설치류의 [18F]Mefway에서 뇌 섭취에 기여하는 P-glycoprotein의 효과

Dept. of Medicine/박사I. INTRODUCTION Efflux transporters in brain capillary endothelial cells impede xenobiotics, including therapeutic drugs and PET radioligands. Of these, P-gp (P-glycoprotein) and Bcrp (Breast cancer resistance protein) are prominent efflux pumps. P-gp is colocalized with Bcrp on the luminal membranes of brain capillary endothelial cells, and they have partly overlapping substrate specificities that include a wide range of compounds. Thus, to investigate the effects of P-gp, it is required to evaluate the impact of Bcrp. The aim of this study was to determine whether the brain uptake of [18F]Mefway is influenced by the action of P-gp and Bcrp in rodents. II. MATERIALS AND METHODS To effectively suppress in vivo defluorination, fluconazole (OneFlu, JW Pharmaceutial, Seoul, Korea) was used. [18F]Mefway was applied to the pharmacological inhibition rats and genetically disrupted mice. In rats, blocking of P-gp activity was performed by tariquidar (TQD). For the pharmacological inhibition experiments male Sprague-Dawley (n = 3 for each group) rats were used. For genetic disruption, male Mdr1a/b(-/-), Bcrp1(-/-), Mdr1a/b(-/-)Bcrp1(-/-), and wild-type (WT) mice were used (n = 4 for each group). III. RESULTS Pretreatment of TQD results in 160% higher hippocampal uptake compared with control rats. In genetically disrupted mice, a maximal brain uptake value of 3.2 SUV in the triple knockout mice (Mdr1a/b(-/-)Bcrp1(-/-) was comparable to that of the double knockout mice (Mdr1a/b(-/-)) and two-fold those of the wild type and Bcrp1(-/-) knockout mice. The differences of binding values were statistically insignificant between control and experimental groups. The brain-to-plasma ratios for triple knockout mice were also 2-5 times higher than those for other groups. IV. CONCLUSION In PET, chemical knockout P-gp disruption induced considerable increase of brain uptake of [18F]Mefway compared with control group or wild type. This result was substantiated by ex vivo experiments showing high brain-to-plasma ratios for P-gp disrupted mice. In conclusion, [18F]Mefway is modulated by P-gp, and not by Bcrp in rodents. I. 목적 뇌 모세혈관 내피 세포에서 유출 운반자들은 치료 약물들과 PET 방사성 리간드들을 포함한 생체이물질들을 방해한다. 이들 중에서 P-glycoprotein (P-gp)와 breast cancer resistance protein (Bcrp)는 가장 중요한 유출 운반자들이다. P-gp는 뇌 모세혈관 내피 세포의 관강내막에 Bcrp와 함께 발현이 되어 있으며 일부 기질 특이성을 공유하고 있다. 따라서 P-gp의 효과를 알아보기 위해서는 Bcrp의 효과를 알아볼 필요성이 있다. 본 연구의 목적은 설치류의 [18F]Mefway에서 P-glycoprotein (P-gp)와 breast cancer resistance protein (Bcrp)의 뇌섭취에 관련된 효과를 알아보고자 하였다. II. 대상 및 방법 탈불소화를 효과적으로 억제시키기 위하여 fluconazole (OneFlu, JW Pharmaceutial, 서울, 대한민국)를 사용하였다. [18F]Mefway를 약물학적으로 억제시킨 쥐와 유전적으로 변형시킨 생쥐에게 적용을 하였다. 쥐에서는 tariquidar(TQD)를 이용하여 P-gp 기능을 억제 시켰다. 약물학적으로 억제시키는 실험을 위해서는 수컷 Sprague-Dawley (각 그룹 당 세 마리) 쥐를 사용하였다. 유전적 변형을 위하여서는 수컷 Mdr1a/b(-/-), Bcrp1(-/-), Mdr1a/b(-/-)Bcrp1(-/-), 그리고 대조군 생쥐가 사용되었다 (각 그룹 당 네 마리). III. 결과 TQD를 전처치함으로써 대조군 쥐와 비교 시 해마의 뇌 섭취가 160% 증가되었다. 유전적으로 변형된 생쥐에서, 최대 뇌 섭취가 3.2 SUV인 삼중 유전자 결손 생쥐 (tKO, Mdr1a/b(-/-)Bcrp1(-/-) 와 비교 시 이중 유전자 결손 생쥐 (dKO, Mdr1a/b(-/-)) 와 최대 뇌 섭취가 비슷하였으며 대조 군과 Bcrp1(-/-) 유전자 결손 생쥐와 비교 시 두 배 높은 최대 뇌 섭취를 보였다. 대조 군과 실험 군간의 결합 가치의 차이는 통계적으로 의미가 없는 것으로 나타났다. [18F]Mefway 농도의 뇌와 혈장간 비율은 다른 그룹과 비교 시 삼중 유전자 결손 생쥐에서 2.5배 높은 것으로 확인되었다. IV. 결론 PET에서 화학적으로 P-gp 결여된 경우 대조군과 비교시 [18F]Mefway의 뇌에서 섭취도가 매우 많이 증가되었다. 이러한 결과는 P-gp 결손 생쥐에서도 높은 뇌-혈장간 비율을 보인 실험 결과에서도 입증되었다. 결론적으로 [18F]Mefway는 설치류에서 Bcrp가 아닌 P-gp에 의해서 조절이 된다.ope

In vivo and in vitro studies of the serotonin 1B receptor in relation to major depressive disorder and treatment with ketamine

Despite the large implications of Major Depressive Disorder (MDD) on disease burden worldwide, current treatment options are suboptimal and a third of patients suffering from this disease do not respond to treatment. Therefore, an unmet need exists for the development of new treatment options and methods to aid appropriate treatment selection in individual patients. Selection of suitable biomarkers and reliable quantification methods are essential steps in this process. In recent research on MDD, more interest has arisen for the serotonin 1B (5-HT1B) receptor and for ketamine as a new antidepressant treatment option. This thesis focuses on the involvement of the 5-HT1B receptor and the related protein p11 in the pathophysiology of MDD and the antidepressant mechanism of action of ketamine. For quantification of 5-HT1B receptor densities, the nuclear imaging techniques Autoradiography (ARG) and Positron Emission Tomography (PET) were used. This work includes the development and application of an improved method for quantification of 5-HT1B receptor binding using PET. Quantification of p11 levels was performed in specific cell populations using Flow Cytometry. In study I, 5-HT1B receptor binding densities and cortical distribution were examined using ARG in anterior cingulate cortex tissue of subjects with MDD, schizophrenia, bipolar disorder and healthy controls. Binding of the radioligand [3H]AZ10419369 in tissue of in total 52 subjects showed no significant differences between the subject groups. A distribution pattern with higher 5-HT1B receptor binding in supragranular layer compared to the infragranular layer was found, which correlated with glutamatergic N-methyl-D-aspartate receptor distribution. Female subjects had lower 5-HT1B receptor densities than male subjects, which was mostly profound in the MDD group. In study II, an improved method was developed for delineation of Volumes of Interest (VOIs) for PET data with the radioligand [11C]AZ10419369. Based on a 3D [3H]AZ10419369 ARG model in post mortem brainstem tissue and literature findings, appropriate VOIs for quantification in PET were selected. Two previously developed semi-automatic VOI delineation methods, based on template or individual data, were evaluated on test-retest data of 8 healthy subjects and showed improved reliability compared to a conventional manual VOI. The VOIs created with PET template data of 52 healthy subjects can be automatically applied to future PET studies measuring 5-HT1B receptor binding in the brainstem. Furthermore, in a randomized placebo-controlled study the effect of ketamine on cerebral [11C]AZ10419369 PET binding (study III) and peripheral p11 protein levels measured with Flow Cytometry (study IV) were examined in patients with Selective Serotonin Reuptake Inhibitor (SSRI) resistant MDD. An increase in 5-HT1B binding in the hippocampus and a decrease in p11 levels in both cytotoxic T cells and T-helper cells populations were seen in the ketamine group (n=20), although both did not differ from changes seen in the placebo group (n=10). Changes in Montgomery-Åsberg Depression Rating Scale (MADRS) score after ketamine treatment correlated significantly with baseline 5-HT1B receptor binding in the ventral striatum and baseline p11 levels in cytotoxic T cells. Future studies should be conducted on the role of 5-HT1B receptors and p11 in the antidepressant mechanism of action of ketamine and should clarify if these proteins could be used as biomarkers to predict ketamine treatment response in subjects with SSRI-resistant MDD

PET studies of the serotonin system in major depression and its treatment

The serotonin system has been implicated in major depression since the 1960s, mainly based on the serotonin enhancing properties of antidepressants. Positron emission tomography, PET, is the in vivo molecular imaging method with the best spatial resolution. There has been a gradual development of suitable radioligands for serotonergic targets since the mid-1990s, widening the scope of PET studies of the serotonin system. MADAM is an established radioligand selective for the serotonin transporter, and AZ10419369 is selective for the 5-HT1B receptor. The aim of this thesis was to study the serotonin system in major depressive disorder and the serotonergic effects of treatment with antidepressive medication or with psychotherapy. In order to understand the results with AZ10419369 better we examined the sensitivity of this radioligand to baseline serotonin levels. In studyI we examined serotonin transporter occupancy with PET and MADAM in responders to treatment with seven different antidepressants in different doses. Two tricyclic antidepressants (TCAs) and four selective serotonin reuptake inhibitors (SSRIs) were examined. Mirtazapine was included as a serotonin transporter “dummy”. Serotonin transporter occupancy could be confirmed in vivo for all TCAs and SSRIs. There was no significant difference in serotonin transporter occupancy between the old antidepressants, TCAs, and the new, SSRIs. Mirtazapine did not occupy the serotonin transporter. The average serotonin transporter occupancy in SSRIs and TCAs was 67 %, which was significantly lower than the 80 % serotonin transporter occupancy previously postulated important for SSRI effect. In study II we investigated the effect ofinternet-delivered cognitive behavioural therapy (CBT) for recurrent major depressive disorder on AZ10419369 binding. Ten patients with an ongoing and untreated major depressive episode finished the study according to protocol and were examined with PET and AZ10419369 before and after CBT. All patients responded to treatment. The binding potential, BPND, was reduced by 33 % in the dorsal brain stem, which included the raphe nuclei, from which the serotonergic neurons project. Since the 5-HT1B receptor acts inhibitory, a reduction of 5-HT1B receptor density in the raphe nuclei would in theory result in a general stimulation of the serotonin system. There were no other significant changes in radioligand binding in the brain with CBT. In study III we wanted to compare AZ10419369 binding in patients with an ongoing and untreated major depressive episode within recurrent major depressive disorder with age- and sex matched controls. Ten patients and ten controls were examined with PET and AZ10419369. AZ10419369 binding was lower in the anterior cingulate cortex (25 % lower) and associated regions (20 % lower in the subgenual prefrontal cortex and 45 % lower in the hippocampus). The difference in the anterior cingulate cortex survived Bonferroni correction for multiple comparisons. The anterior cingulate cortex is an established part of the neurocircuitry of depression. There were no significant differences in the other examined brain regions. 3 In study IV we correlated AZ10419369 binding with concentrations of serotonin and its metabolite 5-Hydroxyindoleacetic Acid (5-HIAA) in the cerebrospinal fluid (CSF) at baseline in healthy subjects. Twelve healthy subjects without psychiatric history were first examined with PET and AZ10419369 and then with lumbar puncture for CSF analysis. The CSF concentrations of serotonin and 5-HIAA were determined with high performance liquid chromatography. There were no significant correlations between levels of serotonin and 5-HIAA in the CSF and AZ10419369 binding in the whole brain, in the caudate nucleus or in the occipital cortex. Since correlations between CSF and brain concentrations of serotonin and 5-HIAA have been demonstrated, AZ10419369 binding at baseline likely reflects 5-HT1B receptor density. This has bearing for the interpretation of study II and III

Molecular and Functional Imaging Studies of Psychedelic Drug Action in Animals and Humans

Hallucinogens are a loosely defined group of compounds including LSD, N,N-dimethyltryptamines, mescaline, psilocybin/psilocin, and 2,5-dimethoxy-4-methamphetamine (DOM), which can evoke intense visual and emotional experiences. We are witnessing a renaissance of research interest in hallucinogens, driven by increasing awareness of their psychotherapeutic potential. As such, we now present a narrative review of the literature on hallucinogen binding in vitro and ex vivo, and the various molecular imaging studies with positron emission tomography (PET) or single photon emission computer tomography (SPECT). In general, molecular imaging can depict the uptake and binding distribution of labelled hallucinogenic compounds or their congeners in the brain, as was shown in an early PET study with N1-([11C]-methyl)-2-bromo-LSD ([11C]-MBL); displacement with the non-radioactive competitor ketanserin confirmed that the majority of [11C]-MBL specific binding was to serotonin 5-HT2A receptors. However, interactions at serotonin 5HT1A and other classes of receptors and pleotropic effects on second messenger pathways may contribute to the particular experiential phenomenologies of LSD and other hallucinogenic compounds. Other salient aspects of hallucinogen action include permeability to the blood–brain barrier, the rates of metabolism and elimination, and the formation of active metabolites. Despite the maturation of radiochemistry and molecular imaging in recent years, there has been only a handful of PET or SPECT studies of radiolabeled hallucinogens, most recently using the 5-HT2A/2C agonist N-(2[11CH3O]-methoxybenzyl)-2,5-dimethoxy- 4-bromophenethylamine ([11C]Cimbi-36). In addition to PET studies of target engagement at neuroreceptors and transporters, there is a small number of studies on the effects of hallucinogenic compounds on cerebral perfusion ([15O]-water) or metabolism ([18F]-fluorodeoxyglucose/FDG). There remains considerable scope for basic imaging research on the sites of interaction of hallucinogens and their cerebrometabolic effects; we expect that hybrid imaging with PET in conjunction with functional magnetic resonance imaging (fMRI) should provide especially useful for the next phase of this research

The role of serotonergic and dopaminergic mechanisms and their interaction in Levodopa-induced dyskinesias

Long–term levodopa treatment in Parkinson’s disease (PD) is commonly associated with troublesome levodopa–induced dyskinesias (LIDs). Striatal serotonergic terminals amid the degenerating dopaminergic ones are proposed to play an important role in LIDs by taking up exogenous levodopa and releasing dopamine in an unregulated fashion. However, to date, the underlying mechanisms of LIDs are not fully understood. By using single photon emission computed tomography (SPECT) with 123I–Ioflupane and positron emission tomography (PET) with 11C–DASB and 11C–PE2I, the clinical studies conducted for this thesis aimed (a) to estimate the role of striatal dopamine transporter (DAT) availability in early PD as a prognostic marker for LIDs, (b) to explore whether striatal DAT availability changes over time are related to the appearance of LIDs, (c) to estimate the role of striatal serotonin-to-dopamine transporter (SERT–to–DAT) binding ratios to LIDs, and (d) to look for a relation between the changes in striatal SERT, DAT and SERT–to–DAT binding ratios over time and the appearance of LIDs. The main findings are as follows: (a) in early PD, striatal DAT availability alone does not predict the appearance of future LIDs, (b) at later stages, the occurrence of LIDs may be dependent on the magnitude of DAT decline in the putamen, (c) the SERT–to–DAT binding ratio in the putamen is increased in PD patients as compared to controls, and within PD, it is higher in patients with LIDs as compared to nondyskinetic patients, (d) as PD continues to progress, putaminal serotonergic terminals remain relatively unchanged in comparison to the dopaminergic ones and the aforementioned imbalance (as reflected by the binding ratio) increases over time. These findings provide fundamental insight in the pathophysiology of LIDs and have direct implications for further research towards novel therapeutics in PD dyskinesia.Open Acces

Development of 18F-labeled agonist radioligands for PET imaging of the high-affinity state of cerebral dopamine D2/3 receptors

Positronemissietomografie (PET) is een krachtige techniek voor medische beeldvorming. Met behulp van radioactief gemerkte verbindingen bekend als tracers maakt PET het mogelijk om fysiologische processen in het levende lichaam te visualiseren en kwantitatief te omschrijven. Dopamine is een belangrijke neurotransmitter - een verbinding gebruikt door hersenneuronen om met elkaar te communiceren. Dergelijke communicatie (signalering) vindt plaats wanneer dopamine uit één neuron vrijkomt en aan de receptoren op een andere neuron bindt. Er wordt verondersteld dat het aantal en de functie van dopaminereceptoren in veel neuropsychiatrische stoornissen zoals de ziekte van Parkinson, schizofrenie en verslaving, anders is dan in de gezonde staat. Daarom kan de beeldvorming van dopaminereceptoren door PET gebruikt worden om neuropsychiatrische aandoeningen te diagnosticeren en / of erachter te komen hoe ze zich ontwikkelen. Dopaminereceptoren die behoren tot de tweede en derde (D2/3) subtypes zijn het vaakst afgebeeld door PET. Receptoren van elk subtype kunnen in twee omkeerbare toestanden bestaan, met hoge en lage affiniteit voor dopamine. Volgens één hypothese is de hoge affiniteitstoestand de "functionele" deelverzameling van alle receptoren, die voornamelijk verantwoordelijk is voor de signalering. In dit geval zou de beeldvorming van alleen de hoge affiniteitstoestand meer waardevolle informatie over de toestand van de hersenen moeten opleveren dan de beeldvorming van beide affiniteitsstaten tegelijk. De meeste bestaande PET-tracers voor dopaminereceptoren kunnen echter tussen de affiniteitstoestanden niet onderscheiden. In dit onderzoeksproject hadden we als doel om nieuwe PET-tracers te ontwikkelen die de hoge affiniteitstoestand van D2/3 dopaminereceptoren zouden herkennen en geschikt zouden zijn voor wijdverspreid klinisch gebruik

Development of novel radiotracers as tools for imaging the human brain

Introduction: Brain imaging using single photon emission computed tomography (SPECT) or positron emission tomography (PET) can be used to study the processes underlying neurological and psychiatric disorders. In addition, in vivo brain imaging using SPECT or PET may provide new approaches for drug target identification, pre-clinical testing and occupancy studies, and therefore improve drug discovery. The utility of in vivo brain imaging using SPECT or PET relies on the ability of different radiotracers (typically organic compounds labelled with radionuclides) to bind to a wide variety of targets, including receptors, transporters and enzymes. Therefore the development of novel radiotracers for in vivo brain imaging using SPECT of PET is of vital importance. This thesis is focused on the process of developing novel radiotracers as tools for imaging the human brain, where the radiotracer discovery and development pipeline is discussed and each step prior to clinical trials investigated. Radiotracer discovery: Previously, discovery of novel brain radiotracers has largely relied on simplistic screening tools. Improved selection methods at the early stages of radiotracer discovery and an increased understanding of the relationships between in vitro physicochemical and in vivo radiotracer properties are needed. This thesis investigated if high performance liquid chromatography (HPLC) methodologies could provide criteria for lead candidate selection by comparing HPLC measurements with radiotracer properties in humans. In this study, ten molecules, previously used as radiotracers in humans, were analysed to obtain the following measures: partition coefficient (Log P); permeability (Pm); percentage of plasma protein binding (%PPB); and membrane partition coefficient (Km). Relationships between brain entry measurements (Log P, Pm and %PPB) and in vivo brain percentage injected dose (%ID); and Km and specific binding in vivo (BPND) were investigated. Results showed that HPLC measurements of Pm, %PPB and Km were potentially useful in predicting in vivo performance and hence allow evaluation and ranking of compound libraries for the selection of lead radiotracer candidates at early stages of radiotracer discovery. The HPLC tool developed provides information on in vivo non-specific binding and binding potential that is not possible using conventional screening methods. Another important finding reported in this thesis is that Log P should not be relied on as a predictor of brain entry. The HPLC tool developed, together with competition binding assays, was used to characterise a newly synthesised library of compounds for imaging of the translocator protein (TSPO) in brain using SPECT. Results showed that compound LS 1 was the most likely to succeed within the library investigated, but the high %PPB observed for LS 1 suggested novel compounds with improved %PPB were needed. Thus, a novel library of compounds for imaging of TSPO in brain using SPECT is currently been developed for future testing using the HPLC tool developed here and competition binding assays. Pre-clinical research: radiotracers for imaging the noradrenaline transporter (NAT) in brain using SPECT. In this thesis, NKJ64, a novel iodinated analogue of reboxetine, was successfully radiolabelled via electrophilic iododestannylation and evaluated as a potential SPECT radiotracer for imaging the NAT in brain using rodents and non-human primates. Biological evaluation of the novel radiotracer, 123/125I-NKJ64, in rodents included: in vitro ligand binding assays; in vitro and ex vivo autoradiography; in vivo biodistribution studies and ex vivo pharmacological blocking studies. In rats, 123/125I-NKJ64 displayed saturable binding with nanomolar affinity for the NAT in cortical homogenates, regional distribution consistent with the known density of NAT in the rodent brain and high maximum brain uptake of around 2.93 % of the injected dose. The specific: non-specific ratio (locus coeruleus:caudate putamen) of 123I-NKJ64 uptake was 2.8 at 30 minutes post intravenous injection and prior administration of reboxetine significantly reduced the accumulation of 123I-NKJ64 in the locus coeruleus (> 50% reduction). Data obtained using rodents indicated that further evaluation of 123I-NKJ64 in non-human primates was needed to determine its utility as a SPECT radiotracer for imaging of NAT in brain. Consequently, in vivo kinetic modelling studies using SPECT imaging with 123I-NKJ64 and two baboons were carried out to determine 123I-NKJ64 brain binding kinetics, brain distribution and plasma metabolism in non-human primates. Even though a high brain uptake of around 3.0% of the injected dose was determined, the high non-specific binding observed throughout the brain, a low binding potential (BPND<2) in NAT rich regions and a brain distribution that was inconsistent with the known NAT distribution in non-human primate brain precludes the translation of 123I-NKJ64 into humans. Another NAT radiotracer, 123I-INER, developed by Tamagnan and colleagues at Yale University and Institute for Degenerative Disorders, New Haven, USA, was also investigated as part of this thesis. Kinetic modelling analysis of 123I-INER in baboon brain was investigated for different models, namely invasive and reference tissue models. Bolus plus constant infusion experiments with displacement at equilibrium using six different doses of atomoxetine and four different doses of reboxetine were carried out in several baboons to obtain occupancy measurements as a function of injected dose (mg/kg) for the two NAT selective drugs. Results showed that reference tissue models were able to determine BPND values of 123I-INER in different brain regions. In addition the volume of distribution could be determined by dividing concentration in tissue by the concentration in venous blood at 3 hours post-injection. After administration of atomoxetine or reboxetine, dose-dependent occupancy was observed in brain regions known to contain high densities of NATs. Results supported the translation of 123I-INER into humans studies, despite the slow kinetics determined over the imaging period. Pharmacokinetic properties of 123I-INER described in this thesis may be used to simplify future data acquisition and image processing. Conclusion In conclusion, this thesis reported: (1) the development of novel radiotracers for brain imaging, namely NAT and TSPO; and (2) the development of a new methodology for aiding lead molecule identification at early stages of radiotracer discovery (i.e. prior to radiolabelling). In addition, an overview of radiotracer discovery and development process is provided in a single document, with a focus on brain radiotracers

Serotonin and motherhood: From molecules to mood

Emerging research points to a valuable role of the monoamine neurotransmitter, serotonin, in the display of maternal behaviors and reproduction-associated plasticity in the maternal brain. Serotonin is also implicated in the pathophysiology of numerous affective disorders and likely plays an important role in the pathophysiology of maternal mental illness. Therefore, the main goals of this review are to detail: 1) how the serotonin system of the female brain changes across pregnancy and postpartum; 2) the role of the central serotonergic system in maternal caregiving and maternal aggression; and 3) how the serotonin system and selective serotonin reuptake inhibitor medications (SSRIs) are involved in the treatment of maternal mental illness. Although there is much work to be done, studying the central serotonin system’s multifaceted role in the maternal brain is vital to our understanding of the processes governing matrescence and the maintenance of motherhood

Molecular imaging of dopamine synthesis and release

Positron emission tomography (PET) can be used to measure striatal dopamine synthesis and release, both of which have been shown to be elevated in schizophrenia. One study has demonstrated that first degree relatives of schizophrenia patients exhibit increased dopamine synthesis capacity, suggesting this could be an endophenotype or susceptibility marker. However, the specific relation to schizophrenia was not tested, as the index cases were not studied. In this thesis, I directly tested the hypothesis that both members of twin pairs discordant for schizophrenia show similar increases in dopamine synthesis capacity. I found that striatal dopamine synthesis capacity is not elevated in individuals at genetic risk of schizophrenia or in stable patients with chronic schizophrenia, suggesting that it is not a vulnerability marker for schizophrenia, and is associated with active psychosis only. I also tested whether dopamine synthesis capacity is elevated in otherwise healthy people who report hallucinations. No elevation was found, suggesting that the underlying neurobiology is distinct from schizophrenia. I then considered whether it would be possible to examine similar relationships with measurements of dopamine release. Methodologies for this measurement were still limited: antagonist radioligands such as [11C] raclopride have been used, but the dynamic range for the measure is small, confounding precision. I hypothesised that agonist radioligands could provide a more sensitive measure. [11C]-(+)-4-propyl-3,4,4a,5,6,10b-hexahydro-2H-naphtho[1,2-b][1,4]oxazin-9-ol ([11C]-(+)-PHNO) is a D2/D3 agonist PET radioligand. I directly compared the sensitivity of [11C]-(+)-PHNO to amphetamine challenge with that of the antagonist ligand [11C] raclopride. Mass carry-over and cerebellar binding were potential problems with [11C]-(+)-PHNO. I therefore designed a study to quantify these factors. I found that [11C]-(+)-PHNO is superior to [11C]raclopride for studying acute fluctuations in dopamine in the striatum. Use of [11C]-(+)-PHNO will allow quantification of smaller changes in dopamine release, although mass effects and displaceable cerebellar binding are potential confounding factors