Simultaneous dynamic glucose-enhanced (DGE) MRI and fiber photometry measurements of glucose in the healthy mouse brain.

Glucose is the main energy source in the brain and its regulated uptake and utilization are important biomarkers of pathological brain function. Glucose Chemical Exchange Saturation Transfer (GlucoCEST) and its time-resolved version Dynamic Glucose-Enhanced MRI (DGE) are promising approaches to monitor glucose and detect tumors, since they are radioactivity-free, do not require 13C labeling and are is easily translatable to the clinics. The main principle of DGE is clear. However, what remains to be established is to which extent the signal reflects vascular, extracellular or intracellular glucose. To elucidate the compartmental contributions to the DGE signal, we coupled it with FRET-based fiber photometry of genetically encoded sensors, a technique that combines quantitative glucose readout with cellular specificity. The glucose sensor FLIIP was used with fiber photometry to measure astrocytic and neuronal glucose changes upon injection of D-glucose, 3OMG and L-glucose, in the anaesthetized murine brain. By correlating the kinetic profiles of the techniques, we demonstrate the presence of a vascular contribution to the signal, especially at early time points after injection. Furthermore, we show that, in the case of the commonly used contrast agent 3OMG, the DGE signal actually anticorrelates with the glucose concentration in neurons and astrocytes.

SAFIR-I: Design and Performance of a High-Rate Preclinical PET Insert for MRI.

(1) Background: Small Animal Fast Insert for MRI detector I (SAFIR-I) is a preclinical Positron Emission Tomography (PET) insert for the Bruker BioSpec 70/30 Ultra Shield Refrigerated (USR) preclinical 7T Magnetic Resonance Imaging (MRI) system. It is designed explicitly for high-rate kinetic studies in mice and rats with injected activities reaching 500MBq, enabling truly simultaneous quantitative PET and Magnetic Resonance (MR) imaging with time frames of a few seconds in length. (2) Methods: SAFIR-I has an axial field of view of 54.2mm and an inner diameter of 114mm. It employs Lutetium Yttrium OxyorthoSilicate (LYSO) crystals and Multi Pixel Photon Counter (MPPC) arrays. The Position-Energy-Timing Application Specific Integrated Circuit, version 6, Single Ended (PETA6SE) digitizes the MPPC signals and provides time stamps and energy information. (3) Results: SAFIR-I is MR-compatible. The system's Coincidence Resolving Time (CRT) and energy resolution are between separate-uncertainty 209.0(3)ps and separate-uncertainty 12.41(02) Full Width at Half Maximum (FWHM) at low activity and separate-uncertainty 326.89(12)ps and separate-uncertainty 20.630(011) FWHM at 550MBq, respectively. The peak sensitivity is ∼1.6. The excellent performance facilitated the successful execution of first in vivo rat studies beyond 300MBq. Based on features visible in the acquired images, we estimate the spatial resolution to be ∼2mm in the center of the Field Of View (FOV). (4) Conclusion: The SAFIR-I PET insert provides excellent performance, permitting simultaneous in vivo small animal PET/MR image acquisitions with time frames of a few seconds in length at activities of up to 500MBq.

NH4(+) triggers the release of astrocytic lactate via mitochondrial pyruvate shunting.

Neural activity is accompanied by a transient mismatch between local glucose and oxygen metabolism, a phenomenon of physiological and pathophysiological importance termed aerobic glycolysis. Previous studies have proposed glutamate and K(+) as the neuronal signals that trigger aerobic glycolysis in astrocytes. Here we used a panel of genetically encoded FRET sensors in vitro and in vivo to investigate the participation of NH4(+), a by-product of catabolism that is also released by active neurons. Astrocytes in mixed cortical cultures responded to physiological levels of NH4(+) with an acute rise in cytosolic lactate followed by lactate release into the extracellular space, as detected by a lactate-sniffer. An acute increase in astrocytic lactate was also observed in acute hippocampal slices exposed to NH4(+) and in the somatosensory cortex of anesthetized mice in response to i.v. NH4(+). Unexpectedly, NH4(+) had no effect on astrocytic glucose consumption. Parallel measurements showed simultaneous cytosolic pyruvate accumulation and NADH depletion, suggesting the involvement of mitochondria. An inhibitor-stop technique confirmed a strong inhibition of mitochondrial pyruvate uptake that can be explained by mitochondrial matrix acidification. These results show that physiological NH4(+) diverts the flux of pyruvate from mitochondria to lactate production and release. Considering that NH4(+) is produced stoichiometrically with glutamate during excitatory neurotransmission, we propose that NH4(+) behaves as an intercellular signal and that pyruvate shunting contributes to aerobic lactate production by astrocytes.

Channel-mediated lactate release by K⁺-stimulated astrocytes.

Excitatory synaptic transmission is accompanied by a local surge in interstitial lactate that occurs despite adequate oxygen availability, a puzzling phenomenon termed aerobic glycolysis. In addition to its role as an energy substrate, recent studies have shown that lactate modulates neuronal excitability acting through various targets, including NMDA receptors and G-protein-coupled receptors specific for lactate, but little is known about the cellular and molecular mechanisms responsible for the increase in interstitial lactate. Using a panel of genetically encoded fluorescence nanosensors for energy metabolites, we show here that mouse astrocytes in culture, in cortical slices, and in vivo maintain a steady-state reservoir of lactate. The reservoir was released to the extracellular space immediately after exposure of astrocytes to a physiological rise in extracellular K(+) or cell depolarization. Cell-attached patch-clamp analysis of cultured astrocytes revealed a 37 pS lactate-permeable ion channel activated by cell depolarization. The channel was modulated by lactate itself, resulting in a positive feedback loop for lactate release. A rapid fall in intracellular lactate levels was also observed in cortical astrocytes of anesthetized mice in response to local field stimulation. The existence of an astrocytic lactate reservoir and its quick mobilization via an ion channel in response to a neuronal cue provides fresh support to lactate roles in neuronal fueling and in gliotransmission.

Astrocytes adopt a progenitor-like migratory strategy for regeneration in adult brain.

Mature astrocytes become activated upon non-specific tissue damage and contribute to glial scar formation. Proliferation and migration of adult reactive astrocytes after injury is considered very limited. However, the regenerative behavior of individual astrocytes following selective astroglial loss, as seen in astrocytopathies, such as neuromyelitis optica spectrum disorder, remains unexplored. Here, we performed longitudinal in vivo imaging of cortical astrocytes after focal astrocyte ablation in mice. We discovered that perilesional astrocytes develop a remarkable plasticity for efficient lesion repopulation. A subset of mature astrocytes transforms into reactive progenitor-like (REPL) astrocytes that not only undergo multiple asymmetric divisions but also remain in a multinucleated interstage. This regenerative response facilitates efficient migration of newly formed daughter cell nuclei towards unoccupied astrocyte territories. Our findings define the cellular principles of astrocyte plasticity upon focal lesion, unravelling the REPL phenotype as a fundamental regenerative strategy of mature astrocytes to restore astrocytic networks in the adult mammalian brain. Promoting this regenerative phenotype bears therapeutic potential for neurological conditions involving glial dysfunction.

Somatotopic astrocytic activity in the somatosensory cortex.

Astrocytes play a crucial role in maintaining neuronal function and monitoring their activity. According to neuronal activity maps, the body is represented topographically in the somatosensory cortex. In rats, neighboring cortical areas receive forelimb (FL) and hindlimb (HL) sensory inputs. Whether astrocytic activity is also restricted to the cortical area receiving the respective peripheral sensory inputs is not known. Using wide field optical imaging we measured changes in the concentration of astrocytic calcium within the FL and HL sensorimotor cortex in response to peripheral sensory inputs. Mapping the calcium signals upon electrical stimulation of the forepaw and hindpaw we found activity largely restricted to the FL and HL area, respectively. In comparison to neuronal activity the time course of the astrocytic calcium activity was considerably slower. The signal took 6 s to peak after the onset of a 2 Hz and 2 s long electrical stimulation of the hindpaw and 8 s for a 4 s stimulation. The astrocytic signals were delayed relative to cerebral blood flow measured using laser speckle imaging. The intensity of both the astrocytic and neuronal signals in the HL sensorimotor cortex declined with increase in stimulation frequency. Moreover, blocking neuronal input by tetrodotoxin abolished astrocytic calcium signals. We suggest that the topographical representation of the body is not only true for cortical neurons but also for astrocytes. The maps and the frequency-dependent activations reflect strong reciprocal neuroglial communication and provide a new experimental approach to explore the role of astrocytes in health and disease.

Multimodal imaging in rats reveals impaired neurovascular coupling in sustained hypertension.

BACKGROUND AND PURPOSE: Arterial hypertension is an important risk factor for cerebrovascular diseases, such as transient ischemic attacks or stroke, and represents a major global health issue. The effects of hypertension on cerebral blood flow, particularly at the microvascular level, remain unknown. METHODS: Using the spontaneously hypertensive rat (SHR) model, we examined cortical hemodynamic responses on whisker stimulation applying a multimodal imaging approach (multiwavelength spectroscopy, laser speckle imaging, and 2-photon microscopy). We assessed the effects of hypertension in 10-, 20-, and 40-week-old male SHRs and age-matched male Wistar Kyoto rats (CTRL) on hemodynamic responses, histology, and biochemical parameters. In 40-week-old animals, losartan or verapamil was administered for 10 weeks to test the reversibility of hypertension-induced impairments. RESULTS: Increased arterial blood pressure was associated with a progressive impairment in functional hyperemia in 20- and 40-week-old SHRs; baseline capillary red blood cell velocity was increased in 40-week-old SHRs compared with age-matched CTRLs. Antihypertensive treatment reduced baseline capillary cerebral blood flow almost to CTRL values, whereas functional hyperemic signals did not improve after 10 weeks of drug therapy. Structural analyses of the microvascular network revealed no differences between normo- and hypertensive animals, whereas expression analyses of cerebral lysates showed signs of increased oxidative stress and signs of impaired endothelial homeostasis upon early hypertension. CONCLUSIONS: Impaired neurovascular coupling in the SHR evolves upon sustained hypertension. Antihypertensive monotherapy using verapamil or losartan is not sufficient to abolish this functional impairment. These deficits in neurovascular coupling in response to sustained hypertension might contribute to accelerate progression of neurodegenerative diseases in chronic hypertension.

In vivo evidence for lactate as a neuronal energy source.

Cerebral energy metabolism is a highly compartmentalized and complex process in which transcellular trafficking of metabolites plays a pivotal role. Over the past decade, a role for lactate in fueling the energetic requirements of neurons has emerged. Furthermore, a neuroprotective effect of lactate during hypoglycemia or cerebral ischemia has been reported. The majority of the current evidence concerning lactate metabolism at the cellular level is based on in vitro data; only a few recent in vivo results have demonstrated that the brain preferentially utilizes lactate over glucose. Using voltage-sensitive dye (VSD) imaging, beta-probe measurements of radiotracer kinetics, and brain activation by sensory stimulation in the anesthetized rat, we investigated several aspects of cerebral lactate metabolism. The present study is the first in vivo demonstration of the maintenance of neuronal activity in the presence of lactate as the primary energy source. The loss of the voltage-sensitive dye signal found during severe insulin-induced hypoglycemia is completely prevented by lactate infusion. Thus, lactate has a direct neuroprotective effect. Furthermore, we demonstrate that the brain readily oxidizes lactate in an activity-dependent manner. The washout of 1-[(11)C]L-lactate, reflecting cerebral lactate oxidation, was observed to increase during brain activation from 0.077 ± 0.009 to 0.105 ± 0.007 min(-1). Finally, our data confirm that the brain prefers lactate over glucose as an energy substrate when both substrates are available. Using [(18)F]fluorodeoxyglucose (FDG) to measure the local cerebral metabolic rate of glucose, we demonstrated a lactate concentration-dependent reduction of cerebral glucose utilization during experimentally increased plasma lactate levels.

Assessment of serotonin release capacity in the human brain using dexfenfluramine challenge and [18F]altanserin positron emission tomography.

Although alterations of serotonin (5-HT) system functioning have been proposed for a variety of psychiatric disorders, a direct method quantitatively assessing 5-HT release capacity in the living human brain is still lacking. Therefore, we evaluated a novel method to assess 5-HT release capacity in vivo using dexfenfluramine challenge and [(18)F]altanserin positron emission tomography (PET). Thirteen healthy male subjects received placebo and single oral doses of 40 mg (n = 6) or 60 mg (n = 7) of the potent 5-HT releaser dexfenfluramine separated by an interval of 14 days. Three further subjects received placebo on both days. Two hours after placebo/drug administration, 250 MBq of the 5-HT(2A) receptor selective PET-radiotracer [(18)F]altanserin was administered intravenously as a 30s bolus. Dynamic PET data were subsequently acquired over 90 min. Moreover, arterial blood samples were drawn for measurement of total activity and metabolite correction of the input function. Dexfenfluramine as well as cortisol and prolactin plasma concentration-time profiles was quantitatively determined. Tracer distribution volumes for five volumes-of-interest (prefrontal and occipital cortex, insula, thalamus, caudatum) were calculated by the Logan plot and a 2-tissue compartment model. Dexfenfluramine dose-dependently decreased the total distribution volume of [(18)F]altanserin in cortical regions independent of the PET modeling approach. Cortisol and prolactin plasma concentrations were dose-dependently increased by dexfenfluramine. The decrease in cortical [(18)F]altanserin receptor binding under dexfenfluramine was correlated with the increase of plasma prolactin. These data suggest that the combination of a dexfenfluramine-induced 5-HT release and subsequent assessment of 5-HT(2A) receptor availability with [(18)F]altanserin PET is suitable to measure cortical 5-HT release capacity in the human brain.

Cerebral glucose and lactate consumption during cerebral activation by physical activity in humans.

At rest, the brain takes up oxygen and carbohydrate at an ~6:1 ratio. Exercise increases systemic lactate availability reducing this to as little as 1.7:1 despite a ~20% increase in cerebral metabolic rate for oxygen (CMRo2), thus indicating a disproportionate increase of carbohydrate metabolism. Underlining mechanisms and metabolic fate for the augmented lactate uptake are unknown. This meta-analysis examines whether adrenergic activation explains the increased lactate uptake, cerebral lactate release following cerebral activation compensates for the extra carbohydrate uptake during exercise, and cerebral lactate uptake spares glucose as fuel. Ten studies (n=96) measuring arteriovenous differences for lactate, glucose, and oxygen and cerebral blood flow were included. Cerebral lactate uptake increased during brain activation by whole-body exercise compared to the resting state. Unlike glucose, lactate uptake is proportional to its arterial concentration but is unaffected by sympathetic activity. Following exercise, significant cerebral lactate released as arterial lactate levels decreased, which may balance the surplus lactate uptake in the brain during physical activity in the long term. Finally, cerebral glucose uptake was reduced by ~25% in relation to CMRo2 when cerebral lactate uptake increased, suggesting, in part, preferential lactate consumption during activation. This meta-analysis favors the notion that cerebral lactate uptake is mainly passively governed by its availability, but when lactate is available, lactate supplements glucose and supports an increase in cerebral energy metabolism in an activity-dependent manner.

Measurement of cerebral oxygen pressure in living mice by two-photon phosphorescence lifetime microscopy.

The ability to quantify partial pressure of oxygen (pO2) is of primary importance for studies of metabolic processes in health and disease. Here, we present a protocol for imaging of oxygen distributions in tissue and vasculature of the cerebral cortex of anesthetized and awake mice. We describe in vivo two-photon phosphorescence lifetime microscopy (2PLM) of oxygen using the probe Oxyphor 2P. This minimally invasive protocol outperforms existing approaches in terms of accuracy, resolution, and imaging depth. For complete details on the use and execution of this protocol, please refer to Esipova et al. (2019).

P2R Inhibitors Prevent Antibody-Mediated Complement Activation in an Animal Model of Neuromyelitis Optica : P2R Inhibitors Prevent Autoantibody Injury.

Purinergic 2 receptors (P2Rs) contribute to disease-related immune cell signaling and are upregulated in various pathological settings, including neuroinflammation. P2R inhibitors have been used to treat inflammatory diseases and can protect against complement-mediated cell injury. However, the mechanisms behind these anti-inflammatory properties of P2R inhibitors are not well understood, and their potential in CNS autoimmunity is underexplored. Here, we tested the effects of P2R inhibitors on glial toxicity in a mouse model of neuromyelitis optica spectrum disorder (NMOSD). NMOSD is a destructive CNS autoimmune disorder, in which autoantibodies against astrocytic surface antigen Aquaporin 4 (AQP4) mediate complement-dependent loss of astrocytes. Using two-photon microscopy in vivo, we found that various classes of P2R inhibitors prevented AQP4-IgG/complement-dependent astrocyte death. In vitro, these drugs inhibited the binding of AQP4-IgG or MOG-IgG to their antigen in a dose-dependent manner. Size-exclusion chromatography and circular dichroism spectroscopy revealed a partial unfolding of antibodies in the presence of various P2R inhibitors, suggesting a shared interference with IgG antibodies leading to their conformational change. Our study demonstrates that P2R inhibitors can disrupt complement activation by direct interaction with IgG. This mechanism is likely to influence the role of P2R inhibitors in autoimmune disease models and their therapeutic impact in human disease.

Neutrophils Obstructing Brain Capillaries Are a Major Cause of No-Reflow in Ischemic Stroke.

Despite successful clot retrieval in large vessel occlusion stroke, ∼50% of patients have an unfavorable clinical outcome. The mechanisms underlying this functional reperfusion failure remain unknown, and therapeutic options are lacking. In the thrombin-model of middle cerebral artery (MCA) stroke in mice, we show that, despite successful thrombolytic recanalization of the proximal MCA, cortical blood flow does not fully recover. Using in vivo two-photon imaging, we demonstrate that this is due to microvascular obstruction of ∼20%-30% of capillaries in the infarct core and penumbra by neutrophils adhering to distal capillary segments. Depletion of circulating neutrophils using an anti-Ly6G antibody restores microvascular perfusion without increasing the rate of hemorrhagic complications. Strikingly, infarct size and functional deficits are smaller in mice treated with anti-Ly6G. Thus, we propose neutrophil stalling of brain capillaries to contribute to reperfusion failure, which offers promising therapeutic avenues for ischemic stroke.

A complete pupillometry toolbox for real-time monitoring of locus coeruleus activity in rodents.

The locus coeruleus (LC) is a region in the brainstem that produces noradrenaline and is involved in both normal and pathological brain function. Pupillometry, the measurement of pupil diameter, provides a powerful readout of LC activity in rodents, primates and humans. The protocol detailed here describes a miniaturized setup that can screen LC activity in rodents in real-time and can be established within 1-2 d. Using low-cost Raspberry Pi computers and cameras, the complete custom-built system costs only ~300 euros, is compatible with stereotaxic surgery frames and seamlessly integrates into complex experimental setups. Tools for pupil tracking and a user-friendly Pupillometry App allow quantification, analysis and visualization of pupil size. Pupillometry can discriminate between different, physiologically relevant firing patterns of the LC and can accurately report LC activation as measured by noradrenaline turnover. Pupillometry provides a rapid, non-invasive readout that can be used to verify accurate placement of electrodes/fibers in vivo, thus allowing decisions about the inclusion/exclusion of individual animals before experiments begin.

Arousal-induced cortical activity triggers lactate release from astrocytes.

It has been suggested that, in states of arousal, release of noradrenaline and ß-adrenergic signalling affect long-term memory formation by stimulating astrocytic lactate production from glycogen. However, the temporal relationship between cortical activity and cellular lactate fluctuations upon changes in arousal remains to be fully established. Also, the role of ß-adrenergic signalling and brain glycogen metabolism on neural lactate dynamics in vivo is still unknown. Here, we show that an arousal-induced increase in cortical activity triggers lactate release into the extracellular space, and this correlates with a fast and prominent lactate dip in astrocytes. The immediate drop in astrocytic lactate concentration and the parallel increase in extracellular lactate levels underline an activity-dependent lactate release from astrocytes. Moreover, when ß-adrenergic signalling is blocked or the brain is depleted of glycogen, the arousal-evoked cellular lactate surges are significantly reduced. We provide in vivo evidence that cortical activation upon arousal triggers lactate release from astrocytes, a rise in intracellular lactate levels mediated by ß-adrenergic signalling and the mobilization of lactate from glycogen stores.

A beta-scintillator for surface measurements of radiotracer kinetics in the intact rodent cortex.

Beta+-sensitive probes are useful tools for the measurement of radiotracer kinetics in small animals. They allow the cost-effective development of new PET tracers and offer the possibility to investigate a variety of cerebral processes. The study's main aim was the in vivo evaluation of a probe system for cerebral surface acquisitions. The detector system is a 0.2-mm thick scintillating disk of 3-mm diameter, positioned close to the cerebral surface. The study consists of 4 subparts: (1) simulation of the detection volume, (2) direct comparison with the classic intracortical beta probe regarding its capability to acquire kinetic data, (3) test of the ability to detect local tracer accumulations during infraorbital nerve (ION) electrostimulation and (4) demonstration of the feasibility to measure tracer kinetics in awake animals. Kinetic data acquired with 18F-fluorodeoxyglucose and 15O-H2O were fitted with standard compartment models. The surface probe measurements were in good agreement with those obtained using the intracortical scintillator. ION electrostimulation induced a marked increase in tracer accumulation adequately detected by the surface probe. In the head-fixed animal, a marked change in FDG kinetics was detected between the awake and anesthetized state. The novel surface probe system proved to be a valuable instrument for in vivo radiotracer studies of the cerebral cortex. Its main advantage is the absence of any tissue damage. In addition, serial acquisitions of tracer kinetics in the awake animal turned out to be feasible.

DCC Is Required for the Development of Nociceptive Topognosis in Mice and Humans.

Avoidance of environmental dangers depends on nociceptive topognosis, or the ability to localize painful stimuli. This is proposed to rely on somatotopic maps arising from topographically organized point-to-point connections between the body surface and the CNS. To determine the role of topographic organization of spinal ascending projections in nociceptive topognosis, we generated a conditional knockout mouse lacking expression of the netrin1 receptor DCC in the spinal cord. These mice have an increased number of ipsilateral spinothalamic connections and exhibit aberrant activation of the somatosensory cortex in response to unilateral stimulation. Furthermore, spinal cord-specific Dcc knockout animals displayed mislocalized licking responses to formalin injection, indicating impaired topognosis. Similarly, humans with DCC mutations experience bilateral sensation evoked by unilateral somatosensory stimulation. Collectively, our results constitute functional evidence of the importance of topographic organization of spinofugal connections for nociceptive topognosis.

Spatial heterogeneity of low-grade gliomas at the capillary level: a PET study on tumor blood flow and amino acid uptake.

UNLABELLED: Many low-grade gliomas (World Health Organization grade II) respond to chemotherapy. Cerebral blood flow (CBF) and microvessel density may be critical for drug delivery. We used PET with (18)F-fluoro-ethyl-l-tyrosine (FET) to measure the spatial distribution of the amino acid carrier, which is located at the brain capillaries, and (15)O-H(2)O to measure tumor CBF. METHODS: Seventeen patients with low-grade glioma were studied. Region-of-interest (ROI) analysis was used to quantify tumor tracer uptake, which was normalized to cerebellar uptake (tumor-to-cerebellum ratio). "Active" tumor was defined as tumor having a radioactivity concentration that was at least 110% of the cerebellar activity. This threshold provided measures of active tumor volume, global and peak tumor CBF, and (18)F-FET uptake. Trace ROIs were applied to create voxelwise profiles of CBF and (18)F-FET uptake across tumor and brain. Standard MRI sequences were used for spatial correlations. RESULTS: Fourteen of 17 tumors showed increased global CBF and (18)F-FET uptake. Active tumor volumes ranged between 3 and 270 cm(3) for (18)F-FET and between 1 and 41 cm(3) for CBF. Global (18)F-FET uptake in tumors corresponded to CBF increases (Spearman rank rho = 0.771, P < 0.01). The volumes of increased CBF and (18)F-FET uptake spatially coincided and were also correlated (rho = 0.944, P < 0.01). Trace ROIs showed that irrespective of increased (18)F-FET uptake at the tumor periphery, CBF increases were more confined to the tumor center. Within individual tumors, spatial heterogeneity was present. Particular tumors infiltrating the corpus callosum showed low CBF and (18)F-FET uptake in this tumor region. The patterns observed with PET were not reflected on MRI of the tumors, all of which presented as homogeneous non-gadolinium-enhancing lesions. CONCLUSION: Low-grade gliomas are heterogeneous tumors with regard to the distribution of amino acid uptake and CBF. Both are coupled in the tumor center. At the tumor periphery, where tumor infiltration of surrounding brain occurs, CBF may be low irrespective of increased (18)F-FET uptake. An ongoing study is investigating the effect of chemotherapy on these observations.

Evaluation of the metabotropic glutamate receptor subtype 5 using PET and 11C-ABP688: assessment of methods.

UNLABELLED: (11)C-ABP688 is a new PET ligand to assess the subtype 5 metabotropic glutamate receptor (mGlu(5)). The purpose of this study was to evaluate different methods for the analysis of human (11)C-ABP688 data acquired from 6 healthy, young volunteers. METHODS: The methods were a 1-tissue-compartment model (K(1), k(2)''), a 2-tissue-compartment model (K1-k4), and the noncompartmental method developed by Logan. Parameters related to receptor density were the total distribution volume (DV), DV'' (= K(1)/k(2)'', 1 tissue compartment); specific DV, DV(C2) (= K(1)/k(2)' x k(3)'/k(4), 2 tissue compartments); and DV(tot) for the noncompartmental method. RESULTS: The 1-tissue-compartment model was too simple to adequately fit the data. DV(C2) calculated with the 2-tissue-compartment model ranged from 5.45 +/- 1.47 (anterior cingulate) to 1.91 +/- 0.32 (cerebellum). The corresponding values for DV(tot), calculated with the 2-tissue-compartment model and the Logan method (in parentheses), were 6.57 +/- 1.45 (6.35 +/- 1.32) and 2.93 +/- 0.53 (2.48 +/- 0.40). There was no clear evidence of a region devoid of mGlu(5) receptors. The first-pass extraction fraction exceeded 95%. The minimal scan duration to obtain stable results was estimated to be 45 min. CONCLUSION: (11)C-ABP688 displays favorable kinetics for assessing mGlu(5) receptors. For tracer kinetic modeling, 2-tissue-compartment models are clearly superior to models with only 1 tissue compartment. In comparison to the compartmental models, the Logan method is equally useful if only DV(tot) values are required and fast pixelwise parametric maps are desired. The lack of regions devoid of receptors limits the use of reference region methods that do not require arterial blood sampling. Another advantage of the tracer is the fast kinetics that allow for relatively short acquisitions.