitro A R G commonly relate their f indings to actual physiological p rocesses and to both in vivo and in vitro studies performed individually at other sites, the validity of these associat ions has not been investigated thoroughly in the s a m e individuals and has never been investigated in non-human pr imates in the s a m e study. Therefore, it is not known how well in vivo and in vitro measurements using P E T tracers correlate. B a s e d on previous f indings d iscussed above, P E T B P va lues and A R G binding va lues measured with the s a m e radiotracers, in the s a m e individuals, are expected to correlate significantly in the present study. 39 Subjects in this study received different doses of M P T P and developed clinical symptoms to varying degrees while control an imals did not receive any M P T P treatment. Therefore, this study group covers a large range of lesion severit ies as measured using a cl inical rating sca le for parkinsonian symptoms. Th is sca le is based on clinical rating sca les used to evaluate d i sease severity in humans and has been adapted for non-human primate P D models, so study results are transferable to the human condit ion. Higher behavioural scores indicate greater severity of symptoms. Furthermore, the subjects in this study did not receive any P D treatments and thus represent a va luable model for investigating physiological changes that naturally accompany the progression of P D . A s previously found in human P D patients and animal mode ls of P D , P E T B P values and A R G binding va lues obtained using presynapt ic t racers are expected to correlate significantly with cl inical severity. Therefore, binding of [ 1 1 C ] D T B Z , [ 1 1 C ] M P , and [ 3H]WIN 35,428 by P E T and by A R G should correlate with behavioural scores. Whi le presynapt ic molecu les in degenerat ing nigrostriatal terminals are good targets for evaluating lesion severity in P D , postsynapt ic molecu les in the relatively spared postsynapt ic neurons may not be. Previous studies have reported inconsistent f indings with regards to the relationship between D1 and D2 binding both in vivo and in vitro and clinical severity of P D symptoms. Due to variation in reported effects of P D progression on D1 binding, P E T B P and A R G binding using radiolabeled S C H - 2 3 3 9 0 are not expected to correlate significantly with behavioural scores . A l though conflicting results have been reported as to the fate of D2 receptors in P D , there is s o m e agreement of D2 upregulation, notably in subjects similar to those investigated in this study. Therefore, P E T and A R G measurements using radiolabeled raclopride are expected to correlate weakly, if at al l , with cl inical assessmen ts . S i g n i f i c a n c e The present study will investigate the correlation between in vivo and in vitro methods of evaluating changes in components of the neural D A sys tem. P E T and A R G are often used individually to explore d i sease p rocesses and mechan i sms of treatment effects. P E T provides a noninvasive method of imaging neural function in living beings and allowing longitudinal analys is in humans, indicating enormous clinical potential. 40 However, the p rocess of interest cannot be isolated from the many other interacting neural sys tems in vivo and P E T data may not reflect solely the desired event. In vitro studies using A R G are often used to isolate single p rocesses and determine actual physiological effects on individual neural components . Understanding the relationship between P E T B P and A R G binding can contribute to accurate interpretation of P E T data. U s e of identical radioactive tracers in vivo and in vitro to obtain quantitative measures of B P and binding will minimize sources of variation that were present in previous studies and provide a clearer picture of the true relationship between P E T and A R G techniques. Elucidating the relat ionships between lesion severity and both P E T and A R G binding techniques also contributes to future clinical appl icat ion. Currently, the most common method of evaluat ing the stage of P D progression in the cl inic is the U P D R S , a rating sca le that a s s e s s e s severity of clinical symptoms. P E T is currently under investigation for use in improving early P D diagnosis and differential d iagnos is of park insonism, as well as identifying biomarkers that may predict patient response to speci f ic treatments. Knowledge of the correlation between behaviour and binding of different radioactive P E T tracers will help identify tracers with the highest potential for indicating d i sease severity and relate cl inical behavioural measures of d i sease severity to actual physiological p rocesses . It has been suggested that differential d iagnosis of P D from atypical park insonism by P E T may require examinat ion of multiple D A system components (Antonini et a l . , 1995a). The use of both presynapt ic and postsynaptic tracers in the s a m e individuals in this study may contribute to this purpose by enhancing understanding of relative changes in these components with d i sease progression. 41 Chapte r 2 M e t h o d s Sub jec ts Thirteen adult rhesus monkeys (Macaca mulatta) of both s e x e s (2 females, 11 males) ranging in age from five to twenty-five years were the subjects of this study. Three animals were normal controls. Four animals had bilateral nigrostriatal lesions induced by intravenous (IV) injection of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine ( M P T P ) hydrochloride (cumulative dose : 2-2.5 mg/kg). The remaining six an imals had unilateral nigrostriatal les ions induced by unilateral intracarotid injection of M P T P (cumulative dose: 0.3-3.2 mg/kg). Data from one hemisphere of each of the control animals and bi lateral ly-lesioned animals and both hemispheres from e a c h of the unilaterally-lesioned animals was used in this study, giving a sample s ize of nineteen (N=19). The animals lived in stable socia l groups and were normally housed two per cage, with individual housing on rare occas ions . S e e table 2.1 for detai ls of death and age at t ime of death. fable 2.1. Summary of subjects invesl tigated in t his study. Animal Hemisphere Status Date of Death Age at Death Cause of Death Bony L Normal 27Jun02 18 Sacrificed Elf L Normal 12Nov02 19 Sacrificed Pittstop L Normal 11Mar01 16 Sacrificed (Paralyzed waist down) Enthalpy R MPTP 13Feb04 22 Sacrificed Ezechial R MPTP 04May00 20 Sacrificed (Type 2 Diabetes) Murray R MPTP 11Dec03 22 Sacrificed Rose(F) L MPTP 13Mar01 5 During surgery Karma (F) L,R Hemi-MPTP 16May01 9 Sacrificed Ken L,R Hemi-MPTP 07Nov03 22 Sacrificed Martin L,R Hemi-MPTP 15Jul02 17 Found dead Mike L,R Hemi-MPTP 07Nov03 22 Sacrificed Robert L,R Hemi-MPTP 28Mar00 17 Found dead Scott L,R Hemi-MPTP 24July06 25 Sacrificed An ima ls lived to different ages (mean 18.0 ± 5.6 years), but ten of the thirteen subjects were sacri f iced between the ages of 16 and 22 years. The other subjects lived to age 5, 9, and 25 years . O n e animal died during surgery and two were found dead in their cages , but the rest were sacr i f iced by overdose of pentobarbital . Brains were removed immediately and blocks of striatum were surgically removed and p laced in a f reezer at 42 -80°C where they were stored until sect ioning. M P T P L e s i o n s Apart from the 3 normal control animals, subjects were treated with M P T P to selectively destroy nigral dopaminerg ic neurons and induce a park insonian syndrome (Langston et al . , 1983;Bal lard et a l . , 1985). At least 4 years prior to P E T scann ing , subjects received injections of M P T P hydrochloride (Sigma) d issolved in sterile sal ine at 2 mg/ml. Hemi -lesioned an imals received infusions in the right internal carotid artery once or twice. Systemical ly (i.e. bilaterally) M P T P - l e s i o n e d monkeys received multiple intravenous injections at 0.2-0.5 mg/kg administered over a period of severa l months. Al l lesions were chronic, but total dose of M P T P varied largely among individuals due to wel l -known variability in susceptibi l i ty to M P T P . Even within the primate spec ies , age, sex, route of administrat ion, dose , and other factors affect the outcome of M P T P administration (Doudet et a l . , 1993). Al l lesions were a s s e s s e d using a behavioural test to confirm a stable and persistent syndrome of park insonism. Symptoms were character ized by severe hypokinesia, bradykinesia with pronounced freezing ep isodes , loss of ba lance, hypomimia, and intention tremor (Doudet et a l . , 1989). Behav ioura l A s s e s s m e n t Subjects were clinically a s s e s s e d for P D severity using a modif ied rating sca le based on the cl inical rating sca les used to a s s e s s P D severity in humans . Tab le 2.2 shows the sca le that was used in this study to obtain behavioural sco res . The behavioural score for each animal was obtained within a few months prior to death. Sco res were determined by summing the subscores for severity of eight P D symptoms. Rigidity, bradykinesia, and tremor were scored separate ly for left and right s ides of hemi- les ioned animals , with behaviour on each s ide of the body contributing to the behavioural score for the contralateral hemisphere. Higher scores signify more severe clinical symptoms, with a max imum score of 26. Behavioura l scores of the subjects in the present study ranged from 1 to 18, with mean 6.2 ± 4 .3 . 43 Table 2.2. Behavioura l assessmen t sca le used to obtain score for degree of severity of parkinsonian syndrome. M o n k e y C l i n i c a l A s s e s s m e n t S c a l e Hemi-MPTP 1. Home Cage Activity 0 = Normal 2 = Slightly reduced 4 = Markedly reduced 6 = Severe Hypokinesia/Akinesia 2. Rigidity 0 = Absent 1 = Present L v.s. R 3. Bradykinesia 0 = None 1 = Mild 2 = Moderate 3 = Severe L v.s. R 4. Facial Expression 0 = Normal 1 = Slight Hypomimia 2 = Moderate Hypomimia 3 = Severe Hypomimia (mask) 5. Freezing 0 = None 1 = Some episodes 2 = Frequent episodes 6. Posture 0 = Normal, erect posture 1 = Some stooping 2 = Severe or persistent stooping 7. Gait/ Balance/ Coordination 0 = Normal 2 = Walks slowly with slight or no loss of balance 4 = Markedly impaired, minor lapses in balance 6 = Severe decrease in mobility with major lapses in balance 8. Tremor (usually intention) 0 = None 1 = Mild- intermittent or slight 2 = Moderate - noticeably present 3 = Severe - persistent L v.s. R P E T Exper imenta l P r o c e d u r e E a c h animal was housed in a squeeze -cage for 1 day and fasted for 18 hours prior to scanning. On the morning of the experiment, the animal w a s immobil ized with an intramuscular (IM) injection of ketamine HCI (10 mg/kg), fo l lowed by injection of atropine sulfate (0.05 mg/kg IM) to dec rease secret ions during scann ing . Ketamine has been shown by P E T to alter multiple aspects of D A t ransmiss ion (Tsukada et a l . , 2000 ;Momosak i et a l . , 2004b) .To minimize the effects of ketamine in this P E T study, scanning was c o m m e n c e d at least two hours after initiating anes thes ia . The legs were shaven for insertion of an intravenous (IV) catheter into a saphenous vein and an anesthet ic dose of sod ium pentobarbital was administered (7-10 mg/kg IV). Fol lowing intubation with an endotracheal tube, the animal was transported from the animal care 44 facility to the nearby Department of Nuclear Medic ine where the P E T scans were performed. The animal was p laced in a stereotaxic f rame in a prone posit ion, allowing acquisit ion of coronal P E T images (Fig. 2.1). This frame was special ly des igned for rhesus monkeys to prevent movement during and between P E T s c a n s and to ensure that each animal was scanned in the s a m e plane during consecut ive scans . The monkey was maintained under light anes thes ia with isoflurane gas (1-1.5%) for the remainder of the study. Body temperature was maintained at 37°C with a water heating blanket and a sal ine IV drip was administered at a low flow rate. Heart rate and respiration were monitored throughout the experiment. S c a n s were performed in two d imensional mode using an E C A T 953-31B tomograph (CTI /S iemens, Knoxvi l le, T N , U S A ) which has an in-plane resolution of 6 mm full width at half max imum and an axial resolution of 5 mm, leading ultimately to a resolution of approximately 9 mm in the corrected subject data. The E C A T al lows s imul taneous acquisit ion of 31 s l ices through the head and brain of the monkey, separated by 3.5 mm center to center. Pr ior to any radiotracer injection, a brief t ransmission scan was performed with a rotating rod source for attenuation correct ion of the experimental scans . The animal w a s then p laced inside the P E T scanner and posit ioned using the scanner 's laser beam sys tem to align all monkeys to the s a m e plane. Radiotracers were synthes ized at the Tri-University M e s o n Facil ity (TRIUMF) near the University of British Co lumb ia where the P E T and A R G exper iments were performed. Tracers were synthes ized according to methods previously descr ibed (Ding et a l . , 1994;Adam et a l . , 1997;Jewett et al . , 1997)). The scann ing sequence consisted of six 30-second scans , two 1-minute scans , five 5-minute scans , two 7.5-minute scans , and one 15-minute scan for a total duration of 60 minutes. Th is 2 D acquisit ion was fol lowed by a 15-minute, high-resolution 3D scan to facilitate structure identification. 45 Fig. 2.1. Stereotaxic f rame used to align all monkeys in P E T scanner in s a m e plane. 46 All scans included in this study were basel ine scans , mean ing they were performed prior to the animal receiving any pharmacological intervention. Most scans were performed within the last year of life for each animal . P E T Data A n a l y s i s For each scan in each monkey, regions of interest (ROIs) were p laced over left and right total striatum (circular ROIs : 37 pixels; pixel s ize: 4 m m 2 ) in four consecut ive s l ices. Al though ROIs were a lso p laced regionally over the caudate and putamen, the B P va lues used in the study were obtained using total striatum ROIs . Figure 2.2 shows an example of ROI p lacement on P E T images. Four ROIs (16 pixels each) were placed over an area of non-speci f ic radiotracer accumulat ion in the cerebel lum in two consecut ive s l ices. Posi t ioning of the ROIs was verified on the 3D scan before each ROI was t ransposed onto each time frame of the 2 D kinetic s c a n . Time-activity curves were obtained for e a c h ROI and averaged for each animal onto left and right striatum and cerebel lum. Data analys is was performed using the Logan graphical reference t issue method of P E T data analys is (Logan et al . , 1996). The bolus and constant infusion tracer administration technique creates a true equil ibrium condit ion, where measurements are independent of variation in cerebral blood flow in both speci f ic and non-speci f ic a reas (Doudet et a l . , 2002a). This condit ion meets the assumpt ions of the Logan t issue-input graphical analysis, which permits measurement of the distribution vo lume ratio (DVR) . The bolus plus constant infusion technique was used for [ 1 1 C]raclopr ide and [ 1 1 C ] S C H - 2 3 3 9 0 exper iments, while the more common bolus only technique was used for [ 1 1 C ] D T B Z and [ 1 1 C ] M P exper iments. S ince D V R is equivalent to binding potential (BP) + 1, this analys is produces a measure of B P , a reflection of both the density ( B m a x ) and apparent affinity (K d ) of the receptors (BP= B m a x / /K d ) . Binding potentials were calculated from the measured D V R (BP=DVR-1) for each tracer in e a c h subject. 47 PET: 1 1C-RAC - Regions of interest Fig. 2.2. Posi t ioning of ROIs on P E T images in four sequent ia l s l ices through the striatum. The ROIs encompass ing total striatum, as shown here in left hemisphere in each image, were used to obtain the binding potential va lues for each subject. Images obtained from an exper iment using [ 1 1 C]raclopr ide (courtesy of Doris Doudet). A R G Exper imenta l P r o c e d u r e In vitro A R G was performed on striatal t issue that was removed and frozen immediately after death. F rozen striatal b locks were partially thawed from -80 °C to -16 °C and sect ioned into s ixteen-micron s l ices using a S h a n d o n Cryotome Cryostat (GMI). Sect ions were thaw-mounted onto g lass sl ides (Superfrost P lus , F ischer Scientif ic), then stored at -80°C until autoradiography was performed. Al l in vitro A R G exper iments fol lowed the s a m e general procedure. Experimental protocols were based on the procedures used by van K a m p e n and Stoess l (2003), with slight modif icat ions. 48 To begin an autoradiography experiment, s l ides were removed from the -80°C freezer and warmed up to room temperature to al low them to dry. S l ides were then loaded into sl ide d ishes and pre- incubated in buffer to remove endogenous l igand. T issue was incubated in buffer with the addition of radiotracer to measure total binding. Nonspeci f ic binding in adjacent s l ides was simultaneously measured by adding a non-radioactive ("cold") drug at a high concentrat ion to block all l igand-speci f ic binding sites. After a speci f ied incubation time that varied by tracer, t issue was r insed twice to remove all unbound l igand. After a brief dip in disti l led, deionized water at 4°C, s l ides were al lowed to dry for a period dependent on the radioisotope used . [ 1 1 C] standards were made as previously descr ibed (Strome et a l . , 2005). For each exper imental run, 5ul_ drops of a set of eight serial dilutions of the tracer were pipetted onto strips of T L C plate ( P E SIL G , Whatman) . O n e strip w a s included with each sc reen s o that the activity in the sect ions apposed to the s a m e screen could be determined from the phosphor image produced by scann ing the sc reen . W h e n dry, [ 1 1 C]- incubated s l ides and activity s tandards were apposed directly to multi-sensit ive phosphor sc reens (PerkinElmer), which were scanned two hours later. [ 3H]-incubated s l ides were al lowed to dry fully on the bench for at least 4 hours before they were post-f ixed with paraformaldehyde (Sigma) vapour in a vacuum in order to prevent contamination of the trit ium-sensitive phosphor sc reens (Kashihara et a l . , 1990;Liberatore et a l . , 1999). After twenty-four hours in the desiccator, [ 3H]-incubated sl ides were apposed to trit ium-sensitive sc reens (Fuji) adjacent to [ 3 H]microscales™ (Amersham) for 5 days to quantify t issue activity. Al l sc reens were apposed to a light box for at least 10 minutes prior to p lacement against s l ides in order to remove any latent images. After the speci f ied time, sc reens were removed from casset tes in the dark and scanned immediately in a Cyclone® phosphor imager (Packard) at 600 dpi resolution. Spec i f ic exper imental protocols for each tracer are descr ibed below. Al l [ 1 1 C]- labeled tracers used in this study were synthes ized at T R I U M F and were prepared by the s a m e method used for P E T tracers (descr ibed above). [ 1 1 C](+)DTBZ was prepared almost identically to [ 1 1 C](±)DTBZ, except that the D T B Z precursor was (+)DTBZ instead of (±)DTBZ. [ 1 1 C ] M P , [ 1 1 C]raclopr ide, and [ 1 1 C ] S C H - 2 3 3 9 0 were prepared in exactly the s a m e way as the tracers used for the in vivo P E T experiments. 49 All [ 3H]-labeled tracers were purchased from Perk inElmer , Inc. and unlabeled drugs were purchased from S i g m a . VMAT2 b ind ing with [ 1 1 C](+)dihydrotetrabenazine (DTBZ) For V M A T 2 binding, sect ions were pre-incubated for 5 minutes at 20°C in a sucrose buffer containing 3 0 0 m M sucrose , 5 0 m M Tr is-HCI, and 1 m M E D T A (Van der Borght et al . , 1995). The pre-wash buffer pH was adjusted to 8.0 by dropwise addition of HCI or N a O H . Sect ions were then incubated in the s a m e buffer with the addition of 5 n M [ 1 1 C](+)DTBZ (SA= 615-991 Ci /mmol) at 20°C for 30 minutes. Nonspeci f ic binding was simultaneously performed on adjacent sect ions with the addition of 5 uM (±)tetrabenazine. Incubation was fol lowed by two 3-minute post -washes in the s a m e buffer (also adjusted to pH 8.0) at 4°C. After a brief dip in disti l led, deionized water at 4°C to remove e x c e s s buffer, s l ides were laid out to dry for 20 minutes in a fumehood. S l ides were then p laced against multisensitive storage phosphor sc reens along with a set of s tandards of known activity in standard film casset tes . Casse t tes were stored behind lead for the 2-hour exposure, before the sc reens were removed from the casset tes and scanned in the phosphor imager. D A T b ind ing with [ 1 1 C]cf-tf?reo-methylphenidate (MP) This exper imental protocol was adapted from [ 3 H]MP studies (Unis et al . , 1985;Schwer i , 1990). For D A T binding, sect ions were pre- incubated for 5 minutes at 4°C in buffer containing 120mM NaCI , 5 0 m M Tr is-HCI, and 5 m M KCI. The pre-incubation buffer was adjusted to pH 7.9 with dropwise addition of HCI or N a O H . Sect ions were then incubated in a different buffer containing 3 0 0 m M NaCI 5 0 m M Tr is-HCI, and 5 m M KCI, plus 15nM [ 1 1 C ] M P (SA= 694-913 Ci /mmol) at 4°C for 40 minutes. Staining d ishes containing the buffer and s l ides were surrounded in ice for the duration of the pre-wash and incubation to keep the buffer at 4°C. Nonspeci f ic binding was simultaneously performed on adjacent sect ions with the addition of 10 u M nomifensine. Incubation was fol lowed by two 1-minute post -washes at 4°C in the s a m e buffer as was used for the incubation. After a brief dip in disti l led, deionized water at 4°C to remove excess buffer, s l ides were laid out to dry for 20 minutes in a fumehood. S l ides were then placed against mult isensit ive storage phosphor sc reens along with a set of s tandards of known activity in standard film casset tes. Casse t tes were stored behind lead for the 2-hour 50 exposure, before the sc reens were removed from the casset tes and scanned in the phosphor imager. D A T b ind ing with [ 3H]WIN 35,428 D A T binding was a lso evaluated with a tritiated tracer. S l ides were removed from -80 °C freezer and laid on the bench to dry. Sect ions were circled with nai lpol ish, which was al lowed to dry before they were pre-incubated in buffer containing 120mM NaCI, 5 0 m M Tris-HCI, and 5 m M KCI for 5 minutes at 4°C and pH adjusted to 7.9 by addition of HCI or N a O H . S l ides were then laid out to dry in incubation trays lined with ice and wet paper towels to keep t issue cold and moist (about 30 minutes). Buffer containing 3 0 0 m M NaCI, 5 0 m M Tr is-HCI, 5 m M KCI, and 15nM [ 3 H]WIN 35,428 (SA= 70.1 Ci/mmol) was pipetted onto sect ions and incubation lasted 40 minutes. The increased salt concentrat ion and lower temperature were required for optimal W IN binding. Nonspeci f ic binding was determined in adjacent sect ions by addition of 10 uM nomifensine to the incubation solution before it was pipetted onto those sect ions. Incubation was fol lowed by two 1-minute post -washes in the incubation buffer at 4°C. After a brief dip in disti l led, deionized water at 4°C, s l ides were laid on the bench to dry for at least 4 hours. S l ides were then placed in a des iccator for at least 24 hours to post-fix t issue. Finally, s l ides were p laced against trit ium-sensitive phosphor sc reens along with a set of s tandards of known activity in standard film casset tes . Sc reens were removed from casset tes after five days and immediately scanned in the phosphor imager. D1 receptor b ind ing with [ 3 H ] S C H 23390 For D1 receptor binding, s l ides were removed from the -80 °C freezer and laid on the bench to dry. Sec t ions were circled with nai lpol ish, which w a s al lowed to dry before sect ions were pre- incubated in buffer containing 120mM NaCI , 5 0 m M Tr is-HCI, 5 m M KCI, 2 m M C a C I 2 > and 1 m M M g C I 2 for 15 minutes at 20°C and pH adjusted to 7.4 by addition of HCI or N a O H , just as in the raclopride binding exper iments. S l ides were then laid out to dry in incubation trays lined with wet paper towels to keep t issue moist (about 30 minutes). Buffer containing 30nM ritanserin to block serotonin receptors and 2 n M [ 3 H]SCH 23390 (SA= 66.3 Ci /mmol) was pipetted onto sect ions and incubation lasted 45 minutes. Nonspec i f ic binding was determined in adjacent sect ions by addition of 10 51 uM (+)-butaclamol to the above solution before it was pipetted onto those sect ions. Incubation was fol lowed by two 3-minute post -washes in the s a m e buffer at 4°C. After a brief dip in disti l led, de ion ized water at 4°C, s l ides were laid on the bench to dry for at least 4 hours. S l ides were then p laced in a des iccator for at least 24 hours to post-fix t issue. Finally, s l ides were apposed to trit ium-sensitive phosphor sc reens along with a set of s tandards of known activity in standard film casset tes . Sc reens were placed against a light-box for at least 10 minutes prior to loading of casset tes to erase any latent images. After five days, sc reens were removed from casset tes and scanned in the phosphor imager. D2 receptor binding with [11C]raclopride For D2 receptor binding, sect ions were pre- incubated for 15 minutes at 20°C in buffer containing 120mM NaCI , 5 0 m M Tr is-HCI, 5 m M KCI, 2 m M C a C I 2 , and 1mM MgCI 2 . Buffer was adjusted to pH 7.4 by dropwise addition of HCI or N a O H . Sect ions were then incubated in the s a m e buffer with the addition of 3 n M [ 1 1 C]raclopr ide (SA= 892-1169 Ci /mmol) at 20°C for 45 minutes. Nonspeci f ic binding was s imul taneously performed on adjacent sect ions with the addition of 10 uM (+)-butaclamol. Incubation was followed by two 1-minute pos t -washes in the s a m e buffer at 4°C. After a brief dip in distil led, deionized water at 4°C to remove excess buffer, s l ides were laid out to dry for 20 minutes in a fumehood. S l ides were then p laced against multisensit ive storage phosphor sc reens along with a set of standards of known activity in standard film casset tes. Casse t tes were stored behind lead for the 2-hour exposure, before the screens were removed from the casset tes and scanned in the phosphor imager. D2 receptor binding with [3H]raclopride D2 receptor binding was also investigated using the radiotracer [ 3H]raclopride to compare 1 1 C - l a b e l e d and 3 H- labe led tracer binding. Di f ferences between establ ished protocols for both raclopride binding exper iments were min imized. S l ides were removed from the -80 °C f reezer and laid on the bench to dry. Sec t ions were circled with nailpolish to help prevent the incubation solution from running off the sl ides and keep the t issue bathed throughout the incubation period. W h e n nailpol ish was dry, sect ions were pre- incubated in buffer containing 120mM NaCI , 5 0 m M Tr is-HCI, 5 m M KCI, 2 m M C a C I 2 , and 1mM M g C I 2 for 15 minutes at 20°C and pH adjusted to 7.4 by addition of 52 HCI or N a O H , just as in the [ 1 1 C]raclopr ide binding experiment. S l ides were then laid out to dry in incubation trays lined with wet paper towels to keep t issue moist (about 30 minutes). Buffer containing 3 n M [ 3H]raclopride (SA=55.29 Ci /mmol) was pipetted onto sect ions and incubation lasted 45 minutes. Nonspeci f ic binding was determined in adjacent sect ions by addit ion of 10 u M (+)-butaclamol to the buffer-[ 3H]raclopride solution before it w a s pipetted onto those sect ions. Incubation was fol lowed by two 1-minute post -washes in the s a m e buffer at 4°C. After a brief dip in distil led, deionized water at 4°C, s l ides were laid on the bench to dry for at least 4 hours. S l ides were then placed in a des iccator for at least 24 hours to post-fix t issue. Finally, s l ides were placed against trit ium-sensitive phosphor sc reens along with a set of s tandards of known activity in standard film casset tes. A s with the mult isensit ive sc reens , tritium-sensitive screens were p laced against a light-box for at least 10 minutes prior to loading of casset tes to e rase any latent images. Sc reens were removed from casset tes after five days and immediately scanned in the phosphor imager. A R G Data A n a l y s i s The Cyc lone phosphor imager produces latent image autoradiographs from the optical density (OD) it measu res in exposed screens . Opt iQuant™ software (Packard) was used to measure the light intensity (in digital light units, DLU) in regions of interest (ROIs) of autoradiographs. D L U measurements were then cal ibrated to radioactivity intensity using the s tandards in order to quantify the distribution of radioactivity in the striatal t issue sect ions. Data analys is fol lowed the method deve loped by Strome et al. (2005), with slight modif icat ions. The activity of each point in the standard must be determined in order to create a standard curve that can be used to quantify t issue activity. Before drawing each ROI encompass ing a point, the autoradiographic image w a s d isp layed relative to that point. This process was necessary to prevent over-estimating or under-est imating activity in a point, which can occur when the image is d isplayed in the default mode and very hot spots spil lover onto neighbouring pixels, while very cold spots are not intense enough to be seen at all (Strome et a l . , 2005). Th is method ensures an accurate est imate for the radiation intensity of each individual spot and thus an accurate standard curve. A n addit ional ROI was drawn on a nearby region of the image that had not been exposed 53 to radioactivity to serve as a background measure. Figure 2.3 shows a [ 1 1 C] standard displayed relative to the activity in a high-activity point and a low-activity point, as well as the p lacement of ROIs on all points. D L U values and a reas were computed for each ROI and the proportional background D L U count was subtracted from the D L U value for each point. S ince the points each consisted of a known amount of radioactivity (in nCi) , that amount of activity was related directly to the D L U value for each point. Standard curves were determined using G r a p h P a d Pr ism v4.00 for W indows (GraphPad Software) and linear regression analysis. c o o O o o • Fig. 2.3. [ 1 1 C] standard d isp layed relative to activity in the 6 t h point (left), 2 n d point (middle), and with ROIs drawn on (right). Standards were made by pipetting 5 uL drops of a set of eight serial dilutions onto a strip of T L C plate. Creat ing a standard curve for [3H] exper iments was much easier . The commercia l [ 3 H]microscales™ were already calibrated to activity per mg t issue wet weight (in nCi/mg), so D L U / m m 2 w a s computed with Opt iQuant and related to the provided values of activity per mg t issue wet weight. Figure 2.4 shows a [ 3 H]microsca le™ in an image from a [3H] binding experiment. Standard curves were determined using the s a m e G r a p h P a d software and linear regression analysis. w • u V -• i • Hi 54 Fig. 2.4. Image from a [ 3H]WIN 35,428 binding experiment, demonstrating a [ 3 H]microscale (Amersham) on the left, which was used to calibrate optical density of images to quantitative measures of activity. To determine tracer uptake in striatal t issue sect ions, ROIs were drawn on each t issue sect ion with an attempt to encompass the entire dorsal striatum and exclude the nucleus accumbens , as was attempted in analysis of the P E T data. S ince the resolution of autoradiographic images is high (2.5 line pairs per millimeter), it is possible to be extremely select ive of the speci f ic region e n c o m p a s s e d by an ROI . A R G studies often use this quality to quantitate radioligand binding in speci f ic striatal subregions, such as the dorsolateral striatum, mediolateral striatum, dorsomedia l striatum, ventromedial striatum, etc. However, the E C A T P E T scanner used in this study produces images at a much lower resolution (6 mm full width at half maximum), which had to be taken into account when placing ROIs on autoradiographic images. Images were initially analyzed in two ways: 1) a single ova l -shaped ROI encompass ing the entire dorsal striatum was placed on each t issue sect ion; 2) two individually-drawn oval ROIs were placed on dorsolateral striatal t issue with an attempt to e n c o m p a s s the striatal regions only and exclude the internal capsu le . The first method replicated the analys is method used for placing ROIs on P E T images while the second method replicated the specif ic analysis technique common to A R G experiments. ROI s ize , shape , and placement were consistent among all t racers for each individual subject. For each sl ide, a background ROI of the s a m e s ize was drawn nearby on an unexposed region of the image. 55 For the [ 1 1 C] exper iments, D L U va lues and ROI areas were computed for each ROI and the background count was subtracted from each of the t issue ROI counts. D L U counts were converted to apparent t issue ligand concentrat ion using the standard curve to determine the relationship between D L U measures and activity (in units of nCi). The activity value was divided by the volume of the ROI and the speci f ic activity of the tracer to obtain binding va lues in units of pmol/cc. For the [ 3H] exper iments, the standards derived from commerc ia l [ 3H] microscales were already cal ibrated to a wet weight t issue equivalent est imate, which simplif ied calculat ions. D L U / m m 2 va lues were computed for each ROI and the background count was subtracted from e a c h of the t issue ROI counts. Dividing these D L U / m m 2 va lues by the speci f ic activity of the tracer produced the desired binding va lues in units of pmol/cc. Ave rages of binding va lues for each subject were calculated using counts from two to four sect ions from each condit ion. T issue sect ions were exc luded from calculat ions of average tracer for reasons such as disintegration of part of the sect ion during incubation, or run-off of tracer during incubation resulting in significantly lower binding compared to adjacent sect ions in the s a m e subject (tritiated tracers only). Speci f ic binding was determined by subtracting average binding va lues of the non-specif ic t issue sect ions from average binding va lues of the total t issue sect ions. Single ROI p lacement encompass ing the entire striatum produced lower binding values than individual ROI p lacement excluding the internal capsu le . Th is finding is logically explained by consider ing that the binding of all D A sys tem tracers used in this study is very low in the internal capsu le , which is composed primarily of white matter axonal tracts of neurons projecting between the cortex and subcort ical nuclei and does not contain many transporters or D A receptors. The inclusion of internal capsule in the single ROI method thus reduced the average binding calculated for the entire ROI volume. However, despi te the fact that the absolute binding va lues obtained with the single ROI method were lower, their correlation with in vivo B P va lues and with behavioural scores w a s not significantly different from the correlation of binding values obtained by the individual ROI p lacement method with in vivo and behavioural measures . S ince the single ROI p lacement method more c losely replicated the ROI 56 placement method used for in vivo P E T analysis, the single ROI method was used to obtain the reported va lues of in vitro binding. Figure 2.5 demonstrates the single ROI placement method on an A R G image from a [ 1 1 C] experiment. i i . -Fig. 2.5. ROI p lacement method used to calculate binding measurements from A R G experiments. Statistical Analysis Statistical analys is of binding data was performed using G r a p h P a d Pr ism v4.00 for W indows (GraphPad Software). Relat ionships between in vivo P E T B P measurements, in vitro A R G binding measurements , and behavioural sco res were analysed using Spearman 's correlat ion, a nonparametr ic test of l inear correlation. Statistical s igni f icance was reported at a=0.05. The select ion of a nonparametr ic test over a parametr ic test to investigate the relationships between parameters in this study was based on multiple observat ions. Firstly, the subject group was relatively smal l (19 subjects), which decreases the probability that the group accurately reflects the true situation in the entire population of M P T P monkeys. The subjects were intentionally se lected to cover a wide range of lesion severity, as measured by behavioural assessmen ts , which violates the 57 requirement for parametr ic tests that subjects be drawn randomly from a normally-distributed populat ion. Furthermore, the distribution of behavioural scores was skewed and did not c losely fol low a G a u s s i a n distribution, which is an assumpt ion that must be met in order to val idate the use of a parametr ic test. P E T B P data obtained from previous studies and used in these investigations were not representat ive of a normally distributed populat ion either. For these reasons, a nonparametr ic test was used to evaluate the relat ionships between P E T B P and A R G binding, behavioural scores and P E T B P , and behavioural scores and A R G binding. It is important to note that nonparametr ic tests are statistically less powerful, or sensit ive, than parametr ic tests. Whi le parametric tests use the actual reported values in their calculat ions, nonparametr ic tests rank the measurements into ordered ser ies and use only ranks in their calculat ions. For example, in this study the B P va lues obtained using P E T would be ranked in order of their numerical va lue, as would the binding values obtained using A R G , and their rank-ordered va lues would be used to compute the Spea rman correlation coefficient, p. Exc lus ion of actual B P and binding va lues el iminates a signif icant amount of information from the correlation calculat ion, thereby decreas ing the sensitivity of the test. S ince this study individually measured in vivo P E T B P , in vitro A R G binding and behavioural scores , it was more appropriate to use correlation than linear regression to examine the nature of their relat ionships. Al l three methods were used independently. No method was manipulated to change the measurements taken by another method; rather, data using each method was obtained separately and compared to data from the other methods afterwards. Moreover, correlation avo ids the assumpt ion that measurements obtained by one method are in s o m e way dependent on measurements obtained by another method. It is possib le that va lues obtained using one method may be dependent on va lues obtained using another method in this study, but this has not been conf i rmed. Therefore, correlation analysis was appropriate because it avoided any expectat ion bias that could have interfered with evaluat ion of relat ionships between methods. 58 Chapte r 3 Resul ts C l in ica l Sever i ty a n d P E T m e a s u r e m e n t s Table 3.1 lists the behavioural scores and P E T data used in correlat ions with in vitro A R G data. Behavioura l scores are representative of cl inical severity of parkinsonian symptoms. B P was obtained in vivo using P E T . This data was col lected over a period of many years. Figure 3.1 shows P E T images produced by reconstruct ion of data col lected by the E C A T tomograph during scanning with each of the four P E T tracers investigated in this study. Tab le 3.1. Behavioura l scores and binding potentials (BP) of radiotracers in striatium used for compar isons with in vitro data. Behavioural sco res were measured using rating sca le of cl inica severity of P D symptoms; 3 P was measured in vivo by P ET . Animal ID Behavioural Score [1 1C]DTBZ BP [11C]MP BP [11C] raclopride BP [11C]SCH-23390 BP Control Bony 1 0.94 1.46 1.77 1.82 Elf 2 0.92 1.12 2.24 1.56 Pittstop 2 0.7 1.04 1.61 1.21 Hemi-MPTP3 Karma-L 6 0.69 0.7 1.82 1.18 Karma-R 11 0.18 0.11 2.35 1.02 Ken-L 1 0.83 1.08 1.99 1.5 Ken-R 2 0.72 0.87 1.88 1.5 Martin-L 4 0.82 0.82 2.1 1.74 Martin-R 8 0.28 0.19 2.65 1.74 Mike-L 4 0.66 0.7 1.14 1.52 Mike-R 8 0.28 0.26 1.3 1.24 Robert-L 6 0.66 0.78 1.41 1.51 Robert-R 11 0.14 0.11 1.97 1.51 Scott-L 8 0.46 0.45 1.8 1.19 Scott-R 11 0.22 0.12 1.96 1.07 Bilateral MPTP Enthalpy 5 0.3 0.37 1.94 0.91 Ezechial 5 0.32 0.35 2.02 1.45 Murray 5 0.36 0.39 1.64 0.89 Rose 18 0.31 0.39 3.1 -a "L" and "R" following animal identification signify left hemisphere and right hemisphere, respectively. All hemi-MPTP animals received unilateral M P T P injections into right internal carotid artery. Behavioural score is opposite, i.e. right striatal lesion leads to left side motor impairments. 59 " C M P "C-DTBZ Fig. 3.1. PET images produced by reconstruction of data collected by PET tomograph during scanning. Images show single coronal plane at same striatal level in same subject from separate experiments using the four radiotracers investigated in this study. The white outline indicates the edges of the brain (courtesy of Doris Doudet). A R G m e a s u r e m e n t s Table 3.2 lists binding values (in units of pmol/cc) obtained for each of the tracers in vitro using ARG. Binding experiments were performed on striatal tissue sections from each subject with three of the same radiotracers used for P E T and with three tritiated tracers. Nonspecific binding represented only 1-2% of total binding on average with [11C]-labeled tracers and approximately 10-20% with [3H]-labeled tracers. 60 Table 3.2. Results of radiotracer binding in striatum, obtained in vitro by autoradiography. Binding values are reported in units of pmol/cc. Animal ID [11C]DTBZ binding [11C]MP binding [3H]WIN 35,428 binding [11C]raclopride binding [3H]raclopride binding [3H]SCH-23390 binding Control Bony 244.68 84.12 41.96 57.67 45.50 42.30 Elf 252.13 72.39 63.48 50.93 44.25 48.75 Pittstop 275.22 136.64 76.36 50.92 41.02 41.06 Hemi -MPTP Karma-L 204.08 186.07 94.67 30.62 34.71 58.95 Karma-R 50.73 11.73 3.29 45.34 54.53 75.12 Ken-L 384.34 193.56 93.32 65.85 64.83 45.24 Ken-R 254.22 198.67 71.99 46.73 44.79 44.76 Martin-L 500.70 199.53 90.16 65.49 56.73 43.96 Martin-R 14.77 1.04 3.10 41.35 50.90 57.81 Mike-L 257.26 261.94 76.19 40.12 40.91 50.66 Mike-R 55.45 35.83 10.51 46.62 42.17 41.15 Robert-L 369.27 126.63 74.21 43.22 29.47 49.01 Robert-R 12.17 0.47 1.76 37.54 23.12 47.47 Scott-L 243.18 137.73 87.45 41.60 40.11 43.13 Scott-R 61.58 23.34 16.80 48.23 47.61 50.79 Bilateral M P T P Enthalpy 61.72 39.61 18.40 54.99 38.11 41.12 Ezechial 105.09 39.30 24.16 47.85 43.09 48.78 Murray 127.50 67.88 40.06 47.16 39.09 42.52 Rose 15.20 3.02 2.09 55.67 53.53 59.30 Correlations Between in vivo PET and in vitro A R G Measurements Presynaptic Tracers There were significant correlations between in vivo PET BP measurements and in vitro ARG binding measurements, behaviour and BP measurements, and behaviour and binding measurements for all presynaptic tracers investigated. [ 1 1C]dihydrotetrabenazine Images of [11C](+)DTBZ binding obtained by in vitro ARG are shown in figure 3.2. Binding values obtained in MPTP-lesioned subjects were lower than binding values obtained in unlesioned subjects, as is visibly perceptible by lighter appearance of images of striatal sections from lesioned subjects relative to darkness of sections from unlesioned subjects. 61 1 2 3 4 5 6 Fig. 3.2. Images of [ 1 1 C ] D T B Z binding obtained by in vitro autoradiography in h e m i - M P T P animal (unlesioned hemisphere; s l ides 1-3) and bilateral M P T P animal (slides 4-6). Total binding w a s measured by incubating t issue sect ions in buffer containing [ 1 1 C](+)DTBZ for 30 minutes (sl ides 1,2,4,5); nonspeci f ic binding w a s measured by addit ion of 5 u M (±)TBZ to incubation solution (sl ides 3,6). Nonspec i f ic binding represented only 1-2% of total binding on average for [ 1 1 C ] D T B Z , thus these sect ions are not visible when images are d isp layed relative to much higher activity in sect ions exposed to radioactive tracer only for measurement of total binding. Binding fol lowed a ventrolateral gradient in les ioned subjects, with the nucleus accumbens showing highest binding, fol lowed by less binding in the caudate and even less binding in the putamen. A significant correlation (p=0.84, P<0.0001) was found between in vivo P E T B P measurements and in vitro A R G binding measurements (Fig. 3.3). Behaviour a lso correlated significantly with both P E T B P measurements (p= -0.87, P<0.0001; Fig. 3.4) and A R G binding measurements (p=-0.78, P<0.0001; F ig . 3.5). The negative correlation between behaviour and both P E T and A R G signif ies that more severe clinical parkinsonian symptoms are assoc ia ted with lower B P and binding. [ 1 1 C]DTBZ o.OH 1 1 1 1 1 1 0 100 200 300 400 500 600 DTBZ binding (pmol/cc) Fig. 3.3. Correlat ion of [ 1 1 C](±)DTBZ B P measurements from in vivo P E T and [ 1 1 C](+)DTBZ binding measurements from in vitro A R G . 63 [11C]DTBZ 20n <D 8 ^ CO > 10H p= -0.87 P<0.0001 •• • 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 DTBZ BP Fig. 3.4. Correlat ion of behavioural scores with [ 1 1 C] (± )DTBZ B P from P E T experiments. r11 [ C]DTBZ 20n p= -0.78 PO.0001 i i 1 1 - i 1 100 200 300 400 500 600 DTBZ binding (pmol/cc) Fig. 3.5. Correlat ion of behavioural scores with [ 1 1 C](+)DTBZ binding measurements from A R G exper iments. 64 [ 1 1C]methylphenidate Images of [ 1 1 C ] M P binding obtained by in vitro A R G are shown in figure 3.6. Binding patterns observed with M P were very similar to those observed with D T B Z , with much lower binding in M P T P - l e s i o n e d animals and a clearly visible ventrolateral gradient of optical density in A R G images. In M P T P - l e s i o n e d subjects, reduct ions in binding were greatest in the putamen and less severe but still present in the caudate. A significant correlation of p=0.74 (P<0.0001) was found between in vivo P E T B P measurements and in vitro A R G binding measurements (Fig. 3.7). Behaviour a lso correlated significantly with both P E T B P measurements (p= -0.85, P<0.0001; Fig. 3.8) and A R G binding measurements (p=-0.70, P=0.0008; F ig . 3.9). 65 1 2 3 4 5 6 F i g . 3 .6 . I m a g e s of [ 1 1 C ]MP b i n d i n g o b t a i n e d by in vi t ro a u t o r a d i o g r a p h y in a h e m i - M P T P m o n k e y ( u n l e s i o n e d h e m i s p h e r e ; s l i d e s 1-3) a n d a b i l a te ra l M P T P m o n k e y ( s l i d e s 4 -6 ) . T o t a l b i n d i n g w a s m e a s u r e d by i n c u b a t i n g s l i d e s for 4 0 m i n u t e s in bu f fe r c o n t a i n i n g 15 n M [ 1 1 C ]MP (s l i des 1, 2 , 4 , 5) ; n o n s p e c i f i c b i n d i n g w a s m e a s u r e d by a d d i t i o n of 1 0 u M n o m i f e n s i n e ( s l i des 3 ,6) . [11C]MP 1.75-1.50-1.25-m 1.00H «| 0.75-0.50-0.25-0.00 p= 0.74 P=0.0003 50 100 150 200 250 MP binding (pmol/cc) 300 Fig. 3.7. Correlat ion of [ 1 1 C ] M P B P measured in vivo using P E T with [ " C ] M P binding measurements obtained in vitro using A R G . 11/ [11C]MP 20n p= -0.85 PO.0001 i i i i i 0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 MP BP Fig. 3.8. Correlat ion of behavioural scores with [ 1 1 C ] M P B P measurements using in vivo P E T . 67 [11C]MP 0 o o </) > 20n 1^ 10H p= -0.70 P=0.0008 50 100 150 200 250 MP binding (pmol/cc) 300 Fig. 3.9. Correlat ion of behavioural scores with [ 1 1 C ] M P binding measurements from in vitro A R G . [3H]WIN 35,428 D A T binding w a s a lso evaluated with the tritiated tracer [ 3 H]WIN 35,428. Images of [ 3H]WIN 35,428 binding obtained in vitro by autoradiography are shown in figure 3.10. A significant correlation of p=0.73 (P=0.0004) was found between in vivo P E T B P measurements and in vitro A R G binding measurements (Fig. 3.11). Behaviour a lso correlated significantly with A R G binding measurements (p=-0.64, P=0.003; Fig. 3.12). 68 Fig. 3.10. Images of [ 3 H]WIN 35,428 binding obtained by in vitro autoradiography in a h e m i - M P T P animal . S l ides 1-3 from unles ioned hemisphere and sl ides 4-6 from lesioned hemisphere. Total binding was measured by incubation in buffer containing 15nM [ 3 H]WIN 35,428 for 40 minutes (slides 1,2,4,5); nonspeci f ic binding was measured by addit ion of 10uM nomifensine to incubation buffer (sl ides 3,6). [3H]WIN 35,428 1.75-1 1.50-Q. 1.25-CQ Q. 1.00-U 0-75-0.50-0.25-0.00-p= 0.73 P=0.0004 0 10 20 30 40 50 60 70 80 90 100110 [3H]WIN 35,428 binding (pmol/cc) Fig. 3.11. Correlat ion of [ 1 1 C ] M P B P measurements from in vivo P E T and [ 3H]WIN 35,428 binding measurements from in vitro A R G . O o o > ( 0 00 20n 15^  10H [3H]WIN 35,428 p= -0.64 P=0.003 • • • • i i i 0 10 20 30 40 50 60 70 80 90 100110 WIN 35,428 binding (pmol/cc) Fig. 3.12. Correlat ion of behavioural scores with [ 3 H]WIN 35,428 binding measurements from in vitro A R G . 70 The correlation coefficient for in vitro A R G binding va lues of the D A T radiotracers [ 1 1 C ] M P and [ 3 H]WIN 35,428 was 0.92 ( P O . 0 0 0 1 ; Fig. 3.13). Th is value is c lose to a perfect correlation of 1.0 and the negligible probability of making a type 1 error, or finding a correlation this strong by chance when it does not actually exist (less than 0.0001), signif ies that the correlation is highly reliable. Binding Comparison [11C]MP and [3H]WIN 35,428 0 10 20 30 40 50 60 70 80 90 100110 [3HJWIN 35,428 binding (pmol/cc) Fig. 3.13. Correlat ion of [ 1 1 C ] M P and [ 3H]WIN 35,428 binding va lues from in vitro A R G . Postsynaptic Tracers Surprisingly, there w a s little or no significant correlation between in vivo P E T B P measurements and in vitro A R G binding measurements using radiotracers specif ic for postsynapt ic e lements . Behavioura l scores were a lso only weakly correlated or not correlated at all to B P and binding measurements . [ 3 H]SCH-23390 Images of [ 3 H]SCH-23390 binding obtained by in vitro autoradiography are shown in figure 3.14. Spec i f ic binding was quite homogenous throughout the striatum. 71 Fig. 3.14. [ 3 H]SCH-23390 binding image obtained by in vitro A R G in unlesioned hemisphere of h e m i - M P T P monkey. Total binding measured by incubating sl ides in buffer containing 2 n M [ 3 H]SCH-23390 (4 dark sect ions); nonspeci f ic binding measured by addition of 10uM (+)butaclamol to incubation solution (2 sect ions at right). There was no signif icant correlation between in vivo P E T B P measurements and in vitro A R G binding measurements (Fig. 3.15). There was no significant correlation between behaviour and in vivo P E T B P (Fig. 3.16), but a signif icant correlation was found between behavioural scores and in vitro A R G using [ 3 H]SCH-23390 binding measurements (p=0.52, P=0.02; Fig. 3.17). However, this correlation is strongly driven by the outlier representing the most severely lesioned subject and exclusion of this point makes the correlation nonsignif icant (Fig. 3.17). 72 [3H]SCH-23390 2.0-i 1.5H Q. c o o O) C O C O ^ 1-OH O CO O 0.5H 0.0-p=0.08 P>0.05 0 10 20 30 40 50 60 70 80 [3H]SCH-23390 binding (pmol/cc) Fig. 3.15. Correlat ion of [ 1 1 C ] S C H - 2 3 3 9 0 B P measurements taken in vivo using P E T and [ 3 H]SCH-23390 binding measurements taken in vitro using A R G . [11C]SCH-23390 12-i 11-a> i _ 10-o 9-o 8-CO ro 7-i _ 3 6-O 5-1 Ar o 3-tn 2-1-0-p=-0.39 P>0.05 • •• - i 1 1 1 1 1 1 1 0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 SCH BP Fig. 3.16. Correlat ion of behavioural scores with in vivo P E T [ 1 1 C ] S C H - 2 3 3 9 0 B P measurements . 73 [3H]SCH-23390 2(H o o CO 1H - p=0.52, P=0.02 • p=0.43, P>0.05 1(H • • • o-0 10 20 30 40 50 60 70 80 [3H]SCH-23390 binding (pmol/cc) Fig. 3.17. Correlat ion of behavioural scores with in vitro A R G [ 3 H]SCH-23390 binding measurements . Sol id line represents correlation including all data points; dotted line represents nonsignif icant correlation after exc lus ion of outlier (highest behavioural score). [ 1 1 C]rac lopr ide a n d [ 3 H]raclopr ide D2 receptor binding was investigated with both [ 1 1 C]- labeled and [ 3H]-labeled raclopride. Images of in vitro A R G binding with [ 1 1 C]raclopr ide and [ 3H]raclopride are shown in figure 3.18. No significant correlation was found between in vivo P E T using [ 1 1 C]raclopr ide B P values and in vitro binding using the s a m e tracer (p=0.32, P>0.05; F ig . 3.19). There was no significant correlation between behaviour and either in vivo B P (p=0.27, P>0.5; F ig . 3.20) or in vitro binding (p=-0.45, P>0.05; F ig . 3.21) va lues using [ 1 1 C]raclopr ide. It is surprising that the trend toward a positive correlation between behavioural scores and in vivo B P va lues is opposi te that between behavioural scores and in vitro binding va lues using the s a m e tracer. The trend is far from significant though (P>0.05) and all va lues of both B P and binding fall within a smal l numerical range, which ampli f ies the effects of other var iables among individuals. 74 1 2 3 4 5 6 cn F i g . 3 . 1 8 . I m a g e s o f in vitro A R G u s i n g [ 1 1 C ] r a c l o p r i d e ( s l i d e s 1-3) a n d [ 3 H ] r a c l o p r i d e ( s l i des 4-6; d a r k e r s t a i n i n g a r o u n d s e c t i o n s is n a i l p o l i s h , u s e d to k e e p t r ace r o n s e c t i o n s t h r o u g h o u t i n c u b a t i o n ) in s a m e h e m i - M P T P a n i m a l . B i n d i n g wi th bo th r a d i o t r a c e r s f o l l o w e d s a m e p r o t o c o l , i nc l ud ing i n c u b a t i o n in bu f fe r c o n t a i n i n g 3 n M r a d i o l a b e l e d r a c l o p r i d e , wi th a d d i t i o n of 1 0 u m (+ )bu tac l amo l fo r n o n s p e c i f i c b i nd i ng ( s l i des 3 a n d 6; b a r e l y v i s i b l e for [ 1 1 C ] r a c l o p r i d e b i n d i n g at top) . N o t e h i g h e r p ropo r t i on of n o n s p e c i f i c b i n d i n g in s l i d e s i n c u b a t e d wi th [ 3 H ] r a c l o p r i d e ( d a r k e r a p p e a r a n c e ) t h a n in s l i d e s i n c u b a t e d wi th [ 1 1 C ] r a c l o p r i d e . [11C]raclopride 4. 4. Q_ CO 3. ~o 3. E. 2. I 2. ^ i " 0. 0. 5n 0-5-0-5-0-5-0-5-p= 0.32 P>0.05 10 20 30 40 50 60 70 [11C]raclopride binding (pmol/cc) Fig. 3.19. Relat ionship between P E T B P and A R G binding using [ 1 1 C]raclopr ide 20n CO (0 > (0 <D CQ 10H [11C]raclopride p= 0.27 P>0.05 [11C]raclopride BP Fig. 3.20. Correlat ion of behavioural scores and in vivo P E T B P va lues obtained using [ 1 1 C]raclopr ide. 76 [11C]raclopride 20n 8 ^ CO 15 3 10-> (0 JO 0) 00 p=-0.45 P>0.05 10 i 20 30 40 50 60 70 r11 [ C]raclopride binding (pmol/cc) Fig. 3.21. Correlat ion of behavioural scores and in vitro A R G binding va lues obtained using [ 1 1 C]raclopr ide. Al though no signif icant correlation was found between P E T B P va lues and A R G binding values using [ 1 1 C]raclopr ide, a significant correlation w a s found between P E T B P va lues using [ 1 1 C]raclopr ide and A R G binding using [ 3H]raclopride (p=0.60, P>0.007; F ig. 3.22). There w a s no signif icant correlation between behaviour and in vitro binding values using [ 3H]raclopride (p=0.12, P>0.05; F ig . 3.23), as w a s observed between behaviour and [ 1 1 C]raclopr ide by in vitro binding. Figure 3.24 shows the correlation between in vitro binding va lues obtained using both raclopride radiotracers. There was a significant correlation between binding values obtained by A R G using [ 1 1 C]raclopr ide and those obtained using [ 3H]raclopride (p=0.62, P=0.005). 77 [3H]raclopride 4.5-1 „ 4.0H CQ 3.5-1 o " O 3.0H Q. 2.5H " 1.3 0.5H 0.0 p= 0.60 P=0.007 10 20 i 30 40 50 60 [H]raclopride binding (pmol/cc) Fig. 3.22. Correlat ion of [ 1 1 C]raclopr ide B P values obtained in vivo using P E T and [ 3H]raclopride binding va lues obtained in vitro using A R G . [3H]raclopride 20-1 8 1*H > CO 0) CO 10H p=-0.12 P>0.05 10 20 30 40 50 60 [H]raclopride binding (pmol/cc) Fig. 3.23. Correlat ion between behavioural scores and in vitro A R G [ 3H]raclopride binding. 78 Binding comparison [ 1 1C] raclopride and [3H] raclopride £ 6<H Q. o)50H c 0 10 20 30 40 p= 0.62 P=0.005 50 60 [ 3H]raclopride binding (pmol/cc) Fig. 3.24. Correlat ion of in vitro A R G binding va lues obtained using [ 1 1 C]raclopr ide and [ 3H]raclopride. In summary, results of in vivo P E T and in vitro A R G using radiotracers specif ic for presynaptic components ([ 1 1C]DTF3Z, [ 1 1 C ] M P , [ 3 H]WIN 35,428) correlated significantly with each other and with behavioural assessmen ts of lesion severity. [ 1 1 C ] D T B Z P E T B P demonstrated the strongest correlations with A R G binding with the s a m e radiotracer and with behavioural scores . With postsynapt ic tracers, however, the only significant correlations observed were between behaviour and A R G binding with [ 3 H]SCH-23390 and between [ 1 1 C]raclopr ide P E T B P and [ 3H]raclopride A R G binding, and both of these correlat ions were much weaker than the correlat ions observed using presynaptic tracers. A R G with [ 3H]raclopride and [ 1 1 C]raclopr ide provided binding va lues that were not equal , but fell within a similar range and were significantly correlated. Correlat ion of in vitro A R G binding va lues obtained using [ 1 1 C ] M P and [ 3 H]WIN 35,428 was highly significant, but absolute [ 1 1 C ] M P binding va lues were approximately double [ 3H]WIN 35,428 binding va lues. Nonspec i f ic binding was visibly and proportionally higher in [ 3H] exper iments than in [ 1 1 C] exper iments. 79 Chapte r 4 D i s c u s s i o n a n d C o n c l u s i o n s The purpose of this study was to elucidate the relat ionships between P E T measurements and A R G measurements , obtained in vivo and in vitro respectively, with the s a m e presynapt ic and postsynapt ic radiotracers. The study a lso examined the correlation between P E T and A R G measurements and lesion severity, as measured by behavioural assessmen t of cl inical parkinsonian symptoms in a group of non-human primates encompass ing a wide range of M P T P - i n d u c e d lesion severity. P E T and A R G measurements obtained using presynaptic tracers ( [ 1 1 C ] D T B Z , [ 1 1 C ] M P , [ 3H]WIN 35,428) correlated significantly with each other and with lesion severity. Postsynapt ic tracer binding performed by A R G did not correlate with P E T and neither binding technique correlated with lesion severity in most c a s e s . T h e only significant correlations observed with postsynapt ic tracers were between [ 1 1 C]raclopr ide P E T and [ 3H]raclopride A R G , and between [ 1 1 C ] S C H - 2 3 3 9 0 P E T and lesion severity. The results of this study contribute to our understanding of the relat ionships between P E T and A R G binding techniques and between both binding techniques and clinical severity. Never the less, there are severa l areas in this study where error may have been introduced, including variation among subjects, uncontrol lable di f ferences between in vivo and in vitro binding condit ions, and intrinsic di f ferences between P E T and A R G experimental procedures. T h e s e potential sources of error must be kept in mind when consider ing exper imental results. S o u r c e s of E r ror A major source of error in results is due to the variability among individual subjects. Attempts were made to age-match subjects and expose them to the s a m e living and experimental condit ions, but there is still variation among individuals that cannot be control led. The relatively smal l sample s ize (N=19) a lso limits the power of the correlations. Inclusion of both hemispheres from hemi- les ioned animals as separate subjects may have introduced bias. It is difficult and expens ive to use a large sample s ize in human and non-human primate studies and consequent ly , previous studies have used both hemispheres as individual subjects. Nonethe less , it is inappropriate to a s s u m e that the two hemispheres in an individual are independent without c loser 80 investigation. Prev ious studies and P E T measurements from this study suggest that the unlesioned hemisphere of unilaterally-lesioned monkeys may be affected by M P T P administration via the contralateral internal carotid artery (Doudet et a l . , 1993). Al though B P data suggest that a dif ference does exist between the unles ioned hemisphere and control hemispheres, A R G binding data does not demonstrate t h e ' s a m e effect. This study included only six hemi- les ioned subjects and three control subjects, so it is possible that no trend w a s observed in A R G data s imply due to high inter-subject variation in a smal l sample . U s e of animals with different degrees of lesion severity further compl icated compar isons between hemi- les ioned subjects and controls. A larger study investigating P E T B P and A R G binding in both lesioned and unlesioned hemispheres of h e m i - M P T P monkeys compared to control hemispheres is required before ruling out the possibil i ty of bilateral effects of unilateral lesions. However, this point is not of major importance in this study because no compar isons were made between normal and MPTP- t rea ted subjects. P E T , A R G and behavioural measures were obtained separate ly for each hemisphere and this study examined the correlation of these parameters within individuals, not between them. Another source of error that likely contributed to variation among individuals is the t issue that was used for in vitro A R G binding exper iments. There are many implications regarding this aspect of the study. Firstly, the striatal monkey t issue was surgically removed and p laced immediately in dry ice wrapped in a luminum foil before being placed into the -80°C freezer. In most studies reported in the literature, t issue is f rozen in isopentane coo led with liquid nitrogen (Mann et a l . , 1978;Rioux et al . , 1997;Moore et al . , 1998;Nader et a l . , 2002;St rome et a l . , 2006) prior to being stored at -80°C to promote immediate and uniform preservat ion. More importantly, the lack of inadequate labeling in the initial preparation of striatal b locks likely contributed to high variation among subjects in A R G exper iments. Orientation of striatal b locks was not speci f ied prior to preservat ion, so t issue was sect ioned at different orientations through different striatal subregions. The effects of M P T P are not uniform throughout the striatum, with receptor and transporter densi t ies demonstrat ing anteroposterior, dorsoventral, and mediolateral gradients (Schneider et al . , 1987 ;German et a l . , 1988). Al though attempts were made to be as consistent as possib le in select ion of sect ions at the s a m e striatal level in all subjects, difficulties in accurately identifying the level of striatum and 81 orientation at which sect ions were taken may have greatly increased variability. Furthermore, striatal b locks were sl iced by two different individuals and t issue from seven of the subjects was far more variable in orientation and striatal subregion than t issue sect ions from the other 13 subjects. Due to the smal l samp le s ize, however, all of the subjects from whom more than half of the sect ion w a s striatum (as determined by pattern of raclopride and D T B Z binding) were included in calculat ions. Only one subject was exc luded due to lack of recognizable striatal t issue in sect ions. Another major source of variation between in vivo P E T and in vitro A R G in this study is that P E T data represents average B P over the entire striatum, whereas A R G data represents binding in only a very smal l region of the str iatum. B P va lues were obtained using distribution vo lume ratios from four antero-posterior P E T s l ices, representing average radiotracer uptake over the entire vo lume of the striatum, caudate and putamen. A R G binding va lues were obtained using t issue from a striatal (caudate and putamen) region less than one tenth of a mill imeter thick. Th is smal l region cannot possibly be representat ive of the entire striatum, which is heterogeneous and contains gradients of distribution of severa l D A system components (German et a l . , 1988 ;Obeso et al . , 2000 ;Rosa -Ne to et a l . , 2004). A s a result, correlation between in vivo B P and in vitro binding va lues is lower than could be expected if A R G va lues were obtained in multiple striatal regions and an average binding value for the entire striatum was compared to average B P va lues. Variat ion induced by this factor is further amplif ied by use of t issue sect ions from different antero-posterior striatal regions in this study. In consequence , the variation among binding va lues obtained by A R G relative to behavioural scores likely var ies more among individuals than B P va lues obtained by P E T . This effect is exhibited by lower correlation coeff icients between behavioural scores and binding va lues than between behavioural sco res and B P values. A g e is another factor that varied among individual subjects in A R G exper iments. Most of the monkeys were between the ages of 16 and 23 years , which is equivalent to ages 50-80 years for a human and severa l studies in humans and monkeys have identified significant changes in D A sys tem components with age (Wang et a l . , 1998;Suzuk i et al . , 2001;Doudet et a l . , 2002a;Doudet et a l . , 2006). However, these age di f ferences are not of major importance in this study because the parameter of interest is the relationship 82 between results obtained by in vivo P E T and in vitro A R G techniques in subjects with different degrees of D A sys tem alterations. A range of cl inical severity thought to result from different degrees of D A neuron degenerat ion was se lected purposely for this study and s ince the D A sys tem changes that occur with age are of a similar nature as the changes following M P T P administrat ion, the speci f ic c a u s e of these changes is not important. On the other hand, the postmortem delay (length of t ime between death and t issue preservation) and the duration of cryopreservat ion prior to A R G experimentat ion could have significantly impacted the results of this study. Most an imals were sacri f iced so that their t issue could be col lected immediately and f rozen with a minimum delay between death and cryopreservat ion. However, two of the an imals were found dead and despite attempts to col lect t issue immediately upon d iscovery of death, time between death and freezing of this t issue was up to twelve hours longer. Th is t issue differs from that col lected from sacr i f iced animals in that it is free of changes induced by pharmacological action from pentobarbital overdose, and it has been subjected to addit ional natural post-mortem changes such as autolysis and enzymat ic degradation (Mann e t a l . , 1978). S ince t issue col lection occurred over severa l years (2000-2006), t issue from s o m e animals was in the f reezer far longer than t issue from other an imals . Al though freezing at -80°C is a good way to preserve t issue for weeks to months, extended freezer storage periods can c a u s e dec reased l igand-receptor binding in A R G experiments (Gonza lez -Maeso et a l . , 2002;Mato and P a z o s , 2004). Pro longed freezer time may be the reason that t issue sect ions from s o m e animals were degraded during A R G exper iments. S o m e t issue was washed off s l ides during the w a s h e s and incubation, which dec reased measured activity va lues in the ROIs p laced on those sect ions. Whi le efforts were made to exc lude sect ions that had obviously deteriorated, all sect ions from some subjects exhibited partial deterioration and had to be used in calculat ions, leading to underest imation of binding in those individuals. Anesthet ics have been shown to affect P E T tracer binding measu res in monkeys and rats (Eckenhoff and F a g a n , 1994; F ink -Jensen e t a l . , 1994 ;Tsukada et a l . , 2001;Shahan i 83 et al . , 2002;Votaw et a l . , 2003 ;Momosak i et al . , 2004a) . The mode of action of severa l anesthet ics has been shown to involve interactions with presynapt ic and postsynaptic targets, but the exact mechan isms are unclear. Moreover , the effects of anesthet ics on radiotracer binding vary by anesthetic, dose , radiotracer, neural target, and animal model , not to mention variation in individual subject response. S ince accurate P E T data require that the subject remain mot ionless for the entire duration of the scan , very few P E T studies have been performed on unanesthet ized an imals (Rinne et al . , 1990;Fink-J e n s e n et a l . , 1994 ;Tsukada et al . , 1999). Isoflurane gas was used in this study to maintain anes thes ia in monkeys throughout P E T s c a n s and between s c a n s if the subject underwent multiple s c a n s on the s a m e day. Isoflurane administrat ion is known to increase striatal extracellular D A concentrat ion and has been shown to affect D A T activity and in vivo M P binding (Eckenhoff and F a g a n , 1994;F ink-Jensen et al . , 1994 ;Tsukada et a l . , 1999;Votaw et a l . , 2003). Both isoflurane and halothane reduce the binding potential of multiple D A T -specif ic radiotracers (Eckenhoff and Fagan , 1994 ;Tsukada et a l . , 1999;Shahani et al . , 2002;Votaw et a l . , 2003), although the mechan ism of action is unclear. Isoflurane does not change the total amount of D A T protein, so it could competit ively inhibit the tracer binding to D A T , change its conformation, or cause internal trafficking of D A T (Votaw et al . , 2003). The prevail ing hypothesis is that D A T is trafficked into the cell without changing the total amount of D A T in the striatum, which could be observed as a decrease in apparent affinity of D A T for speci f ic radiol igands. D A T is not the only D A sys tem component affected by isoflurane. P E T studies have reported reduced [ 1 1 C]raclopr ide binding to D2 receptors in rhesus monkeys following isoflurane administrat ion (Tsukada et al . , 1999). Th is observat ion is thought to be an indirect effect l inked to the s a m e mechan ism responsib le for D A T alterations, perhaps an increase in synapt ic D A levels. Taken together, these studies suggest that isoflurane administration affects both D A T and D2 receptor binding, which should be taken into account when examining the P E T B P data obtained in this study from monkeys anesthet ized with isoflurane during data col lection. 84 Ketamine, on the other hand, increases the B P of [ 1 1 C ] C F T for D A T , which has been attributed to an increase in D A T density (Tsukada et a l . , 2001). Ketamine works by noncompetit ively inhibiting the N-methyl-D-aspartate (NMDA) receptor, which modif ies glutamate signal ing and D A synthesis. Findings from P E T studies using [ 1 1 C]raclopr ide and microdialysis vary as to the effect of N M D A antagonists such as ketamine on striatal extracel lular dopamine concentrat ions. Increases (Moghaddam et a l . , 1990;French, 1994 ;Verma and Moghaddam, 1996;Smith et a l . , 1998), dec reases (Kashihara et a l . , 1990;Smith et al . , 1998;Tsukada et a l . , 2000), and no change (Koshikawa et a l . , 1988 ;Onoe et al . , 1994) in [ 1 1 C]raclopr ide B P and synapt ic dopamine concentrat ions have been reported. Ketamine has a lso been shown to almost double D1 binding in rats relative to unanesthet ized animals in [ 1 1 C ] S C H - 2 3 3 9 0 P E T studies (Momosak i et a l . , 2004a) . Al though subjects in the present study did receive ketamine prior to P E T scann ing , this anesthet ic has short-lasting effects and the two-hour t ime delay between injection and scanning is long enough that the effects of ketamine on radiotracer binding should be negligible. An ima ls received a smal l dose of pentobarbital for transport between the animal a rea and the P E T facility, about one hour prior to tracer injection. They received a much larger pentobarbital dose immediately prior to death. Very few studies have investigated the effects of pentobarbital on D A system radiotracer binding either in vivo or in vitro, but it has been shown by P E T to drastically affect in vivo [ 1 1 C ] S C H - 2 3 3 9 0 binding to D1 receptor in rats (Momosak i et a l . , 2004a). B P of [ 1 1 C ] S C H - 2 3 3 9 0 in rats anesthet ized with pentobarbital was reduced by an average of 4 1 % relative to B P values in unanesthet ized rats, with large variation among individuals. The mechan ism of action of pentobarbital is unknown, but it a lso reduces cerebral blood flow and g lucose metabol ism in non-human primates (Branston et a l . , 1979;Dormehl et al . , 1992). S ince much of the pentobarbital would have been washed out prior to P E T scanning, this could help explain the lack of correlation between in vivo B P and in vitro binding with [ 1 1 C ] S C H - 2 3 3 9 0 and [ 3 H]SCH-23390 , respectively. For P E T , monkeys received only an anesthet ic dose of pentobarbital, most of which would have been washed out prior to scanning and data col lect ion! In contrast, in vitro s tudies were performed on t issue obtained immediately following pentobarbital overdose, so t issue likely retained alterations induced by the anesthet ic. 85 Overal l , differential exposure to anesthet ics assoc ia ted with col lection of in vivo P E T data and in vitro autoradiography data likely dec reased the correlations observed between B P and binding va lues for all radiotracers. Therefore, it is remarkable that such strong correlat ions were observed in spite of the confounding effects of anesthes ia. Compared to other anesthet ics, isoflurane appears to have the least effect on u P E T B P values and postmortem A R G binding in the D A sys tem in rats (Kofke et a l . , 1987;Hansen et a l . , 1989). The fact that components of the D A system are highly conserved in m a m m a l s supports the assumpt ion that anesthet ics will have similar effects on D A sys tem radiotracers in monkeys as they did in rats (Neckameyer and Weinste in , 2005;Draper et al . , 2007). Therefore, the use of isof lurane as the primary anesthet ic was the best cho ice for this study to minimize the differential effect of anesthes ia on binding in vivo and in vitro. S ince the an imals must be anesthet ized to obtain accurate P E T s c a n s and must be sacri f iced by pentobarbital overdose for ethical reasons, the di f ferences between in vivo P E T and in vitro A R G caused by anesthet ics are unavoidable. Propert ies intrinsic to exper imental equipment and data analys is techniques may also have contributed to lower correlations between in vivo and in vitro binding measurements than would be expected for binding with the s a m e l igand. Images obtained by P E T with the E C A T scanner have a theoretical resolution of 5 mm, which provides an actual resolution of only 9 mm in the filtered images after data corrections. Phys ica l properties of scattering and attenuation of g a m m a emiss ions must be accounted for, which reduces the accuracy of P E T . Th is leads to lower specificity and more uniform B P va lues among individuals than the binding va lues obtained by A R G , which has a resolution of approximately 0.05 mm. A R G binding va lues spanned a larger l inear range than P E T B P va lues, which demonstrates the ability of A R G to provide more speci f ic data that reveals the dif ferences between individuals more effectively than P E T . A main source of error in accurate estimation of radiotracer B P using P E T is violation of assumpt ions including that of t issue homogeneity and that of equil ibrium binding 86 condit ions. Striatal t issue is heterogeneous, consist ing of both white matter (internal capsule) and grey matter (containing subregions with different protein distributions) (Rappaport et a l . , 1993;Jan et a l . , 2003;Glynn and A h m a d , 2003). Equil ibrium condit ions are very difficult to attain in P E T studies and data are rarely col lected at a true equil ibrium (Doudet et al . , 2002b). Inability to meet these assumpt ions introduces noise into results and dec reases accuracy of P E T data relative to A R G data resulting from binding measurements obtained under true equil ibrium condit ions. Storage phosphor sc reens a lso present limitations. Mult i-sensit ive sc reens used to detect decay of [ 1 1 C]- labeled l igands are different than the trit ium-sensitive screens. Nonspeci f ic binding is higher in trit ium-sensitive sc reens than in multi-sensit ive sc reens and correlat ions between in vitro binding with [ 1 1 C]- labeled and [ 3H]-labeled tracers is imperfect in part due to intrinsic di f ferences between phosphor sc reens . A possib le source of error in the A R G exper iments using [ 3H]-labeled tracers is the age of the commerc ia l s tandards used to calibrate image optical densi ty to amount of activity. The standards were six years old and s ince the half-life of tritium is twelve years, 2 5 % of the radioisotope had decayed , resulting in standard curves that were not perfectly linear. This probably affected cal ibrated binding va lues, especia l ly at low and high extremes where s o m e va lues were not within the linear dynamic range, and could have been prevented by using new commerc ia l s tandards (LAnnunz ia ta , 1998). Hinds ight is 20/20 If I were able to perform this study again, there are severa l changes I would make that I expect would produce stronger correlations between in vivo P E T and in vitro A R G measurements using the s a m e radiotracers. Most of these improvements would aim to reduce variability among individual measurements . Assum ing unlimited resources were avai lable, I would use a much larger sample s ize to increase power, with unles ioned control monkeys represent ing at least one third of the subjects to increase the validity of the results. I would a lso include more animals with greater cl inical severity, which would provide a sample more representative of the entire range of lesion severi t ies and minimize bias in the correlation induced by the greater number of subjects with mild lesions. T issue would be obtained from age-matched 87 animals to el iminate any potential variation between D A sys tem changes due to age and changes induced by M P T P administration. In collecting t issue for postmortem A R G exper iments, I would remove and cryopreserve entire hemispheres to retain landmarks for t issue orientation. T i ssue would remain in the f reezer for a limited t ime period (as short as possib le, no more than one year) to prevent interfering effects of extended freezer storage time. W h o l e hemispheres would be sect ioned on a def ined orientation that would be identical for all an imals, as is often done in human A R G studies and avoids effects of variation due to heterogeneity of striatal t issue (Tupala et al . , 2001 ;Schou et a l . , 2005;Storvik et al . , 2006). I would take sect ions from the entire length of the hemisphere and perform A R G on sl ices from multiple regions. Th is would provide consistent, identifiable brain regions from all subjects and would al low calculat ion of average binding va lues throughout the entire striatum instead of just a smal l region. P E T B P was reported as an average from the entire striatum and these changes would more c losely match P E T condit ions, providing an average of A R G binding throughout the striatum. Discussion It is noteworthy that signif icant correlations were observed between in vivo and in vitro binding techniques and with behavioural scores , in light of the many sources of error and large variability among individuals investigated in this study. Variat ion among individual measurements can dec rease the signi f icance of a correlation, but the correlations obtained in this study with presynapt ic t racers were highly significant (P^O.003, meaning that the chance of making a type one error, or finding a correlation that does not really exist, w a s less than 0.3%). The fact that observed correlations were significant to this degree despi te variability among subjects supports the interpretation that these correlat ions are real. A s expected, in vivo P E T B P measurements obtained with presynapt ic radiotracers ( [ 1 1 C]DTBZ, [ 1 1 C ] M P ) correlated significantly with binding measurements obtained in vitro by A R G using the s a m e radiotracers and [ 3 H]WIN 35, 428 . The notably strong and significant correlation between A R G binding va lues obtained using [ 3H]WIN 35, 428 and those obtained using [ 1 1 C ] M P was also expected. T h e error due to inter-subject 88 variability and physical di f ferences between multi-sensit ive and trit ium-sensitive storage phosphor sc reens may partially account for dev iance from a perfect correlation (p=1.0). Dif ferences intrinsic to the tracers may have a lso contributed, s ince absolute values of specif ic binding obtained with either tracer produced unexpected results; va lues obtained with [ 1 1 C ] M P were twice as high as those obtained with [ 3H]WIN 35,428. Th is finding conflicts with that of a study in mice, where [ 3 H]WIN 35, 428 binding values were twice as high as [ 1 1 C ] M P binding va lues (Gatley et a l . , 1995). Diverging results may be due to di f ferences in exper imental methods. Gat ley et a l . (1995) injected live mice with radioactive tracer, s o binding occurred in vivo, where the tracer had a c c e s s only to those transporters located on cell sur faces. In the present study, however, binding was performed in vitro and tracer had a c c e s s to all transporters exposed by sect ioning. S ince internalization is the primary method of D A T regulation, sect ioning may have exposed internalized transporters that exhibited different l igand affinity in vivo (Little et al . , 2002). Th is hypothesis is supported by the fact that pH and ion concentration are different inside the cell than outside and these factors affect l igand binding kinetics (Phelps et al . , 1986;Pol lak and Whar ton, 1993). It is poss ib le that internalized D A T s have lower l igand affinity, which might lead to dec reased in vivo binding of the highly reversible M P , but increased A R G binding under consistent, optimal in vitro binding condit ions. A n alternate explanat ion is the difference in incubation concentrat ion relative to the binding affinities of W I N and M P for DAT . It has been shown that M P and WIN have different affinities for D A T , but the reason behind this di f ference is unknown (Schweri et al . , 1992;Gat ley et a l . , 1995). They may bind distinct si tes on DAT , or their chemica l structures may lead to different binding kinetics. The latter hypothesis is supported by the finding that M P binding is more reversible than W I N 35,428 binding to D A T (Gatley et al . , 1995). S i nce A R G exper iments in this study used both tracers at the s a m e incubation concentrat ion, their relative incubation concentrat ion to affinity ratios were different. S ince M P and W I N 35,428 are bel ieved to have different affinities for DAT , binding va lues obtained by incubating t issue in the s a m e concentrat ion of each tracer should be different, as w a s observed. Unfortunately this point w a s not real ized until after experimentat ion and thus the present study cannot provide conc lus ive results about the relationship between M P and W I N binding in vitro. Future A R G studies using incubation 89 concentrat ions relative to the binding affinity of each tracer would help clarify the relationship between M P and WIN 35, 428 binding in vitro. A l though it is often assumed in the literature that these two methods produce equivalent data, this assumpt ion has not been val idated (Kaufman and Madras , 1992a;Frost et a l . , 1993;Gat ley et a l . , 1995;Ding et a l . , 1995). Performing in vivo or in vitro saturation a s s a y s with [ 1 1 C ] M P and [ 1 1 C]WIN 35, 428 or their tritiated counterparts would avoid conflicting data from radioistope-induced variation and might clarify the binding relationship between these two l igands. A s predicted, presynapt ic tracer binding both in vivo and in vitro correlated significantly with clinical severity. Behavioura l scores correlated more strongly with P E T B P values than with A R G binding va lues, which is logical consider ing the condit ions under which radiotracer interactions occur in both situations. P E T and behavioural assessmen ts are both performed in living subjects and measurements reflect interactions of several neural sys tems. A R G , on the other hand, examines tracer binding in isolated postmortem t issue sect ions in artificial environments. F e w studies have compared in vivo and in vitro binding using P E T tracers with behavioural data, but, in agreement with the present study, strong correlat ions have been observed between [ 1 1 C ] D T B Z A R G binding and behaviour in 6 - O H D A rats (Strome et a l . , 2006). P E T data using presynaptic tracers has a lso been shown to correlate strongly with clinical severity in human P D patients, M P T P non-human primates, and 6 - O H D A rats (Eberl ing et al . , 1999;Lee et a l . , 2 0 0 0 A u et al . , 2005;lnaj i et al . , 2005 ;Bohnen et a l . , 2006). The present study suggests that use of in vivo P E T and in vitro binding va lues using [ 1 1 C ] D T B Z and [ 1 1 C ] M P and in vitro using [ 3H]WIN 35, 428 are valid techniques for assess ing clinical severity. This study supports the prevail ing opinion in the literature that binding with [ 1 1 C ] D T B Z is the best indicator of P D symptom severity. This finding is consistent with human studies in P D patients, as well as with rodent and non-human primate models of P D (Lee et al . , 2000;Doudet et a l . , 2006;St rome et al . , 2006 ;Bohnen et a l . , 2006 ;Soss i et al . , 2007). Cl in ical severity of P D symptoms was more highly correlated to [ 1 1 C ] D T B Z binding in vivo and in vitro than to binding with any other radiotracer. Prev ious studies have establ ished correlat ions between defined behavioural groups; for example , control with 90 early and late stage P D groups, or control with symptomat ic and asymptomat ic groups (Doudet et a l . , 2006 ;Bohnen et al . , 2006). The present study extends these findings to include M P T P monkeys exhibiting a wide range of lesion severity. Binding exper iments both in vivo and in vitro with radiotracers targeting D1 and D2 receptors demonstrated drastically different effects of M P T P administration on the postsynapt ic D A sys tem. The lack of significant correlation between P E T B P values and A R G binding va lues for all postsynapt ic tracers except [ 3H]raclopride was unexpected. One likely contributing c a u s e of failure to detect signif icant correlat ions is that B P and binding va lues obtained in vivo and in vitro respectively with radiolabeled raclopride and S C H - 2 3 3 9 0 fell within smal l numerical ranges. Therefore, it is poss ib le that a significant correlation does exist and was simply undetected because it is relatively weak and the smal l di f ferences in binding between severely- les ioned subjects and unlesioned subjects were not large enough to overcome the effects of significant inter-subject variability, anes thes ia , and the presence of endogenous l igand. Th is explanation for lack of correlation with in vivo and in vitro [ 1 1 C]raclopr ide binding is supported by the [ 3H]raclopride binding results. [ 3H]raclopride binding va lues measured by A R G did correlate significantly with [ 1 1 C]raclopr ide P E T B P va lues and compar ison of [ 1 1 C]raclopr ide binding in vivo and in vitro showed a similar trend. The weak correlation may a lso have been reduced further by the loss of presynaptical ly- located D2 autoreceptors with increased degenerat ion of the nigrostriatal neurons in which they reside. S ince greater nigrostriatal neuron degenerat ion is related to greater clinical severity, the slight upregulation of postsynaptic D2 receptors in subjects with greater lesion severity could have been partly masked by the loss of presynapt ic D2 receptors in measurements from either binding technique. Another cause of the di f ferences in P E T and A R G results using raclopride and S C H -23390 is the p resence of endogenous DA. It is well known that raclopride binding is inf luenced by extracel lular D A levels, but the sensitivity of S C H - 2 3 3 9 0 to synaptic D A levels is uncertain (Marshal l et a l . , 1989; lwata et a l . , 1996;Abi -Dargham et a l . , 1999;Laruel le, 2000 ;Sasak i et a l . , 2002). The results of this study suggest that individual response to M P T P administrat ion is highly variable with regards to the postsynapt ic terminal, especia l ly D1 receptor. However, the concentrat ion of extracellular D A may 91 play a role in D A receptor activity and cannot be ignored. Furthermore, endogenous D A and radiol igand must compete for D A receptor binding sites in vivo, whereas endogenous D A is washed away prior to binding in vitro, so this difference likely decreased correlat ions between P E T and A R G data. Wh i le D2 binding shows trends of correlating with cl inical severity that simply do not reach signi f icance, trends in the relationship between D1 binding and clinical severeity are inconsistent in vivo and in vitro. The lack of correlation between behavioural scores and B P measured in vivo by P E T using [ 1 1 C ] S C H - 2 3 3 9 0 for D1 is consistent with the literature suggest ing that terminals are relatively spared in MPTP- t rea ted animals (Bernheimer et a l . , 1973 ;German et al . , 1996). It is likely that the significant correlation detected between behaviour and binding values obtained by A R G using [ 3 H]SCH-23390 is an artifact caused by a single outlier representing the most severely lesioned subject. Th is subject was not exc luded because the a im of this study was to investigate as large a range of lesion severity as possib le. However, the data suggest that [ 3 H]SCH-23390 binding in the majority of subjects, especia l ly mi ldly- lesioned to moderately- lesioned subjects, is not linearly correlated to lesion severity. The distribution of binding va lues for the rest of the subjects fell within a smal l range of binding va lues, simi lar to the situation with raclopride binding. Rac lopr ide binding both in vivo and in vitro was expected to correlate weakly with behavioural scores , but no significant correlat ions were observed. This is also likely the result of the narrow dynamic range of raclopride binding and inability for binding di f ferences to overcome inter-subject variability to confirm a legitimate correlation in this smal l sample . The present study was performed to investigate the relationship between in vivo P E T and in vitro A R G and the ability of both techniques to a s s e s s the severity of clinical P D symptoms in the M P T P non-human primate model of P D . Whi le P E T evaluates a molecule under realistic condit ions in vivo, it is a lso affected by other variables that cannot be control led or measured , so cannot isolate the behaviour of the molecule of interest. A R G isolates the molecule and can provide an idea of what is happening to that molecule without interference from other transmitter sys tems. T h e s e techniques complement each other and defining the relationship between them can help 92 researchers gain more information in future exper iments using the techniques individually. The noninvasive nature of P E T is a major advantage that makes it extremely valuable for cl inical purposes. It can be used comfortably in humans with neurodegenerat ive d i sease to track their d i sease progression and their response to treatment interventions in order to identify best treatment opt ions. It would be beneficial to be able to measure actual physiological responses with a noninvasive technique such as P E T , but P E T data from a single scan represents multiple p rocesses and prior to this study, it was unknown how well P E T B P va lues correlated with actual physiological responses as measured by postmortem A R G using the s a m e presynaptic and postsynapt ic radiotracers in the s a m e individuals. The results of this study suggest that P E T data obtained using the presynaptic tracers [ 1 1 C ] D T B Z and [ 1 1 C ] M P are reasonably representat ive of actual physiological p rocesses as measured by A R G in the M P T P non-human primate model . The highly significant correlat ions between P E T and A R G data support the idea that either technique could be used on its own to provide similar information about the presynaptic molecule of interest. The highly significant correlat ions between in vivo and in vitro binding of these presynapt ic tracers and lesion severity in the M P T P monkey model of P D a lso support the potential use of P E T with [ 1 1 C ] D T B Z and [ 1 1 C ] M P to d iagnose D A terminal integrity and d i sease severity, as has been proposed in the literature, with [ 1 1 C ] D T B Z being the best option (Lee et a l . , 2000 ;Au et a l . , 2005;St rome et al. , 2006). However, further studies involving cell counts of surviving nigrostriatal D A neurons must be performed in order to confirm this correlation in humans and M P T P non-human primates. Whi le [ 3 H]MTBZ A R G has been shown to correlate with S N c neuron density in 6 - O H D A rats, this hypothesis has never been tested in humans or non-human primates (Vander Borght et a l . , 1995b). Unfortunately most of the S N t issue from the present subjects was lost in f reezer acc idents , but D A neuron counts (by T H immunohistochemistry) in the S N might validate the use of P E T with presynaptic tracers to evaluate the degree of D A neuron degenerat ion in P D patients. S N cell counts would be expected to correlate with striatal presynapt ic tracer binding values, s ince presynapt ic tracers are thought to bind the s a m e D A cel ls that have their cell bodies in the S N . If this correlation was observed, 93 1 it would have important implications in P D diagnosis and treatment. It would validate an objective method of determining the amount of surviving D A terminals in P D patients, tracking the progression of their d i sease with time, and evaluat ing the eff icacy of new P D treatments. Overal l , this information would support the validity of P E T using speci f ic presynaptic tracers of the D A system for evaluating the integrity of the DA system during progression of P D . Accura te assessmen t of d i sease stage would improve select ion of the best treatment option for max imum treatment benefit for individual patients (Romrel l et a l . , 2003;Tui te and R iss , 2003 ;Lees , 2005). S ince the correlation observed between P E T and behavioural evaluat ion techniques was not perfectly l inear throughout d isease progression, this study suggests that caution must be taken when extrapolating from measurements using a single technique to predict the actual physiological meaning of the data. Th is warning is especial ly important at the ext remes, such as at very high cl inical severity, where the correlation between [ 1 1 C ] D T B Z P E T and behavioural scores was different than at low to moderate clinical severity. Few studies have performed A R G using positron-emitting tracers to obtain quantitative data (Gatley et a l . , 1998;Strome et al . , 2005;St rome et a l . , 2006). Th is study supports the previous f indings in 6 - O H D A rats and humans that P E T tracers can be used in vitro to obtain quantitative data by A R G and extends the subject group to include M P T P monkeys. Methodologica l di f ferences exist among sites and s ince A R G binding values can differ by up to a full order of magnitude, they are difficult to compare directly across sites (Hall et a l . , 1988;Farde et al . , 1989). Resul ts of this study differ from results obtained at other sites and a lso highlight the large variation among individuals, thereby emphas iz ing that caut ion must be taken when compar ing results of P E T and A R G studies across si tes. The present study makes an obvious contribution to P D research by supporting the use of P E T with [ 1 1 C ] D T B Z and [ 1 1 C ] M P for assess ing cl inical severity of P D . It a lso supports the use of this technique throughout the course of d i sease progression, s ince significant correlat ions were detected between P E T B P va lues and a large range of clinical severi t ies. 94 The potential uses of P E T for early d iagnosis of brain d i s e a s e s as well as differential d iagnosis of d i seases that present with similar cl inical symptoms are presently intense topics in the field of P E T . Th is application could lead to recognit ion of d i seases before the onset of cl inical symptoms, allowing earlier treatment and potentially slowing or preventing the progression of d i sease in the future. It is important to understand exactly what P E T data represents and how it should be interpreted before it can be analyzed accurately, which is where A R G contributes. This study suggests that significant l inear relationships do exist between P E T and A R G for s o m e radiotracers, but that these relationships differ by tracer and by target molecule. It highlights the importance of fully investigating tracer binding in vivo and in vitro for e a c h individual tracer, preferably in the s a m e individual to limit sources of error and variability. S ince the use of both techniques in the s a m e individuals in this study did not el iminate all variation and correlation coeff icients never reached 1.0, it is appropriate to conc lude that P E T and A R G have inherent di f ferences and do not measure identical p rocesses . Nevertheless, the significant correlation between P E T and A R G measurements with some radiotracers supports the utility of A R G as a tool for identifying new P E T tracers. Al though this study demonstrates that in vitro binding by A R G is not a lways representative of the results that will be obtained by in vivo P E T with that tracer, as in the case of D1 and D2 receptors, the benefits of A R G in reducing time, cost, and number of an imals used outweigh the d isadvantage of not fully replicating in vivo condit ions. S o m e tracers, such as the presynaptic tracers investigated in this study, do behave similarly in vitro and in vivo too (Bergstrom et a l . , 2003). Development of new P E T tracers is occurr ing rapidly at present and is expected to al low application of P E T technology to addit ional cl inical and research fields in the future. For example, support for the use of P E T in drug development to confirm drug binding targets in the living human brain, a s s e s s the relationship between the degree of drug binding and pharmacodynamic effects, and ensure sufficient drug exposure to the brain is rapidly increasing (Lee and Farde, 2006;Lever , 2007). Another potential application of P E T is detection of b iomarkers of d isease . D T B Z - P E T has al ready been proposed as a biomarker for P D , s ince it al lows detection of substant ial precl inical pathology (Bohnen et al . , 2006). [ 1 8 F ] F D O P A is used clinically as a P E T biomarker for cancer and its use 95 has also been proposed for neurodegenerat ive d i seases including P D , A D , and HD (Snow et al . , 1993;Antonini et a l . , 1995b;Antonini et a l . , 1996;Kung et al . , 2004;Duet et al . , 2007; lsrae l and Kuten, 2007 ;Schoder and G o n e n , 2007). P E T has also been proposed for use in combinat ion with molecular genet ics techniques to provide indices of probable individual response to certain drugs such as ant i -depressants (Lee and Farde, 2006). A R G presents an invaluable tool for prel iminary evaluat ion of potential tracers for these purposes. However, this study a lso e m p h a s i z e s the importance of fully characterizing the behaviour of P E T radiotracers in vivo as part of the validation process, s ince s o m e tracers display significantly different ou tcomes in vivo than they do in vitro. Although neurodegenerat ive d i seases dominate a large portion of P E T research, other human condit ions involving the D A system can also benefit from the findings of this study. The most c o m m o n target for P E T radiol igands is the D2 receptor, which is thought to be involved in sch izophren ia , attention deficit hyperactivity disorder (ADHD) , depress ion, subs tance abuse , rest less legs syndrome, and many other condit ions (Farde et a l . , 1987;Vo lkow et a l . , 1997b;Volkow et a l . , 1999 ;Fuente -Fernandez et a l . , 2004 ;Townsend et a l . , 2004 ;Cervenka et a l . , 2006;Blodget t et a l . , 2007). This study reveals that P E T and A R G provide diverging results for D1 and D2 radiotracers in s o m e cases , though, especia l ly when the ligand or target is sensi t ive to endogenous l igand. It demonstrates the importance of characterizing the relationship between in vivo and in vitro binding before it is poss ib le to accurately interpret the results of either technique alone. This point can be appl ied to all P E T and A R G radiotracers. The utility of P E T is limited by its spatial resolution, so investigation of smal l subcort ical regions, as in rest less leg syndrome, is somet imes better served by A R G due to its high spatial resolution. However, P E T technology is rapidly progressing and with improvements in resolut ion, it may be possib le to investigate smal ler brain regions by P E T . A recent attempt to apply P E T to the study of rest less leg syndrome identified significant di f ferences between control and exper imental groups, supporting future potential for the use of P E T in investigation of smal l brain regions (Cervenka et al . , 2006). 96 Conclusions The present study contributes to the growing body of in vivo P E T and in vitro A R G studies investigating components of the D A system by directly compar ing results of both techniques in the s a m e individuals using identical radiotracers. Despi te the intrinsic dif ferences of P E T and A R G techniques, they provide comparab le measures that convey important information about the functioning of neural sys tems. The significant positive correlat ions observed between results of P E T and A R G using radiotracers speci f ic for presynapt ic components of the D A sys tem suggest that these techniques are measur ing similar p rocesses . They provide support for the use of P E T on its own with presynapt ic tracers to evaluate actual physiological p rocesses , which normally requires the concurrent use of A R G to confirm accurate interpretation of P E T findings. This study a lso supports the prevalent opinion in the literature that D T B Z binding is the best indicator of cl inical severity of all radiotracers investigated here. However, this study a lso emphas izes the importance of including postmortem techniques in the val idation of P E T image analys is . Signif icant dif ferences occur between in vivo and in vitro results obtained with s o m e tracers, such as the postsynapt ic tracers investigated in this study. T h e s e di f ferences are likely multifactorial, relating to severa l aspec ts of the distinct in vivo and in vitro environments, as well as experimental limitations such as the use of anesthet ics during P E T . T h e s e dif ferences also suggest that large variations exist among subjects and that response to M P T P varies greatly among individuals, which leads to significantly different experimental results from P E T and A R G exper iments and demands large samp le s izes in order to observe significant effects of M P T P treatment. The M P T P non-human primate model of P D is used extensively in P D research in the pursuit of understanding the d i sease and in the search for potential treatments. Th is study extends the utility of the M P T P non-human primate model to include evaluation of animals with a large range of lesion severity. The non invas iveness of P E T and its ability to provide longitudinal information about brain function in living, consc ious humans presents an invaluable tool for cl inicians. Th is study shows that A R G and P E T using the s a m e radiotracer show similar regional 97 distribution and highly correlated quantitative binding results for some tracers, supporting the use of ARG in development of novel PET tracers. With the rapid progression of PET technology, potential applications of this technique are vast and ARG will play an important role in translating this potential to the clinic in numerous fields. 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