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Synaptic signaling depends on ATP generated by mitochondria. Dysfunctional mitochondria shift the redox balance towards a more oxidative environment. Due to extensive connectivity, the striatum is especially vulnerable to mitochondrial dysfunction. We found that neuronal calcium-binding protein 2 (NECAB2) plays a role in striatal function and mitochondrial homeostasis. NECAB2 is a predominantly endosomal striatal protein which partially colocalizes with mitochondria. This colocalization is enhanced by mild oxidative stress. Global knockout of Necab2 in the mouse results in increased superoxide levels, increased DNA oxidation and reduced levels of the antioxidant glutathione which correlates with an altered mitochondrial shape and function. Striatal mitochondria from Necab2 knockout mice are more abundant and smaller and characterized by a reduced spare capacity suggestive of intrinsic uncoupling respectively mitochondrial dysfunction. In line with this, we also found an altered stress-induced interaction of endosomes with mitochondria in Necab2 knockout striatal cultures. The predominance of dysfunctional mitochondria and the pro-oxidative redox milieu correlates with a loss of striatal synapses and behavioral changes characteristic of striatal dysfunction like reduced motivation and altered sensory gating. Together this suggests an involvement of NECAB2 in an endosomal pathway of mitochondrial stress response important for striatal function.
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Antioxidantes , Corpo Estriado , Estresse Oxidativo , Animais , Camundongos , Antioxidantes/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas do Olho/metabolismo , Camundongos Knockout , Mitocôndrias/genética , Mitocôndrias/metabolismo , Neurônios/metabolismo , Oxirredução , Estresse Oxidativo/fisiologia , Corpo Estriado/fisiologiaRESUMO
Since animal experiments cannot be completely avoided, the pain, suffering, and distress of laboratory animals must be minimized. To this end, a major prerequisite is reliable assessment of pain and distress. Usually, evaluation of animal welfare is done by visual inspection and score sheets. However, relatively little is known about whether standardized, but subjective, score sheets are able to reliably reflect the status of the animals. The current study aimed to compare visual assessment scores and changes in body weight with concentrations of fecal corticosterone metabolites (FCMs) in a neuroscientific experimental setup. Additionally, effects of refinement procedures were investigated. Eight male adult Sprague-Dawley rats underwent several experimental interventions, including electroencephalograph electrode implantation and subsequent recording, positron emission tomography (PET), and sleep deprivation (SD) by motorized activity wheels. Additional 16 rats were either used as controls without any treatment or to evaluate refinement strategies. Stress responses were determined on a daily basis by means of measuring FCMs, body weight, and evaluation of the animals' welfare by standardized score sheets. Surgery provoked a significant elevation of FCM levels for up to five days. Increases in FCMs due to PET procedures or SD in activity wheels were also highly significant, while visual assessment scores did not indicate elevated stress levels and body weights remained constant. Visual assessment scores correlate with neither changes in body weight nor increases in FCM levels. Habituation procedures to activity wheels used for SD had no impact on corticosterone release. Our results revealed that actual score sheets for visual assessment of animal welfare did not mirror physiological stress responses assessed by FCM measurements. Moreover, small changes in body weight did not correlate with FCM concentration either. In conclusion, as visual assessment is a method allowing immediate interventions on suffering animals to alleviate burden, timely stress assessment in experimental rodents via score sheets should be ideally complemented by validated objective measures (e.g., fecal FCM measured by well-established assays for reliable detection of FCMs). This will complete a comprehensive appraisal of the animals' welfare status in a retrospective manner and refine stressor procedures in the long run.
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INTRODUCTION: The suitability of novel positron emission tomography (PET) radioligands for quantitative in vivo imaging is affected by various physicochemical and pharmacological parameters. In this study, the combined effect of binding affinity, lipophilicity, protein binding and blood plasma level on cerebral pharmacokinetics and PET imaging characteristics of three xanthine-derived A1 adenosine receptor (A1AR) radioligands was investigated in rats. METHODS: A comparative evaluation of two novel cyclobutyl-substituted xanthine derivatives, 8-cyclobutyl-3-(3-[18F]fluoropropyl)-1-propylxanthine ([18F]CBX) and 3-(3-[18F]fluoropropyl)-8-(1-methylcyclobutyl)-1-propylxanthine ([18F]MCBX), with the reference A1AR radioligand 8-cyclopentyl-3-(3-[18F]fluoropropyl)-1-propylxanthine ([18F]CPFPX) was conducted. This evaluation included in vitro competition binding assays, in vitro autoradiography and in vivo PET imaging. Differences in cerebral pharmacokinetics and minimal scan duration required for quantification of cerebral distribution volume (VT) were assessed. RESULTS: Measured Ki values of non-labeled CBX, MCBX and CPFPX were 10.0 ± 0.52 nM, 3.3 ± 0.30 nM and 1.4 ± 0.15 nM, respectively (n = 3-4). In vitro autoradiographic binding patterns in rat brain were comparable between the radioligands, as well as the fraction of non-specific binding (1.0-1.9%). In vivo cerebral pharmacokinetics of the novel cyclobutyl-substituted xanthines differed considerably from that of [18F]CPFPX. Brain uptake and VT of [18F]CBX were substantially lower despite the higher concentration of radiotracer in plasma. [18F]MCBX showed comparable uptake and VT, but faster cerebral kinetics than [18F]CPFPX. However, the faster kinetics of [18F]MCBX did not enable the quantification of cerebral VT in a shorter scan time. CONCLUSIONS: The combined effect of individual physicochemical and pharmacological properties of a radiotracer on its PET imaging characteristics cannot be readily predicted. In vivo performance of the xanthine A1AR radioligands was mainly influenced by binding affinity; plasma concentrations and cerebral kinetics were of secondary importance.
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Encéfalo/metabolismo , Tomografia por Emissão de Pósitrons , Receptor A1 de Adenosina/metabolismo , Xantina/farmacocinética , Animais , Encéfalo/diagnóstico por imagem , Interações Hidrofóbicas e Hidrofílicas , Ligantes , Masculino , Ratos , Ratos Sprague-Dawley , Xantina/sangue , Xantina/química , Xantina/metabolismoRESUMO
The prediction of in vivo clearance from in vitro metabolism models such as liver microsomes is an established procedure in drug discovery. The potentials and limitations of this approach have been extensively evaluated in the pharmaceutical sector; however, this is not the case for the field of positron emission tomography (PET) radiotracer development. The application of PET radiotracers and classical drugs differs greatly with regard to the amount of substance administered. In typical PET imaging sessions, subnanomolar quantities of the radiotracer are injected, resulting in body concentrations that cannot be readily simulated in analytical assays. This raises concerns regarding the predictability of radiotracer clearance from in vitro data. We assessed the accuracy of clearance prediction for three prototypical PET radiotracers developed for imaging the A1 adenosine receptor (A1AR). Using the half-life (t1/2) approach and physiologically based scaling, in vivo clearance in the rat model was predicted from microsomal stability data. Actual clearance could be accurately predicted with an average fold error (AFE) of 0.78 and a root mean square error (RMSE) of 1.6. The observed slight underprediction (1.3-fold) is in accordance with the prediction accuracy reported for classical drugs. This result indicates that the prediction of radiotracer clearance is possible despite concentration differences of more than three orders of magnitude between in vitro and in vivo conditions. Consequently, in vitro metabolism models represent a valuable tool for PET radiotracer development.
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PURPOSE: In vivo imaging for the A1 adenosine receptors (A1ARs) with positron emission tomography (PET) using 8-cyclopentyl-3-(3-[18F]fluoropropyl)-1-propylxan- thine ([18F]CPFPX) has become an important tool for studying physiological processes quantitatively in mice. However, the measurement of arterial input functions (AIFs) on mice is a method with restricted applicability because of the small total blood volume and the related difficulties in withdrawing blood. Therefore, the aim of this study was to extract an appropriate [18F]CPFPX image-derived input function (IDIF) from dynamic PET images of mice. PROCEDURES: In this study, five mice were scanned with [18F]CPFPX for 60 min. Arterial blood samples (n = 7 per animal) were collected from the femoral artery and corrected for metabolites. To generate IDIFs, three different approaches were selected: (A) volume of interest (VOI) placed over the heart (cube, 10 mm); (B) VOI set over abdominal vena cava/aorta region with a cuboid (5 × 5 × 15 mm); and (C) with 1 × 1 × 1 mm voxels on five consecutive slices. A calculated scaling factor (α) was used to correct for partial volume effect; the method of obtaining the total metabolite correction of [18F]CPFPX for IDIFs was developed. Three IDIFs were validated by comparison with AIF. Validation included the following: visual performance; computing area under the curve (AUC) ratios (IDIF/AIF) of whole-blood curves and parent curves; and the mean distribution volume (V T) ratios (IDIF/AIF) of A1ARs calculated by Logan plot and two-tissue compartment model. RESULTS: Compared with the AIF, the IDIF with VOI over heart showed the best performance among the three IDIFs after scaling by 1.77 (α) in terms of visual analysis, AUC ratios (IDIF/AIF; whole-blood AUC ratio, 1.03 ± 0.06; parent curve AUC ratio, 1.01 ± 0.10) and V T ratios (IDIF/AIF; Logan V T ratio, 1.00 ± 0.17; two-tissue compartment model V T ratio, 1.00 ± 0.13) evaluation. The A1ARs distribution of average parametric images was in good accordance to autoradiography of the mouse brain. CONCLUSION: The proposed study provides evidence that IDIF with VOI over heart can replace AIF effectively for quantification of A1ARs using PET and [18F]CPFPX in mice brains.
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PURPOSE: Caffeine, a nonselective antagonist of adenosine receptors, is the most popular psychostimulant worldwide. Recently, a protective role of moderate chronic caffeine consumption against neurodegenerative diseases such as Alzheimer's and Parkinson's disease has been discussed. Thus, aim of the present study was an in vivo investigation of effects of long-term caffeine consumption on the adenosine A1 receptor (A1AR) in the rat brain. PROCEDURES: Sixteen adult, male rats underwent five positron emission tomography (PET) scans with the highly selective A1AR radioligand [18F]CPFPX in order to determine A1AR availability. After the first baseline PET scan, the animals were assigned to two groups: Caffeine treatment and control group. The caffeine-treated animals received caffeinated tap water (30 mg/kg bodyweight/day, corresponding to 4-5 cups of coffee per day in humans) for 12 weeks. Subsequently, caffeine was withdrawn and repeated PET measurements were performed on day 1, 2, 4, and 7 of caffeine withdrawal. The control animals were measured according to the same time schedule. RESULTS: At day 1, after 4.4 h of caffeine withdrawal, a significant decrease (- 34.5%, p < 0.001) of whole brain A1AR availability was observed. Unlike all other investigated brain regions in caffeine-treated rats, the hypothalamus and nucleus accumbens showed no significant intraindividual differences between baseline and first withdrawal PET scan. After approximately 27 h of caffeine withdrawal, the region- and group-specific effects disappeared and A1AR availability settled around baseline. CONCLUSIONS: The present study provides evidence that chronic caffeine consumption does not lead to persistent changes in functional availability of cerebral A1ARs which have previously been associated with neuroprotective effects of caffeine. The acute and region-specific decrease in cerebral A1AR availability directly after caffeine withdrawal is most likely caused by residual amounts of caffeine metabolites disguising an unchanged A1AR expression at this early time-point.
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Encéfalo/metabolismo , Cafeína/farmacologia , Tomografia por Emissão de Pósitrons , Receptor A1 de Adenosina/metabolismo , Animais , Cafeína/administração & dosagem , Processamento de Imagem Assistida por Computador , Masculino , Ratos Sprague-DawleyRESUMO
Cerebral administration of botulinum neurotoxin A (BoNT-A) has been shown to improve disease-specific motor behavior in a rat model of Parkinson disease (PD). Since the dopaminergic system of the basal ganglia fundamentally contributes to motor function, we investigated the impact of BoNT-A on striatal dopamine receptor expression using in vitro and in vivo imaging techniques (positron emission tomography and quantitative autoradiography, respectively). Seventeen male Wistar rats were unilaterally lesioned with 6-hydroxydopamine (6-OHDA) and assigned to two treatment groups 7 weeks later: 10 rats were treated ipsilaterally with an intrastriatal injection of 1 ng BoNT-A, while the others received vehicle (n = 7). All animals were tested for asymmetric motor behavior (apomorphine-induced rotations and forelimb usage) and for striatal expression of dopamine receptors and transporters (D1 R, D2 R, and DAT). The striatal D2 R availability was also quantified longitudinally (1.5, 3, and 5 months after intervention) in 5 animals per treatment group. The 6-OHDA lesion alone induced a unilateral PD-like phenotype and a 13% increase of striatal D2 R. BoNT-A treatment reduced the asymmetry in both apomorphine-induced rotational behavior and D2 R expression, with the latter returning to normal values 5 months after intervention. D1 R expression was significantly reduced, while DAT concentrations showed no alteration. Independent of the treatment, higher interhemispheric symmetry in raclopride binding to D2 R was generally associated with reduced forelimb akinesia. Our findings indicate that striatal BoNT-A treatment diminishes motor impairment and induces changes in D1 and D2 binding site density in the 6-OHDA rat model of PD.
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Toxinas Botulínicas Tipo A/administração & dosagem , Corpo Estriado/metabolismo , Transtornos Parkinsonianos/tratamento farmacológico , Transtornos Parkinsonianos/metabolismo , Receptores de Dopamina D1/metabolismo , Receptores de Dopamina D2/metabolismo , Animais , Corpo Estriado/diagnóstico por imagem , Corpo Estriado/efeitos dos fármacos , Injeções Intraventriculares , Masculino , Transtornos Parkinsonianos/diagnóstico por imagem , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/fisiologia , Ratos , Ratos WistarRESUMO
The metabotrophic subtype 5 glutamate receptor (mGluR5) plays a critical role in synaptic plasticity besides its involvement in numerous neurological disorders, such as depression. As mGluR5 availability in humans is altered in sleep deprivation, we hypothesized that mGluR5 availability underlies a circadian variation. To investigate whether mGluR5 underlies potential circadian changes we measured its density in a randomized fashion at six different daytimes in 11 adult Sprague-Dawley rats. mGluR5 density was quantified by positron emission tomography (PET) using the radioactive ligand [11 C]ABP688. [11 C]ABP688 uptake was quantified in nine regions of interest with a reference tissue model. Significant differences in the binding potential (BPND ) and therefore mGluR5 availability between the different circadian times were found in cortex, cingulate cortex, amygdala, caudate putamen and nucleus accumbens. Further post-hoc statistical analysis (Tukey-Kramer test) of the different time-points revealed significant changes in BPND between 07:00 hours (start of light-on phase) and 15:00 hours (last time-point of the light-on phase) in the caudate putamen. This study shows that mGluR5 availability is increased during the light-on, or sleep phase, of rodents by approximately 10%. Given that altered mGluR5 densities play a role in psychiatric disorders, further investigation is warranted to evaluate their circadian involvement in mood changes in humans.