Biodistribution and dosimetry in human healthy volunteers of the PET radioligands [11C]CHDI-00485180-R and [11C]CHDI-00485626, designed for quantification of cerebral aggregated mutant huntingtin.

PURPOSE: Huntington's disease is caused by a trinucleotide expansion in the HTT gene, which leads to aggregation of mutant huntingtin (mHTT) protein in the brain and neurotoxicity. Direct in vivo measurement of mHTT aggregates in human brain parenchyma is not yet possible. In this first-in-human study, we investigated biodistribution and dosimetry in healthy volunteers of [11C]CHDI-00485180-R ([11C]CHDI-180R) and [11C]CHDI-00485626 ([11C]CHDI-626), two tracers designed for PET imaging of aggregated mHTT in the brain that have been validated in preclinical models. METHODS: Biodistribution and radiation dosimetry studies were performed in 3 healthy volunteers (age 25.7 ± 0.5 years; 2 F) for [11C]CHDI-180R and in 3 healthy volunteers (age 35.3 ± 6.8 years; 2 F) for [11C]CHDI-626 using sequential whole-body PET-CT. Source organs were delineated in 3D using combined PET and CT data. Individual organ doses and effective doses were determined using OLINDA 2.1. RESULTS: There were no clinically relevant adverse events. The mean effective dose (ED) for [11C]CHDI-180R was 4.58 ± 0.65 µSv/MBq, with highest absorbed doses for liver (16.9 µGy/MBq), heart wall (15.9 µGy/MBq) and small intestine (15.8 µGy/MBq). Mean ED for [11C]CHDI-626 was 5.09 ± 0.06 µSv/MBq with the highest absorbed doses for the gallbladder (26.5 µGy/MBq), small intestine (20.4 µGy/MBq) and liver (19.6 µGy/MBq). Decay-corrected brain uptake curves showed promising kinetics for [11C]CHDI-180R, but for [11C]CHDI-626 an increasing signal over time was found, probably due to accumulation of a brain-penetrant metabolite. CONCLUSION: [11C]CHDI-180R and [11C]CHDI-626 are safe for in vivo PET imaging in humans. The estimated radiation burden is in line with most 11C-ligands. While [11C]CHDI-180R has promising kinetic properties in the brain, [11C]CHDI-626 is not suitable for human in vivo mHTT PET due to the possibility of a radiometabolite accumulating in brain parenchyma. TRIAL REGISTRATION: EudraCT number 2020-002129-27. CLINICALTRIALS: gov NCT05224115 (retrospectively registered).

Development of a ligand for in vivo imaging of mutant huntingtin in Huntington's disease.

Huntington's disease (HD) is a dominantly inherited neurodegenerative disorder caused by a CAG trinucleotide expansion in the huntingtin (HTT) gene that encodes the pathologic mutant HTT (mHTT) protein with an expanded polyglutamine (polyQ) tract. Whereas several therapeutic programs targeting mHTT expression have advanced to clinical evaluation, methods to visualize mHTT protein species in the living brain are lacking. Here, we demonstrate the development and characterization of a positron emission tomography (PET) imaging radioligand with high affinity and selectivity for mHTT aggregates. This small molecule radiolabeled with 11C ([11C]CHDI-180R) allowed noninvasive monitoring of mHTT pathology in the brain and could track region- and time-dependent suppression of mHTT in response to therapeutic interventions targeting mHTT expression in a rodent model. We further showed that in these animals, therapeutic agents that lowered mHTT in the striatum had a functional restorative effect that could be measured by preservation of striatal imaging markers, enabling a translational path to assess the functional effect of mHTT lowering.

Synaptic Vesicle Glycoprotein 2A Is Affected in the Central Nervous System of Mice with Huntington Disease and in the Brain of a Human with Huntington Disease Postmortem.

Synaptic dysfunction is a primary mechanism underlying Huntington disease (HD) progression. This study investigated changes in synaptic vesicle glycoprotein 2A (SV2A) density by means of 11C-UCB-J small-animal PET imaging in the central nervous system of mice with HD. Methods: Dynamic 11C-UCB-J small-animal PET imaging was performed at clinically relevant disease stages (at 3, 7, 10, and 16 mo) in the heterozygous knock-in Q175DN mouse model of HD and wild-type littermates (16-18 mice per genotype and time point). Cerebral 11C-UCB-J analyses were performed to assess genotypic differences during presymptomatic (3 mo) and symptomatic (7-16 mo) disease stages. 11C-UCB-J binding in the spinal cord was quantified at 16 mo. 3H-UCB-J autoradiography and SV2A immunofluorescence were performed postmortem in mouse and human brain tissues. Results:11C-UCB-J binding was lower in symptomatic heterozygous mice than in wild-type littermates in parallel with disease progression (7 and 10 mo: P < 0.01; 16 mo: P < 0.0001). Specific 11C-UCB-J binding was detectable in the spinal cord, with symptomatic heterozygous mice displaying a significant reduction (P < 0.0001). 3H-UCB-J autoradiography and SV2A immunofluorescence corroborated the in vivo measurements demonstrating lower SV2A in heterozygous mice (P < 0.05). Finally, preliminary analysis of SV2A in the human brain postmortem suggested lower SV2A in HD gene carriers than in controls without dementia. Conclusion:11C-UCB-J PET detected SV2A deficits during symptomatic disease in heterozygous mice in both the brain and the spinal cord and therefore may be suitable as a novel marker of synaptic integrity widely distributed in the central nervous system. On clinical application, 11C-UCB-J PET imaging may have promise for SV2A measurement in patients with HD during disease progression and after disease-modifying therapeutic strategies.

Longitudinal preclinical evaluation of the novel radioligand [11C]CHDI-626 for PET imaging of mutant huntingtin aggregates in Huntington's disease.

PURPOSE: As several therapies aimed at lowering mutant huntingtin (mHTT) brain levels in Huntington's disease (HD) are currently being investigated, noninvasive positron emission tomography (PET) imaging of mHTT could be utilized to directly evaluate therapeutic efficacy and monitor disease progression. Here we characterized and longitudinally assessed the novel radioligand [11C]CHDI-626 for mHTT PET imaging in the zQ175DN mouse model of HD. METHODS: After evaluating radiometabolites and radioligand kinetics, we conducted longitudinal dynamic PET imaging at 3, 6, 9, and 13 months of age (M) in wild-type (WT, n = 17) and heterozygous (HET, n = 23) zQ175DN mice. Statistical analysis was performed to evaluate temporal and genotypic differences. Cross-sectional cohorts at each longitudinal time point were included for post-mortem [3H]CHDI-626 autoradiography. RESULTS: Despite fast metabolism and kinetics, the radioligand was suitable for PET imaging of mHTT. Longitudinal quantification could discriminate between genotypes already at premanifest stage (3 M), showing an age-associated increase in signal in HET mice in parallel with mHTT aggregate load progression, as supported by the post-mortem [3H]CHDI-626 autoradiography. CONCLUSION: With clinical evaluation underway, [11C]CHDI-626 PET imaging appears to be a suitable preclinical candidate marker to monitor natural HD progression and for the evaluation of mHTT-lowering therapies.

Kinetic Modelling and Test-Retest Reproducibility for the Dopamine D1R Radioligand [11C]SCH23390 in Healthy and Diseased Mice.

PURPOSE: Our aim in this study was to compare different non-invasive pharmacokinetic models and assess test-retest reproducibility of the radioligand [11C]SCH23390 for the quantification of dopamine D1-like receptor (D1R) in both wild-type (WT) mice and heterozygous (HET) Q175DN mice as Huntington's disease (HD) model. PROCEDURES: Adult WT (n = 9) and HET (n = 14) mice underwent a 90-min [11C]SCH23390 positron emission tomography (PET) scan followed by computed tomography (CT) to evaluate the pharmacokinetic modelling in healthy and diseased conditions. Additionally, 5 WT mice and 7 HET animals received a second [11C]SCH23390 PET scan for test-retest reproducibility. Parallel assessment of the simplified reference tissue model (SRTM), the multilinear reference tissue model (MRTM) and the Logan reference tissue model (Logan Ref) using the striatum as a receptor-rich region and the cerebellum as a receptor-free (reference) region was performed to define the most suitable method for regional- and voxel-based quantification of the binding potential (BPND). Finally, standardised uptake value ratio (SUVR-1) was assessed as a potential simplified measurement. RESULTS: For all models, we measured a significant decline in dopamine D1R density (e.g. SRTM = - 38.5 ± 5.0 %, p < 0.0001) in HET mice compared to WT littermates. Shortening the 90-min scan duration resulted in large underestimation of striatal BPND in both WT mice (SRTM 60 min: - 17.7 ± 2.8 %, p = 0.0078) and diseased HET (SRTM 60 min: - 13.1 ± 4.1 %, p = 0.0001). Striatal BPND measurements were very reproducible with an average test-retest variability below 5 % when using both MRTM and SRTM. Parametric BPND maps generated with SRTM were highly reliable, showing nearly perfect agreement to the regional analysis (r2 = 0.99, p < 0.0001). Finally, SRTM provided the most accurate estimate for relative tracer delivery R1 with both regional- and voxel-based analyses. SUVR-1 at different time intervals were not sufficiently reliable when compared to BPND (r2 < 0.66). CONCLUSIONS: Ninety-minute acquisition and the use of SRTM for pharmacokinetic modelling is recommended. [11C]SCH23390 PET imaging demonstrates optimal characteristics for the study of dopamine D1R density in models of psychiatric and neurological disorders as exemplified in the Q175DN mouse model of HD.

Elevated Type 1 Metabotropic Glutamate Receptor Availability in a Mouse Model of Huntington's Disease: a Longitudinal PET Study.

Impairment of group I metabotropic glutamate receptors (mGluRs) results in altered glutamate signalling, which is associated with several neurological disorders including Huntington's Disease (HD), an autosomal neurodegenerative disease. In this study, we assessed in vivo pathological changes in mGluR1 availability in the Q175DN mouse model of HD using longitudinal positron emission tomography (PET) imaging with the radioligand [11C]ITDM. Ninety-minute dynamic PET imaging scans were performed in 22 heterozygous (HET) Q175DN mice and 22 wild-type (WT) littermates longitudinally at 6, 12, and 16 months of age. Analyses of regional volume of distribution with an image-derived input function (VT (IDIF)) and voxel-wise parametric VT (IDIF) maps were performed to assess differences between genotypes. Post-mortem evaluation at 16 months was done to support in vivo findings. [11C]ITDM VT (IDIF) quantification revealed higher mGluR1 availability in the brain of HET mice compared to WT littermates (e.g. cerebellum: + 15.0%, + 17.9%, and + 17.6% at 6, 12, and 16 months, respectively; p < 0.001). In addition, an age-related decline in [11C]ITDM binding independent of genotype was observed between 6 and 12 months. Voxel-wise analysis of parametric maps and post-mortem quantifications confirmed the elevated mGluR1 availability in HET mice compared to WT littermates. In conclusion, in vivo measurement of mGluR1 availability using longitudinal [11C]ITDM PET imaging demonstrated higher [11C]ITDM binding in extra-striatal brain regions during the course of disease in the Q175DN mouse model.

Validation and noninvasive kinetic modeling of [11C]UCB-J PET imaging in mice.

Synaptic pathology is associated with several brain disorders, thus positron emission tomography (PET) imaging of synaptic vesicle glycoprotein 2A (SV2A) using the radioligand [11C]UCB-J may provide a tool to measure synaptic alterations. Given the pivotal role of mouse models in understanding neuropsychiatric and neurodegenerative disorders, this study aims to validate and characterize [11C]UCB-J in mice. We performed a blocking study to verify the specificity of the radiotracer to SV2A, examined kinetic models using an image-derived input function (IDIF) for quantification of the radiotracer, and investigated the in vivo metabolism. Regional TACs during baseline showed rapid uptake of [11C]UCB-J into the brain. Pretreatment with levetiracetam confirmed target engagement in a dose-dependent manner. VT (IDIF) values estimated with one- and two-tissue compartmental models (1TCM and 2TCM) were highly comparable (r=0.999, p < 0.0001), with 1TCM performing better than 2TCM for K1 (IDIF). A scan duration of 60 min was sufficient for reliable VT (IDIF) and K1 (IDIF) estimations. In vivo metabolism of [11C]UCB-J was relatively rapid, with a parent fraction of 22.5 ± 4.2% at 15 min p.i. In conclusion, our findings show that [11C]UCB-J selectively binds to SV2A with optimal kinetics in the mouse representing a promising tool to noninvasively quantify synaptic density in comparative or therapeutic studies in neuropsychiatric and neurodegenerative disorder models.

In vitro and In vivo Assessment of Suitable Reference Region and Kinetic Modelling for the mGluR1 Radioligand [11C]ITDM in Mice.

PURPOSE: This study aimed at investigating binding specificity, suitability of reference region-based kinetic modelling, and pharmacokinetics of the metabotropic glutamate receptor 1 (mGluR1) radioligand [11C]ITDM in mice. PROCEDURES: We performed in vivo blocking as well as displacement of [11C]ITDM during positron emission tomography (PET) imaging using the specific mGluR1 antagonist YM-202074. Additionally, we assessed in vitro blocking of [3H]ITDM at two different doses of YM-202074. As an alternative to reference region models, we validated the use of a noninvasive image-derived input function (IDIF) compared to an arterial input function measured with an invasive arteriovenous (AV) shunt using a population-based curve for radiometabolite correction and characterized the pharmacokinetic modelling of [11C]ITDM in the mouse brain. Finally, we also assessed semi-quantitative approaches. RESULTS: In vivo blocking with YM-202074 resulted in a decreased [11C]ITDM binding, ranging from - 35.8 ± 8.0 % in pons to - 65.8 ± 3.0 % in thalamus. Displacement was also markedly observed in all tested regions. In addition, in vitro [3H]ITDM binding could be blocked in a dose-dependent manner. The volume of distribution (VT) based on the noninvasive IDIF (VT (IDIF)) showed excellent agreement with the VT values based on the metabolite-corrected plasma input function regardless of the metabolite correction (r2 > 0.943, p < 0.0001). Two-tissue compartmental model (2TCM) was found to be the preferred model and showed optimal agreement with Logan plot (r2 > 0.960, p < 0.0001). A minimum scan duration of 80 min was required for proper parameter estimation. SUV was not reliable (r2 = 0.379, p = 0.0011), unlike the SUV ratio to the SUV of the input function, which showed to be a valid approach. CONCLUSIONS: No suitable reference region could be identified for [11C]ITDM as strongly supported by in vivo and in vitro evidence of specific binding in all brain regions. However, by applying appropriate kinetic models, [11C]ITDM PET imaging represents a promising tool to visualize mGluR1 in the mouse brain.

The dopamine dysfunction in schizophrenia revisited: new insights into topography and course.

Schizophrenia has long been associated with an imbalance in dopamine (DA) neurotransmission, and brain imaging has played an important role in advancing our knowledge and providing evidence for the dopaminergic abnormalities. This chapter reviews the evidence for DA dysfunction in different brain regions in schizophrenia, in particular striatal, extrastriatal, and prefrontal regions, with emphasis on recently published findings. As opposed to the traditional view that most striatal dopaminergic excess, associated with the positive symptoms of schizophrenia, involves the dopaminergic mesolimbic pathway, recent evidence points to the nigrostriatal pathway as the area of highest dysregulation. Furthermore, evidence from translational research suggests that dopaminergic excess may be present in the prodromal phase, and may by itself, as suggested by the phenotype observed in transgenic mice with developmental overexpression of dorso-striatal D(2) receptors, be an early pathogenic condition, leading to irreversible cortical dysfunction.

Imaging the high-affinity state of the dopamine D2 receptor in vivo: fact or fiction?

Positron Emission Tomography (PET) has been used for more than three decades to image and quantify dopamine D2 receptors (D2R) in vivo with antagonist radioligands but in the recent years agonist radioligands have also been employed. In vitro competition studies have demonstrated that agonists bind to both a high and a low-affinity state of the D2Rs, of which the high affinity state reflects receptors that are coupled to G-proteins and the low-affinity state reflects receptors uncoupled from G-proteins. In contrast, antagonists bind with uniform affinity to the total pool of receptors. Results of these studies led to the proposal that D2Rs exist in high and low-affinity states for agonists in vivo and sparked the development and use of agonist radioligands for PET imaging with the primary purpose of measuring the proportion of receptors in the high-affinity (activating) state. Although several lines of research support the presence of high and low-affinity states of D2Rs and their detection by in vivo imaging paradigms, a growing body of controversial data has now called this into question. These include both in vivo and ex vivo studies of anesthesia effects, rodent models with increased proportions of high-affinity state D2Rs as well as the molecular evidence for stable receptor-G-protein complexes. In this commentary we review these data and discuss the evidence for the in vivo existence of D2Rs configured in high and low-affinity states and whether or not the high-affinity state of the D2R can, in fact, be imaged in vivo with agonist radioligands.

Imaging changes in glutamate transmission in vivo with the metabotropic glutamate receptor 5 tracer [11C] ABP688 and N-acetylcysteine challenge.

BACKGROUND: An imaging method to probe glutamate levels in vivo would allow the study of glutamate transmission in disease states and in response to therapeutic interventions. Here we demonstrate the feasibility of this approach for the first time using positron emission tomography and [(11)C] ABP688, a radiotracer for an allosteric site on the metabotropic glutamate receptor 5. METHODS: We conducted two sets of experiments in anesthetized baboons: test and retest without pharmacologic challenge and in combination with N-acetylcysteine (NAC), a promoter of the cystine-glutamate antiporter that increases extrasynaptic glutamate release. The goal was to assess whether NAC-induced changes in [(11)C] ABP688 binding potential, ΔBP(ND), could be detected above the noise in the measurement. RESULTS: Linear mixed modeling comparing ΔBP(ND) from test-retest to ΔBP(ND) from NAC challenge across all brain regions showed a highly significant effect of treatment [F(1,40) = 21.2, p < .001]. ΔBP(ND) was significantly different from zero following NAC [F(1,20) = 76.6, p < .001] but not after test-retest studies. CONCLUSIONS: NAC induced decrease in [(11)C] ABP688 ΔBP(ND) may be the result of allosteric modulation, although other mechanisms may be at play. We outline steps needed to replicate and validate this method as a new tool to measure in vivo glutamate transmission.

Presynaptic dopamine in schizophrenia.

Presynaptic dopamine (DA) transmission has been measured in schizophrenia using different paradigms aimed at providing estimates of the integrity or the activity of the presynaptic dopaminergic neuron. RESEARCHERS HAVE MEASURED: (1) DA synthesis capacity with [(18) F]DOPA, a measure of the activity of dopa decarboxylase, (2) DA release with studies measuring the impact of a DA releasing stimulant challenge on the binding of a D(2) receptor radiotracer, (3) D(2) baseline occupancy by DA, a measure of baseline intrasynaptic DA, assessed by the changes in binding of D(2) radiotracer induced by DA depletion, and (4) the DA and the vesicular monoamine transporters, to assess the integrity of presynaptic terminals. The relationship between DA release and D(2) receptor occupancy at baseline by DA has also been assessed in the same patients. Overall, these different imaging modalities have converged to show a dysregulation of presynaptic dopaminergic activity in schizophrenia, leading to excessive DA release in the striatum, particularly in the projection to the associative striatum, an area of integration between cognitive and limbic cortical inputs. Excessive striatal presynaptic DA is linked to the emergence of acute psychotic symptoms and to their response to treatment in schizophrenia. Understanding the etiology of this dysregulation and its consequences on the rest of the circuitry is important for future drug development.

Dopamine beta-hydroxylase-deficient mice have normal densities of D(2) dopamine receptors in the high-affinity state based on in vivo PET imaging and in vitro radioligand binding.

In vitro, D(2) dopamine receptors (DAR) can exist in low- and high-affinity states for agonists and increases of D(2) receptors in high-affinity state have been proposed to underlie DA receptor supersensitivity in vivo. Deletion of the gene for dopamine beta-hydroxylase (DBH) causes mice to become hypersensitive to the effects of psychostimulants, and in vitro radioligand binding results suggest an increased percentage of D(2) receptors in a high-affinity state. To determine whether DBH knockout mice display an increase of high-affinity state D(2) receptors in vivo, we scanned DBH knockout and control mice with the agonist PET radioligand [(11)C]MNPA, which is thought to bind preferentially to the high-affinity state of the D(2) receptor. In addition, we performed in vitro binding experiments on striatal homogenates with [(3)H]methylspiperone to measure B(max) values and the percentages of high- and low-affinity states of the D(2) receptor. We found that the in vivo striatal binding of [(11)C]MNPA was similar in DBH knockout mice and heterozygous controls and the in vitro B(max) values and percentages of D(2) receptors in the high-affinity state, were not significantly different between these two groups. In summary, our results suggest that DBH knockout mice have normal levels of D(2) receptors in the high-affinity state and that additional mechanisms contribute to their behavioral sensitivity to psychostimulants.

D2 dopamine receptor internalization prolongs the decrease of radioligand binding after amphetamine: a PET study in a receptor internalization-deficient mouse model.

Dopamine released by amphetamine decreases the in vivo binding of PET radioligands to the dopamine D(2) receptor. Although concentrations of extracellular dopamine largely return to baseline within 1 to 2 h after amphetamine treatment, radioligand binding remains decreased for several hours. The purpose of this study was to determine whether the prolonged decrease of radioligand binding after amphetamine administration is caused by receptor internalization. To distinguish dopamine displacement from receptor internalization, we used wild-type and arrestin3 (arr3) knockout mice, which are incapable of internalizing D(2) receptors. In addition, we used both the D(2) selective agonist [(11)C]MNPA (which is thought to bind to the high affinity state of the receptor) and the D(2) selective antagonist [(18)F]fallypride (which does not differentiate between high and low affinity state). After an initial baseline scan, animals were divided in three groups for a second scan: either 30 min or 4 h after amphetamine administration (3 mg/kg, i.p.) or as retest. At 30 min, [(11)C]MNPA showed greater displacement than [(18)F]fallypride, but each radioligand gave similar displacement in knockout and wild-type mice. At 4 h, the binding of both radioligands returned to baseline in arr3 knockout mice, but remained decreased in wild-type mice. Radioligand binding was unaltered on retest scanning. Our results suggest that the prolonged decrease of radioligand binding after amphetamine is mainly due to internalization of the D(2) receptor rather than dopamine displacement. In addition, this study demonstrates the utility of small animal PET to study receptor trafficking in vivo in genetically modified mice.

Pharmacological characterization of 2-methoxy-N-propylnorapomorphine's interactions with D2 and D3 dopamine receptors.

Dopaminergic signaling pathways have been extensively investigated using PET imaging, primarily with antagonist radioligands of D(2) and D(3) dopamine receptors (DARs). Recently, agonist radioligands of D(2)/D(3) DARs have begun to be developed and employed. One such agonist is (R)-2-(11)CH(3)O-N-n-propylnorapomorphine (MNPA). Here, we perform a pharmacological characterization of MNPA using recombinant D(2) and D(3) DARs expressed in HEK293 cells. MNPA was found to robustly inhibit forskolin-stimulated cAMP accumulation to the same extent as dopamine in D(2) or D(3) DAR-transfected cells, indicating that it is a full agonist at both receptors. MNPA is approximately 50-fold more potent than dopamine at the D(2) DAR, but equally potent as dopamine at the D(3) DAR. MNPA competition binding curves in membrane preparations expressing D(2) DARs revealed two binding states of high and low-affinity. In the presence of GTP, only one binding state of low affinity was observed. Direct saturation binding assays using [(3)H]MNPA revealed similar results as with the competition experiments leading to the conclusion that MNPA binds to the D(2) DAR in an agonist-specific fashion. In contrast to membrane preparations, using intact cell binding assays, only one site of low affinity was observed for MNPA and other agonists binding to the D(2) DAR. MNPA was also found to induce D(2) DAR internalization to an even greater extent than dopamine as determined using both cell surface receptor binding assays and confocal fluorescence microscopy. Taken together, our data indicate that the PET tracer, MNPA, is a full and potent agonist at both D(2) and D(3) receptors.

Reciprocal modulation of function between the D1 and D2 dopamine receptors and the Na+,K+-ATPase.

It is well documented that dopamine can increase or decrease the activity of the Na+,K+-ATPase (NKA, sodium pump) in an organ-specific fashion. This regulation can occur, at least partially, via receptor-mediated second messenger activation and can promote NKA insertion or removal from the plasma membrane. Using co-immunoprecipitation and mass spectrometry, we now show that, in both brain and HEK293T cells, D1 and D2 dopamine receptors (DARs) can exist in a complex with the sodium pump. To determine the impact of NKA on DAR function, biological assays were conducted with NKA and DARs co-expressed in HEK293T cells. In this system, expression of NKA dramatically decreased D1 and D2 DAR densities with a concomitant functional decrease in DAR-mediated regulation of cAMP levels. Interestingly, pharmacological inhibition of endogenous or overexpressed NKA enhanced DAR function without altering receptor number or localization. Similarly, DAR function was also augmented by small interfering RNA reduction of the endogenous NKA. These data suggest that, under basal conditions, NKA negatively regulates DAR function via protein-protein interactions. In reciprocal fashion, expression of DARs decreases endogenous NKA function in the absence of dopamine, implicating DAR proteins as regulators of NKA activity. Notably, dopamine stimulation or pertussis toxin inhibition of D2 receptor signaling did not alter NKA activity, indicating that the D2-mediated decrease in NKA function is dependent upon protein-protein interactions rather than signaling molecules. This evidence for reciprocal regulation between DARs and NKA provides a novel control mechanism for both DAR signaling and cellular ion balance.

Occupancy of dopamine D2/3 receptors in rat brain by endogenous dopamine measured with the agonist positron emission tomography radioligand [11C]MNPA.

Estimates of dopamine D(2/3) receptor occupancy by endogenous dopamine using positron emission tomography (PET) in animals have varied almost threefold. This variability may have been caused by incomplete depletion of dopamine or by the use of antagonist radioligands, which appear less sensitive than agonist radioligands to changes in endogenous dopamine. PET scans were performed in rats with the agonist PET radioligand [(11)C]MNPA ([O-methyl-(11)C]2-methoxy-N-propylnorapomorphine). [(11)C]MNPA was injected as a bolus plus constant infusion to achieve steady-state concentration in the body and equilibrium receptor binding in the brain. Radioligand binding was compared at baseline and after treatment with reserpine plus alpha-methyl-para-tyrosine, which cause approximately 95% depletion of endogenous dopamine. Depletion of dopamine increased radioligand binding in striatum but had little effect in cerebellum. Striatal [(11)C]MNPA binding potential was 0.93 +/- 0.12 at baseline and increased to 1.99 +/- 0.25 after dopamine depletion. Occupancy of D(2/3) receptors by endogenous dopamine at baseline was calculated to be approximately 53%. Striatal binding was displaceable with raclopride, but not with BP 897 (a selective D(3) compound), thus confirming the D(2) receptor specificity of [(11)C]MNPA binding. Radioactivity extracted from rat brain contained only 8-10% radiometabolites and was insignificantly altered by administration of reserpine plus alpha-methyl-para-tyrosine. Hence, dopamine depletion did not increase the PET measurements via an effect on radiotracer metabolism. Our in vivo estimate of dopamine's occupancy of D(2/3) receptors at baseline is higher than that previously reported using antagonist radioligands and PET, but is similar to that reported using agonist radioligands and ex vivo measurements.

Kinetic brain analysis and whole-body imaging in monkey of [11C]MNPA: a dopamine agonist radioligand.

With a view to future extension of the use of the agonist radioligand [(11)C]MNPA ([O-methyl-(11)C]2-methoxy-N-propylnorapomorphine) from animals to humans, we performed two positron emission tomography (PET) studies in monkeys. First, we assessed the ability to quantify the brain uptake of [(11)C]MNPA with compartmental modeling. Second, we estimated the radiation exposure of [(11)C]MNPA to human subjects based on whole-body imaging in monkeys. Brain PET scans were acquired for 90 min and included concurrent measurements of the plasma concentration of unchanged radioligand. Time-activity data from striatum and cerebellum were quantified with two methods, a reference tissue model and distribution volume. Whole-body PET scans were acquired for 120 min using four bed positions from head to mid thigh. Regions of interest were drawn on compressed planar whole-body images to identify organs with the highest radiation exposures. After injection of [(11)C]MNPA, the highest concentration of radioactivity in brain was in striatum, with lowest levels in cerebellum. Distribution volume was well identified with a two-tissue compartmental model and was quite stable from 60 to 90 min. Whole-body PET scans showed the organ with the highest radiation burden (muSv/MBq) was the urinary bladder wall (26.0), followed by lungs (22.5), gallbladder wall (21.9), and heart wall (16.1). With a 2.4-h voiding interval, the effective dose was 6.4 muSv/MBq (23.5 mrem/mCi). In conclusion, brain uptake of [(11)C]MNPA reflected the density of D(2/3) receptors, quantified relative to serial arterial measurements, and caused moderate to low radiation exposure.