David J. Williamson

Baseline-dependent effects of cocaine pre-exposure on impulsivity and D2/3 receptor availability in the rat striatum: possible relevance to the attention-deficit hyperactivity syndrome.

We have previously shown that impulsivity in rats predicts the emergence of compulsive cocaine seeking and taking, and is coupled to decreased D2/3 receptor availability in the ventral striatum. As withdrawal from cocaine normalises high impulsivity in rats, we investigated, using positron emission tomography (PET), the effects of response-contingent cocaine administration on D2/3 receptor availability in the striatum. Rats were screened for impulsive behavior on the five-choice serial reaction time task. After a baseline PET scan with the D2/3 ligand [18F]fallypride, rats were trained to self-administer cocaine for 15 days under a long-access schedule. As a follow-up, rats were assessed for impulsivity and underwent a second [18F]fallypride PET scan. At baseline, we found that D2/3 receptor availability was significantly lower in the left, but not right, ventral striatum of high-impulsive rats compared with low-impulsive rats. While the number of self-administered cocaine infusions was not different between the two impulsivity groups, impulsivity selectively decreased in high-impulsive rats withdrawn from cocaine. This effect was accompanied by a significant increase in D2/3 receptor availability in the left, but not right, ventral striatum. We further report that D2/3 receptor availability was inversely related to baseline D2/3 receptor availability in the ventral striatum of high-impulsive rats, as well as to the left and right dorsal striatum of both low-impulsive and high-impulsive rats. These findings indicate that the reduction in impulsivity in high-impulsive rats by prior cocaine exposure may be mediated by a selective correction of deficient D2/3 receptor availability in the ventral striatum. A similar baseline-dependent mechanism may account for the therapeutic effects of stimulant drugs in clinical disorders such as ADHD.

Dissociable Rate-Dependent Effects of Oral Methylphenidate on Impulsivity and D2/3 Receptor Availability in the Striatum

We have previously shown that impulsivity in rats is linked to decreased dopamine D2/3 receptor availability in the ventral striatum. In the present study, we investigated, using longitudinal positron emission tomography (PET), the effects of orally administered methylphenidate (MPH), a first-line treatment for attention deficit hyperactivity disorder, on D2/3 receptor availability in the dorsal and ventral striatum and related these changes to impulsivity. Rats were screened for impulsive behavior on a five-choice serial reaction time task. After a baseline PET scan with the D2/3 ligand [18F]fallypride, rats received 6 mg/kg MPH, orally, twice each day for 28 d. Rats were then reassessed for impulsivity and underwent a second [18F]fallypride PET scan. Before MPH treatment, we found that D2/3 receptor availability was significantly decreased in the left but not the right ventral striatum of high-impulse (HI) rats compared with low-impulse (LI) rats. MPH treatment increased impulsivity in LI rats, and modulated impulsivity and D2/3 receptor availability in the dorsal and ventral striatum of HI rats through inverse relationships with baseline levels of impulsivity and D2/3 receptor availability, respectively. However, we found no relationship between the effects of MPH on impulsivity and D2/3 receptor availability in any of the striatal subregions investigated. These findings indicate that trait-like impulsivity is associated with decreased D2/3 receptor availability in the left ventral striatum, and that stimulant drugs modulate impulsivity and striatal D2/3 receptor availability through independent mechanisms.

Synthesis and Initial in Vivo Studies with [11C]SB-216763: The First Radiolabeled Brain Penetrative Inhibitor of GSK-3

Quantifying glycogen synthase kinase-3 (GSK-3) activity in vivo using positron emission tomography (PET) imaging is of interest because dysregulation of GSK-3 is implicated in numerous diseases and neurological disorders for which GSK-3 inhibitors are being considered as therapeutic strategies. Previous PET radiotracers for GSK-3 have been reported, but none of the published examples cross the blood-brain barrier. Therefore, we have an ongoing interest in developing a brain penetrating radiotracer for GSK-3. To this end, we were interested in synthesis and preclinical evaluation of [(11)C]SB-216763, a high-affinity inhibitor of GSK-3 (K i = 9 nM; IC50 = 34 nM). Initial radiosyntheses of [(11)C]SB-216763 proved ineffective in our hands because of competing [3 + 3] sigmatropic shifts. Therefore, we have developed a novel one-pot two-step synthesis of [(11)C]SB-216763 from a 2,4-dimethoxybenzyl-protected maleimide precursor, which provided high specific activity [(11)C]SB-216763 in 1% noncorrected radiochemical yield (based upon [(11)C]CH3I) and 97-100% radiochemical purity (n = 7). Initial preclinical evaluation in rodent and nonhuman primate PET imaging studies revealed high initial brain uptake (peak rodent SUV = 2.5 @ 3 min postinjection; peak nonhuman primate SUV = 1.9 @ 5 min postinjection) followed by washout. Brain uptake was highest in thalamus, striatum, cortex, and cerebellum, areas known to be rich in GSK-3. These results make the arylindolemaleimide skeleton our lead scaffold for developing a PET radiotracer for quantification of GSK-3 density in vivo and ultimately translating it into clinical use.

Cortical Selective Neuronal Loss, Impaired Behavior, and Normal Magnetic Resonance Imaging in a New Rat Model of True Transient Ischemic Attacks

Background and Purpose— New-definition transient ischemic attacks (TIAs) are frequent but difficult to diagnose because magnetic resonance imaging (MRI)-negative by definition. However, hidden underlying cell damage might be present and account for the reported long-lasting cognitive impairment after TIAs. Most prior rodent models of true TIA targeted the striatum or have not been fully characterized. Here we present the MRI, behavioral, and quantitative cell changes characterizing a new rodent model of true TIA targeting the more behaviorally relevant cerebral cortex. Methods— Fifteen-minute distal middle cerebral artery occlusion was performed in 29 spontaneously hypertensive rats allowed to survive for 7 to 60 days. Behavior was assessed serially using both global neurological and fine sensorimotor tests. Diffusion- and T2-weighted MRI was obtained 20 min postreperfusion and again 7 to 60 days later, and then changes in neurons and microglia were quantified across the middle cerebral artery territory using immunohistochemistry. Results— No MRI changes or pan-necrosis were observed at any time point, but patchy cortical selective neuronal loss affected 28/29 rats, regardless of survival interval, together with topographically congruent microglial activation that gradually declined over time. The Neuroscore was unchanged, but there was marked contralateral sensorimotor impairment, still recovering by day 28. Conclusions— Our new rodent model mimicking true cortical TIA is characterized by normal MRI, but consistent cortical selective neuronal loss and microglial activation and long-lasting sensorimotor deficits. By causing selective neuronal loss, TIAs and silent microemboli might affect neuronal reserve, thereby increasing long-term cognitive impairment risk. Selective neuronal loss and microglial activation might represent novel therapeutic targets that could be detectable in vivo after TIAs using appropriate imaging tracers.

Regional distribution of selective neuronal loss and microglial activation across the MCA territory after transient focal ischemia: quantitative versus semiquantitative systematic immunohistochemical assessment.

Histopathologic assessment in transient middle cerebral artery occlusion (MCAo) rodent models generally lacks comprehensiveness and exposes to interobserver bias. Here we compared a novel quantitative assessment of regional infarction, selective neuronal loss (SNL) and microglial activation (MA) across the MCA territory to a previously published semiquantitative visual protocol. NeuN and OX42 immunohistochemistry was applied after either 15 or 45 minutes distal MCAo to maximize SNL and infarction, respectively. Survival times varied from 28 to 60 days to cover potential biases such as delayed tissue shrinkage. Damage was assessed using a template of 44 cytoarchitectonic regions of interest (ROIs) mapped onto a subset of digitized coronal sections spanning the MCA territory. For each ROI were obtained a semiquantitative visually determined index of histopathologic changes (method 1), and lpsilateral/contralesional ratios of remaining neurons and activated microglia cell counts (method 2). There was excellent agreement between the two methods for 28-day survival for both MCAo durations, whereas method 2 more sensitively detected subtle SNL and MA at 45 days and 60 days after 15-minute MCAo. Thus the visual method is accurate for usual degrees of ischemic damage, but absolute cell quantification is superior to detect subtle changes and should therefore be preferred in brief MCAo models, although requires optimal staining quality.

Radiosynthesis and characterization of astemizole derivatives as lead compounds toward PET imaging of τ-pathology

Formation of neurofibrillary tangles, comprising of microtubule-associated tau protein, is a hallmark of a group of neurodegenerative diseases, including Alzheimers disease. In consequence, in vivo imaging of neurofibrillary tangles is a current focus of positron emission tomography research. Herein, development of an in vitro radioligand binding assay which uses synthetic aggregates as a model of neurofibrillary tangles is reported, together with evaluation of novel derivatives of the tau protein ligand astemizole.

Validation and quantification of [18F]altanserin binding in the rat brain using blood input and reference tissue modeling.

The 5-hydroxytryptamine type 2a (5-HT2A) selective radiotracer [18F]altanserin has been subjected to a quantitative micro-positron emission tomography study in Lister Hooded rats. Metabolite-corrected plasma input modeling was compared with reference tissue modeling using the cerebellum as reference tissue. [18F]altanserin showed sufficient brain uptake in a distribution pattern consistent with the known distribution of 5-HT2A receptors. Full binding saturation and displacement was documented, and no significant uptake of radioactive metabolites was detected in the brain. Blood input as well as reference tissue models were equally appropriate to describe the radiotracer kinetics. [18F]altanserin is suitable for quantification of 5-HT2A receptor availability in rats.

A comparison of four PET tracers for brain hypoxia mapping in a rodent model of stroke.

INTRODUCTION Severe brain hypoxia in the territory of the occluded artery is a key feature of ischemic stroke. This region can be imaged using positron emission tomography (PET) and the standard hypoxia radiotracer (18)F-fluoromisonidazole ((18)F-FMISO). However, the utility of (18)F-FMISO is limited by its slow accumulation in the lesion. Therefore, this study investigated three hypoxia-sensitive radiotracers, namely the nitroimidazole (18)F-fluoroazomycin arabinoside ((18)F-FAZA) and two (64)Cu bis(thiosemicarbazone) complexes ((64)Cu-ATSM and (64)Cu-ATSE), expected to have improved pharmacokinetic profiles relative to (18)F-FMISO, in a rodent model of ischemic stroke. METHODS In anaesthetised Wistar rats, the distal middle cerebral artery was permanently occluded by electrocoagulation, the radiotracers administered intravenously and animals PET scanned for up to 3hours, followed by T2-weighted magnetic resonance imaging to map the infarct. RESULTS As expected, late and prominent (18)F-FMISO retention was observed despite lower tracer delivery into the affected region. Time-activity curves revealed that both (64)Cu-ATSM and (64)Cu-ATSE showed rapid entry and efflux from the brain, but did not show significant accumulation in the lesion. (18)F-FAZA showed limited brain penetration, and accumulation in the lesion was inconsistent, low and as slow as (18)F-FMISO. CONCLUSIONS This study suggests further development of these radiotracers as hypoxia markers for ischemic stroke may not be warranted.

Synthesis and Evaluation of 18F-FE-PEO in Rodents: An 18F-Labeled Full Agonist for Opioid Receptor Imaging

We have investigated the opioid receptor (OR) agonist (20R)-4,5-α-epoxy-6-(2-18F-fluoroethoxy)-3-hydroxy-α,17-dimethyl-α-(2-phenyleth-1-yl)-6,14-ethenomorphinan-7-methanol (18F-FE-PEO) as a candidate OR PET ligand. This tracer is attractive because it combines 18F labeling, is suited to the slow kinetics of high-affinity ligands, and has agonist binding, which has been shown to be more sensitive to changes in OR occupation than is antagonist binding. Methods: Agonist potency and off-target binding were investigated in vitro, and autoradiographic studies on rat brain sections were used to assess binding patterns. Quantification of the tracer in vivo was investigated using small-animal PET in rats with blood sampling. Results: 18F-FE-PEO was obtained by direct nucleophilic radiofluorination and subsequent deprotection with a yield of 28% ± 15%, a specific activity of 52–224 MBq/nmol, and a radiochemical purity of more than 97% (90 min from end of bombardment). In vitro studies showed it to be a full agonist ligand, which selectively binds to OR with high affinity, although it is not selective to a single OR subtype (inhibition constant, 0.4–1.6 nM across OR subtypes). Autoradiography binding patterns were consistent with the known distribution of OR, although nondisplaceable signal typically constituted one third of the signal in OR-dense regions. Although metabolites were present in blood (∼40% of plasma radioactivity was nonparent 3 h after injection), no significant metabolite fraction was found in brain tissue, aiding PET quantification. A plasma input 2-tissue-compartment model provided good fits to the PET data, and regional distribution volumes from the latter correlated well with those from Logan plot analysis (r2 = 0.98). The cerebellum had the lowest distribution volume, but the time–activity curve data could not be adequately fitted with a 1-tissue-compartment model. Reference tissue models using the cerebellum as the reference region did not provide good fits to the data, so blood-based kinetic analysis is recommended. Conclusion: As the first 18F-labeled OR agonist ligand, 18F-FE-PEO is a useful addition to the existing OR ligand portfolio.

Brain hypoxia mapping in acute stroke: Back-to-back T2′ MR versus 18F-fluoromisonidazole PET in rodents

Background Mapping the hypoxic brain in acute ischemic stroke has considerable potential for both diagnosis and treatment monitoring. PET using 18F-fluoro-misonidazole (FMISO) is the reference method; however, it lacks clinical accessibility and involves radiation exposure. MR-based T2′ mapping may identify tissue hypoxia and holds clinical potential. However, its validation against FMISO imaging is lacking. Here we implemented back-to-back FMISO-PET and T2′ MR in rodents subjected to acute middle cerebral artery occlusion. For direct clinical relevance, regions of interest delineating reduced T2′ signal areas were manually drawn. Methods Wistar rats were subjected to filament middle cerebral artery occlusion, immediately followed by intravenous FMISO injection. Multi-echo T2 and T2* sequences were acquired twice during FMISO brain uptake, interleaved with diffusion-weighted imaging. Perfusion-weighted MR was also acquired whenever feasible. Immediately following MR, PET data reflecting the history of FMISO brain uptake during MR acquisition were acquired. T2′ maps were generated voxel-wise from T2 and T2*. Two raters independently drew T2′ lesion regions of interest. FMISO uptake and perfusion data were obtained within T2′ consensus regions of interest, and their overlap with the automatically generated FMISO lesion and apparent diffusion coefficient lesion regions of interest was computed. Results As predicted, consensus T2′ lesion regions of interest exhibited high FMISO uptake as well as substantial overlap with the FMISO lesion and significant hypoperfusion, but only small overlap with the apparent diffusion coefficient lesion. Overlap of the T2′ lesion regions of interest between the two raters was ∼50%. Conclusions This study provides formal validation of T2′ to map non-core hypoxic tissue in acute stroke. T2′ lesion delineation reproducibility was suboptimal, reflecting unclear lesion borders.