In Vivo Preclinical Molecular Imaging of Repeated Exposure to an N-methyl-d-aspartate Antagonist and a Glutaminase Inhibitor as Potential Glutamatergic Modulators.

Glutamate is the principal excitatory neurotransmitter in the brain and is at the base of a wide variety of neuropathologies, including epilepsy, autism, Fragile X, and obsessive compulsive disorder. Glutamate has also become the target for novel drugs in treatment and in fundamental research settings. However, much remains unknown on the working mechanisms of these drugs and the effects of chronic administration on the glutamatergic system. This study investigated the chronic effects of two glutamate-modulating drugs with imaging techniques to further clarify their working mechanisms for future research opportunities. Animals were exposed to saline (1 ml/kg), (5S,10R)-(+)-5-Methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine (MK-801) (0.3 mg/kg), or ebselen (10 mg/kg) for 7 consecutive days. At the sixth injection, animals underwent a positron emission tomography (PET)/computed tomography (CT) with (3-(6-methyl-pyridin-2-ylethynyl)-cyclohex-2-enone-O-11C-methyl-oxime) (ABP-688) to visualize the metabotropic G protein-coupled glutamate receptor 5 (mGluR5). After the seventh injection, animals underwent a magnetic resonance spectroscopy (MRS) scan to visualize glutamate and glutamine content. Afterward, results were verified by mGluR5 immunohistochemistry (IHC). PET/CT analysis revealed that animals receiving chronic MK-801 or ebselen had a significant (P < 0.05) higher binding potential (2.90 ± 0.47 and 2.87 ± 0.46, respectively) when compared with saline (1.97 ± 0.39) in the caudate putamen. This was confirmed by mGluR5 IHC, with 60.83% ± 6.30% of the area being highlighted for ebselen and 57.14% ± 9.23% for MK-801 versus 50.21% ± 5.71% for the saline group. MRS displayed significant changes on the glutamine level when comparing chronic ebselen (2.20 ± 0.40 µmol/g) to control (2.72 ± 0.34 µmol/g). Therefore, although no direct effects on glutamate were visualized, the changes in glutamine suggest changes in the total glutamate-glutamine pool. This highlights the potential of both drugs to modulate glutamatergic pathologies.

Quantification of Metabotropic Glutamate Receptor 5 Availability With Both [11C]ABP688 and [18F]FPEB Positron Emission Tomography in the Sapap3 Knockout Mouse Model for Obsessive-Compulsive-like Behavior.

BACKGROUND: This study provides a first direct comparison between positron emission tomography radioligands targeting the allosteric site of the metabotropic glutamate receptor 5 (mGluR5): [11C]ABP688 and [18F]FPEB. A blocking paradigm was set up to substantiate the common binding site of both radioligands. Second, both radioligands were applied in Sapap3 knockout (KO) mice showing compulsive-like behavior characterized by a lower in vivo mGluR5 availability. METHODS: First, wild-type mice (n = 7) received four position emission tomography/computed tomography scans: a [11C]ABP688 scan, a [18F]FPEB scan, and two blocking scans using cold FPEB and cold ABP688, respectively. A second experiment compared both radioligands in wild-type (n = 7) and KO (n = 10) mice. The simplified reference tissue model was used to calculate the nondisplaceable binding potential representing the in vivo availability of mGluR5 in the brain. RESULTS: Using cold FPEB as a blocking compound for [11C]ABP688 micro-positron emission tomography and vice versa, we observed averaged global reductions in mGluR5 availability of circa 98% for [11C]ABP688 and 82%-96% for [18F]FPEB. For KOs, the [11C]ABP688 nondisplaceable binding potential was on average 25% lower compared with wild-type control mice (p < .0001-.001), while this was about 17% for [18F]FPEB (p < .05). CONCLUSIONS: The current findings substantiate a common binding site and suggest a strong relationship between mGluR5 availability levels measured with both radioligands. In Sapap3 KO mice, a reduced mGluR5 availability could therefore be demonstrated with both radioligands. With [11C]ABP688, higher significance levels were achieved in more brain regions. These findings suggest [11C]ABP688 as a preferable radiotracer to quantify mGluR5 availability, as exemplified here in a model for compulsive-like behavior.

Validation of a spatially variant resolution model for small animal brain PET studies.

In small animal positron emission tomography (PET) studies, given the spatial resolution of preclinical PET scanners, quantification in small regions can be challenging. Moreover, in scans where animals are placed away from the center of the field of view (CFOV), e.g. in simultaneous scans of multiple animals, quantification accuracy can be compromised due to the loss of spatial resolution towards the edge of the FOV. Here, we implemented a spatially variant resolution model to improve quantification in small regions and to allow simultaneous scanning of multiple animals without compromising quantification accuracy. The scanner's point spread function (PSF) was characterized across the FOV and modelled using a spatially variant and asymmetric Gaussian function. The spatially variant PSF (SVPSF) was then used for resolution modelling in the iterative reconstruction. To assess the image quality, a line source phantom in a cold and warm background, as well as mouse brain [18F]FDG scans, were performed. The SVPSF and the vendor's maximum a posteriori (MAP3D) reconstructions produced uniform spatial resolution across the scanner FOV, but MAP3D resulted in lower spatial resolution. The line sources recovery coefficient using SVPSF was similar at the CFOV and at the edge of the FOV. In contrast, the other tested reconstructions produced lower recovery coefficient at the edge of the FOV. In mouse brain reconstructions, less spill-over from hot regions to cold regions, as well as more symmetric regional brain uptake was observed using SVPSF. The contrast in brain images was the highest using SVPSF, in mice scanned at the CFOV and off-center. Incorporation of a spatially variant resolution model for small animal brain PET improves quantification accuracy in small regions and produces consistent image spatial resolution across the FOV. Therefore, simultaneous scanning of multiple animals can benefit by using spatially variant resolution modelling.

Sapap3 deletion causes dynamic synaptic density abnormalities: a longitudinal [11C]UCB-J PET study in a model of obsessive-compulsive disorder-like behaviour.

BACKGROUND: Currently, the evidence on synaptic abnormalities in neuropsychiatric disorders-including obsessive-compulsive disorder (OCD)-is emerging. The newly established positron emission tomography (PET) ligand ((R)-1-((3-((11)C-methyl-(11)C)pyridin-4-yl)methyl)-4-(3,4,5-trifluorophenyl)pyrrolidin-2-one) ([11C]UCB-J) provides the opportunity to visualize synaptic density changes in vivo, by targeting the synaptic vesicle protein 2A (SV2A). Here, we aim to evaluate such alterations in the brain of the SAP90/PSD-95-associated protein 3 (Sapap3) knockout (ko) mouse model, showing an abnormal corticostriatal neurotransmission resulting in OCD-like behaviour. METHODS: Longitudinal [11C]UCB-J µPET/CT scans were acquired in Sapap3 ko and wildtype (wt) control mice (n = 9/group) to study SV2A availability. Based on the Logan reference method, we calculated the volume of distribution (VT(IDIF)) for [11C]UCB-J. Both cross-sectional (wt vs. ko) and longitudinal (3 vs. 9 months) volume-of-interest-based statistical analysis and voxel-based statistical parametric mapping were performed. Both [11C]UCB-J ex vivo autoradiography and [3H]UCB-J in vitro autoradiography were used for the validation of the µPET data. RESULTS: At the age of 3 months, Sapap3 ko mice are already characterized by a significantly lower SV2A availability compared to wt littermates (i.a. cortex - 12.69%, p < 0.01; striatum - 14.12%, p < 0.001, thalamus - 13.11%, p < 0.001, and hippocampus - 12.99%, p < 0.001). Healthy ageing in control mice was associated with a diffuse and significant (p < 0.001) decline throughout the brain, whereas in Sapap3 ko mice this decline was more confined to the corticostriatal level. A strong linear relationship (p < 0.0001) was established between the outcome parameters of [11C]UCB-J µPET and [11C]UCB-J ex vivo autoradiography, while such relationship was absent for [3H]UCB-J in vitro autoradiography. CONCLUSIONS: [11C]UCB-J PET is a potential marker for synaptic density deficits in the Sapap3 ko mouse model for OCD, parallel to disease progression. Our data suggest that [11C]UCB-J ex vivo autoradiography is a suitable proxy for [11C]UCB-J PET data in mice.

Progression of obsessive compulsive disorder-like grooming in Sapap3 knockout mice: A longitudinal [11C]ABP688 PET study.

We aimed to evaluate [3-(6-methyl-pyridin-2-ylethynyl)-cyclohex-2-enone-0-11C-methyloxime] ([11C]ABP688) small animal positron emission tomography (µPET) as a biomarker to visualize possible longitudinal changes in metabotropic glutamate receptor 5 (mGluR5) availability in the brain of SAP90/PSD-95 associated protein 3 (Sapap3) knockout (ko) mice, showing obsessive compulsive disorder (OCD)-like behavior. METHODS: Alongside the assessment of grooming, we performed [11C]ABP688 µPET/CT imaging in wildtype (wt; n=10) and ko (n=11) mice both at 3 and 9 months. Using the simplified reference tissue method (SRTM), the nondisplaceable binding potential (BPND) was calculated representing the in vivo availability of the metabotropic glutamate receptor 5 (mGluR5) in the brain with the cerebellum as a reference region. Longitudinal voxel-based statistical parametric mapping (SPM) was performed on BPND images. Results were verified using [11C]ABP688 ex vivo autoradiography, [3H]ABP688 in vitro autoradiography, and mGluR5 immunohistochemistry. RESULTS: Cross-sectional comparisons revealed significantly increased grooming parameters in ko animals, at both time points. A significant longitudinal increase in % grooming duration (+268.25%; p<0.05) reflected aggravation of this behavior in ko mice. [11C]ABP688 µPET revealed significantly lower mGluR5 availability in the cortex, striatum, hippocampus, and amygdala of ko mice at both ages. A significant longitudinal BPND decline was present for ko mice (p<0.01: cortex -17.14%, striatum -19.82%, amygdala -23.57%; p<0.05: hippocampus -15.53%), which was confirmed by SPM (p<0.01). CONCLUSION: Sapap3 ko mice show a decline in mGluR5 availability in OCD relevant brain regions parallel to the worsening of OCD-like behavior. This demonstrates a potential role for [11C]ABP688 PET as a biomarker to monitor disease progression in vivo.

Neuroreceptor kinetics in rats repeatedly exposed to quinpirole as a model for OCD.

BACKGROUND: Obsessive-compulsive disorder (OCD) is a chronic, incapacitating, early onset psychiatric disorder that is characterized by obsessions and compulsions originating from a disturbance in the cortico-striato-thalamico-cortical circuit. We implemented the preclinical quinpirole (QP) rat model for compulsive checking in OCD to analyse the behaviour and visualize the D2R, mGluR5 and GLT1 density in order to contribute to the understanding of the neuroreceptor kinetics. METHODS: Animals (n = 14) were exposed to either saline (1 mL/kg) or QP (dopamine D2-agonist, 0.5 mg/kg) twice-weekly during 7 weeks. After each injection animals were placed on an open field test. After model setup, animals were placed in a behavioural cage equipped with tracking software and hardware in order to analyse the behaviour. Subsequently, sagittal slides were made of the CP in the right hemisphere and a staining was done with the D2R, mGluR5 and GLT-1 antibody to visualize the corresponding receptor. RESULTS: The QP animals displayed a strong increase in travelled distance (+596.70%) and in the number of homebase visits (+1222.90%) compared to the control animals. After chronic exposure to QP, animals had a significantly (p < 0.05) higher percentage of D2R density in the CP (7.92% ± 0.48%) versus 6.66% ± 0.28% in animals treated with saline. There were no differences for mGluR5 and GLT1 receptor density. CONCLUSIONS: Chronic exposure to QP leads to hyperlocomotion and an increase in D2R density. Furthermore, as mGluR5 and GLT1 density did not seem to be directly affected, decreased levels of glutamate might have influenced the binding potential in earlier reports.

Awake 18F-FDG PET Imaging of Memantine-Induced Brain Activation and Test-Retest in Freely Running Mice.

PET scans of the mouse brain are usually performed with anesthesia to immobilize the animal. However, it is desirable to avoid the confounding factor of anesthesia in mouse-brain response. Methods: We developed and validated brain PET imaging of awake, freely moving mice. Head-motion tracking was performed using radioactive point-source markers, and we used the tracking information for PET-image motion correction. Regional 18F-FDG brain uptake in a test, retest, and memantine-challenge study was measured in awake (n = 8) and anesthetized (n = 8) C57BL/6 mice. An awake uptake period was considered for the anesthesia scans. Results: Awake (motion-corrected) PET images showed an 18F-FDG uptake pattern comparable to the pattern of anesthetized mice. The test-retest variability (represented by the intraclass correlation coefficient) of the regional SUV quantification in the awake animals (0.424-0.555) was marginally lower than that in the anesthetized animals (intraclass correlation coefficient, 0.491-0.629) over the different regions. The increased memantine-induced 18F-FDG uptake was more pronounced in awake (+63.6%) than in anesthetized (+24.2%) animals. Additional behavioral information, acquired for awake animals, showed increased motor activity on a memantine challenge (total distance traveled, 18.2 ± 5.28 m) compared with test-retest (6.49 ± 2.21 m). Conclusion: The present method enables brain PET imaging on awake mice, thereby avoiding the confounding effects of anesthesia on the PET reading. It allows the simultaneous measurement of behavioral information during PET acquisitions. The method does not require any additional hardware, and it can be deployed in typical high-throughput scan protocols.

MicroPET Outperforms Beta-Microprobes in Determining Neuroreceptor Availability under Pharmacological Restriction for Cold Mass Occupancy.

Both non-invasive micro-positron emission tomography (µPET) and in situ beta-microprobes have the ability to determine radiotracer kinetics and neuroreceptor availability in vivo. Beta-microprobes were proposed as a cost-effective alternative to µPET, but literature revealed conflicting results most likely due to methodological differences and inflicted tissue damage. The current study has three main objectives: (i) evaluate the theoretical advantages of beta-microprobes; (ii) perform µPET imaging to assess the impact of (beta-micro)probe implantation on relative tracer delivery (R1) and receptor occupancy (non-displaceable binding potential, BPND) in the rat brain; and (iii) investigate whether beta-microprobe recordings produce robust results when a pharmacological restriction for cold mass dose (tracer dose condition) is imposed. We performed acquisitions (n = 61) in naive animals, dummy probe implanted animals (outer diameter: 0.75 and 1.00 mm) and beta-microprobe implanted animals (outer diameter: 0.75 mm) using two different radiotracers with high affinity for the striatum: [11C]raclopride (n = 29) and [11C]ABP688 (n = 32). In addition, acquisitions were completed with or without an imposed restriction for cold mass occupancy. We estimated BPND and R1 values using the simplified reference tissue method (SRTM). [11C]raclopride dummy µPET BPND (0.75 mm: -13.01 ± 0.94%; 1.00 mm: -13.89 ± 1.20%) and R1 values (0.75 mm: -29.67 ± 4.94%; 1.00 mm: -39.07 ± 3.17%) significantly decreased at the implant side vs. the contralateral intact side. A similar comparison for [11C]ABP688 dummy µPET, demonstrated significantly (p < 0.05) decreased BPND (-19.09 ± 2.45%) and R1 values (-38.12 ± 6.58%) in the striatum with a 1.00 mm implant, but not with a 0.75 mm implant. Particularly in tracer dose conditions, despite lower impact of partial volume effects, beta-microprobes proved unfit to produce representative results due to tissue destruction associated with probe insertion. We advise to use tracer dose µPET to obtain accurate results concerning receptor availability and tracer delivery, keeping in mind associated partial volume effects for which it is possible to correct.