TSPO and amyloid deposits in sub-regions of the hippocampus in the 3xTgAD mouse model of Alzheimer's disease.

The involvement of the 18kDa translocator protein (TSPO), a marker of neuroinflammation, in Alzheimer's disease (AD) remains controversial. In the present report, we used [125I]-CLINDE, a SPECT TSPO radiotracer never before used in AD, and we investigated the relationship between TSPO and amyloid plaque density (using [125I]-DRM106) in a triple transgenic mouse model of AD (3xTgAD, APPSWE, PS1M146V and TauP301L). Our results show that TSPO increases appear before those of amyloid deposits. Moreover, the different parts of the hippocampus are differentially affected. Indeed, for both TSPO and amyloid, the subiculum is affected earlier and the ventral hippocampus later than the dorsal hippocampus. In the subiculum and the dorsal hippocampus of 3xTgAD mice, a positive correlation between TSPO and of amyloid deposit levels is observed. This data supports the hypothesis that TSPO could be used as a predictive marker of amyloid pathology. In addition, our immunohistochemical data shows a segregation of TSPO in the hippocampus and immunofluorescence imaging revealed a mainly microglial origin of the TSPO expression. Thus, imaging TSPO with CLINDE may be a good alternative to PET radiotracers.

5-HT2A receptor SPECT imaging with [¹²³I]R91150 under P-gp inhibition with tariquidar: More is better?

INTRODUCTION: Pharmacological P-glycoprotein (P-gp) inhibition with tariquidar (TQD) is considered a promising strategy for the augmentation of radiotracer brain uptake. However, a region-dependent effect may compromise the robustness of quantitative studies. For this reason, we studied the effect of a TQD pretreatment on 5-HT2A imaging with [(123)I]R91150 and compared results with those obtained in Mdr1a knock-out (KO) rats. METHODS: Ex vivo autoradiography was performed in TQD (15 mg/kg) pretreated wild-type (WT-TQD), Mdr1a knock-out (KO) and untreated WT rats for Specific Binding Ratio (SBR) estimation. In vivo dynamic SPECT imaging with serial arterial blood sampling was performed in the former two groups of rats and kinetic analysis was performed with a one tissue-compartment (1TC) model and the Specific Uptake Ratio (SUR). Results were analyzed statistically using repeated measures ANOVA. RESULTS: SBR values differed between WT-TQD, Mdr1a KO and WT rats in a region-dependent manner (p<0.0001). In vivo brain uptake of radiotracer did not differ between groups. Similarly, kinetic analysis provided distribution volume (V(T)) values that did not differ significantly between groups. SUR binding potential (BPND) values from both groups highly correlated with corresponding V(T) (r=0.970, p<0.0001 and r=0.962, p<0.0001, respectively). However, SUR measured over averaged images between 100 and 120 min, using cerebellum as reference region, demonstrated values that were, by average, 2.99±0.53 times higher in the WT-TQD group, with the difference between groups being region-dependent (p<0.001). In addition, coefficient of variation of the SUR BPND values across brain regions was significantly higher in the WT-TQD rats (41.25%±9.63% versus 11.13%±5.59%, p<0.0001). CONCLUSION: P-gp inhibition with TQD leads to region-dependent effect in the rat brain, with probably sub-optimal effect in cerebellum. This warrants attention when it is used as a reference region for quantitative studies.

In Vivo Quantification of 5-HT2A Brain Receptors in Mdr1a KO Rats with 123I-R91150 Single-Photon Emission Computed Tomography.

Our goal was to identify suitable image quantification methods to image 5-hydroxytryptamine2A (5-HT2A) receptors in vivo in Mdr1a knockout (KO) rats (i.e., P-glycoprotein KO) using 123I-R91150 single-photon emission computed tomography (SPECT). The 123I-R91150 binding parameters estimated with different reference tissue models (simplified reference tissue model [SRTM], Logan reference tissue model, and tissue ratio [TR] method) were compared to the estimates obtained with a comprehensive three-tissue/seven-parameter (3T/7k)-based model. The SRTM and Logan reference tissue model estimates of 5-HT2A receptor (5-HT2AR) nondisplaceable binding potential (BPND) correlated well with the absolute receptor density measured with the 3T/7k gold standard (r > .89). Quantification of 5-HT2AR using the Logan reference tissue model required at least 90 minutes of scanning, whereas the SRTM required at least 110 minutes. The TR method estimates were also highly correlated to the 5-HT2AR density (r > .91) and only required a single 20-minute scan between 100 and 120 minutes postinjection. However, a systematic overestimation of the BPND values was observed. The Logan reference tissue method is more convenient than the SRTM for the quantification of 5-HT2AR in Mdr1a KO rats using 123I-R91150 SPECT. The TR method is an interesting and simple alternative, despite its bias, as it still provides a valid index of 5-HT2AR density.

SPECT imaging of glioma with radioiodinated CLINDE: evidence from a mouse GL26 glioma model.

BACKGROUND: Recent research has demonstrated the potential of 18-kDa translocator protein (TSPO) to serve as a target for nuclear imaging of gliomas. The aim of this study was to evaluate SPECT imaging of GL26 mouse glioma using radioiodinated CLINDE, a TSPO-specific tracer. METHODS: GL26 cells, previously transfected with an enhanced green fluorescent protein (EGFP)-expressing lentivirus, were stereotactically implanted in the striatum of C57/Bl6 mice. At 4 weeks post-injection, dynamic SPECT scans with [(123)I]CLINDE were performed. A displacement study assessed specificity of tracer binding. SPECT images were compared to results of autoradiography, fluorescence microscopy, in situ nucleic acid hybridization, histology, and immunohistochemistry. Western blotting was performed to verify TSPO production by the tumor. RESULTS: Specific uptake of tracer by the tumor is observed with a high signal-to-noise ratio. Tracer uptake by the tumor is indeed 3.26 ± 0.32 times higher than that of the contralateral striatum, and 78% of the activity is displaceable by unlabeled CLINDE. Finally, TSPO is abundantly expressed by the GL26 cells. CONCLUSIONS: The present study demonstrates the feasibility of [(123)I]CLINDE SPECT in translational studies and underlines its potential for clinical glioma SPECT imaging.

A modified simplified reference tissue model for the quantification of dopamine D2/3 receptors with [18F]fallypride images.

Defluorination of [18F]fallypride and accumulation of 18F in skull and glands leads to the contamination of brain structures with spillover activity due to partial volume effects, leading to considerable errors in binding potential estimations. Here we propose a modification of the simplified reference tissue model (SRTM) to take into account the contribution of skull activity to the radioactivity kinetic pattern in cerebellum and target regions. It consists of the introduction of an additional parameter for each volume of interest (sT) and one for the cerebellum (sR), corresponding to the fraction of skull activity contaminating these structures. Using five rat positron emission tomography experiments, we applied the modified SRTM (SRTMc), which resulted in excellent fits. As a relative means of comparison of results, we applied factor analysis (FA) to decompose dynamic data into images corresponding to brain and skull activity. With the skull factor images, we estimated the "true" sT and sR values, ultimately permitting us to fix the sR value. Parameters obtained with the SRTMc were closely correlated with values obtained from FA-corrected data. In conclusion, we propose an efficient method for reliable quantification of dopamine D2/3 receptors with single-injection [18F]fallypride scans that is potentially applicable to human studies where 18F skull accumulation compromises binding parameter estimation.

Small-animal single-photon emission computed tomographic imaging of the brain serotoninergic systems in wild-type and mdr1a knockout rats.

The pharmacokinetic properties of radiotracers are crucial for successful in vivo single-photon emission computed tomographic (SPECT) imaging. Our goal was to determine if MDR1A-deficient animals could allow better SPECT imaging outcomes than wild-type (WT) animals for a selection of serotoninergic radioligands. Thus, we compared the performances of 123I-p-MPPI, 123I-R91150, 123I-SB207710, and 123I-ADAM radioligands, for imaging of their respective targets (5-hydroxytryptamine [5-HT]1A, 5-HT2A, 5-HT4, and serotonin transporter [SERT]), in WT and Mdr1a knockout (KO) rats. With 123I-SB207710, virtually no SPECT signal was recorded in the brain of WT or KO animals. For 123I-p-MPPI, low nondisplaceable binding potentials (BPND, mean ± SD) were observed in WT (0.49 ± 0.25) and KO (0.89 ± 0.52) animals. For 123I-ADAM, modest imaging contrast was observed in WT (1.27 ± 0.02) and KO (1.31 ± 0.09) animals. For 123I-R91150, the BPND were significantly higher in Mdr1a KO (3.98 ± 0.65) animals compared to WT animals (1.22 ± 0.26). The pharmacokinetics of 123I-SB207710 and 123I-p-MPPI do not make them ideal tracers for preclinical SPECT neuroimaging. 123I-ADAM showed adequate brain uptake regardless of Mdr1a expression and appeared suitable for preclinical SPECT neuroimaging in both animal strains. The use of Mdr1a KO animals significantly improved the brain penetration of 123I-R91150, making this animal strain an interesting option when considering SPECT neuroimaging of 5-HT2A receptors in rat.

Quantification of GABAA receptors in the rat brain with [(123)I]Iomazenil SPECT from factor analysis-denoised images.

PURPOSE: In vivo imaging of GABAA receptors is essential for the comprehension of psychiatric disorders in which the GABAergic system is implicated. Small animal SPECT provides a modality for in vivo imaging of the GABAergic system in rodents using [(123)I]Iomazenil, an antagonist of the GABAA receptor. The goal of this work is to describe and evaluate different quantitative reference tissue methods that enable reliable binding potential (BP) estimations in the rat brain to be obtained. METHODS: Five male Sprague-Dawley rats were used for [(123)I]Iomazenil brain SPECT scans. Binding parameters were obtained with a one-tissue compartment model (1TC), a constrained two-tissue compartment model (2TCc), the two-step Simplified Reference Tissue Model (SRTM2), Logan graphical analysis and analysis of delayed-activity images. In addition, we employed factor analysis (FA) to deal with noise in data. RESULTS: BPND obtained with SRTM2, Logan graphical analysis and delayed-activity analysis was highly correlated with BPF values obtained with 2TCc (r=0.954 and 0.945 respectively, p<0.0001). Equally significant correlations were found between values obtained with 2TCc and SRTM2 in raw and FA-denoised images (r=0.961 and 0.909 respectively, p<0.0001). Scans of at least 100min are required to obtain stable BPND values from raw images while scans of only 70min are sufficient from FA-denoised images. These images are also associated with significantly lower standard errors of 2TCc and SRTM2 BP values. CONCLUSION: Reference tissue methods such as SRTM2 and Logan graphical analysis can provide equally reliable BPND values from rat brain [(123)I]Iomazenil SPECT. Acquisitions, however, can be much less time-consuming either with analysis of delayed activity obtained from a 20-minute scan 50min after tracer injection or with FA-denoising of images.

Innately low D2 receptor availability is associated with high novelty-seeking and enhanced behavioural sensitization to amphetamine.

High novelty-seeking has been related to an increased risk for developing addiction, but the neurobiological mechanism underlying this relationship is unclear. We investigated whether differences in dopamine (DA) D2/3-receptor (D2/3R) function underlie phenotypic divergence in novelty-seeking and vulnerability to addiction. Measures of D2/3R availability using the D2R-preferring antagonist [18F]Fallypride, and the D3R-preferring agonist [3H]-(+)-PHNO and of DA-related gene expression and behaviours were used to characterize DA signalling in Roman high- (RHA) and low-avoidance (RLA) rats, which respectively display high and low behavioural responsiveness both to novelty and psychostimulant exposure. When compared to RLA rats, high novelty-responding RHAs had lower levels of D2R, but not D3R, binding and mRNA in substantia nigra/ventral tegmental area (SN/VTA) and showed behavioural evidence of D2-autoreceptor subsensitivity. RHA rats also showed a higher expression of the tyrosine hydroxylase gene in SN/VTA, higher levels of extracellular DA in striatum and augmentation of the DA-releasing effects of amphetamine (Amph), suggesting hyperfunctioning of midbrain DA neurons. RHA rats also exhibited lower availabilities and functional sensitivity of D2R, but not D3R, in striatum, which were inversely correlated with individual scores of novelty-seeking, which, in turn, predicted the magnitude of Amph-induced behavioural sensitization. These results indicate that innately low levels of D2R in SN/VTA and striatum, whether they are a cause or consequence of the concomitantly observed elevated DA tone, result in a specific pattern of DA signalling that may subserve novelty-seeking and vulnerability to drug use. This suggests that D2R deficits in SN/VTA and striatum could both constitute neurochemical markers of an addiction-prone phenotype.

Maladaptive exploratory behavior and neuropathology of the PS-1 P117L Alzheimer transgenic mice.

Patients with the early-onset Alzheimer's disease P117L mutation in the presenilin-1 gene (PS-1) present pathological hallmarks in the hippocampus, the frontal cortex and the basal ganglia. In the present work we determined by immunohistochemistry which brain regions were injured in the transgenic PS-1 P117L mice, in comparison to their littermates, the B6D2 mice. Furthermore, as these regions are involved in novelty detection, we investigated the behavior of these mice in tests for object and place novelty recognition. Limited numbers of senile plaques and neurofibrillary tangles were detected in aged PS-1 P117L mice in the CA1 only, indicating that the disease is restrained to an initial neuropathological stage. Western blots showed a change in PSD-95 expression (p=0.03), not in NR2A subunit, NR2B subunit and synaptophysin expressions in the frontal cortex, suggesting specific synaptic alterations. The behavioral tests repeatedly revealed, despite a non-significant preference for object or place novelty, maladaptive exploratory behavior of the PS-1 P117L mice in novel environmental conditions, not due to locomotor problems. These mice, unlike the B6D2 mice, were less inhibited to visit the center of the cages (p=0.01) and they continued to move excessively in the presence of a displaced object (p=0.021). Overall, the PS-1 P117L mice appear to be in an initial Alzheimer's disease-like neuropathological stage, and they showed a lack of reaction toward novel environmental conditions.

Quantification of dopamine D(2/3) receptors in rat brain using factor analysis corrected [18F]Fallypride images.

The goal of this work is to quantify the binding parameters of [(18)F]Fallypride in the striatal and extrastriatal regions of the rat brain using factor analysis (FA) to correct small animal PET kinetic imaging for spillover defluorination radioactivity. Eleven rats were employed for YAP-(S)PET acquisitions and metabolite studies. All kinetic parameters including B'(max) and K(d)V(R) were estimated with a three-tissue compartment seven-parameter model (3T-7k) on the basis of all the FA-corrected data from the multi-injection protocol. Binding potential (BP(ND)) was calculated with Logan's graphical analysis taking cerebellum as the reference region and using the first injection raw (BP(ND-RAW)) and FA-corrected (BP(ND-FA)) data. Three distinct factors corresponding to free+non-specific binding, specific binding and skull and gland accumulation were recovered from FA with their corresponding spatial distributions. The resulting reconstructed images without skull and gland accumulation were improved to provide a better contrast between specific and non-specific regions. Very bad fits were obtained when using time-activity curves (TACs) calculated from the raw [(18)F]Fallypride data, whereas all TACs were well fitted by the 3T-7k model after FA correction. FA-corrected data enables the cerebellar region to be used as reference for the Logan approach. The magnitude of the BP(ND-FA) values was increased from 21% to 108% across regions and the rank order of BP(ND-FA) values (Cx

Chronic Δ9-tetrahydrocannabinol exposure induces a sensitization of dopamine D2/3 receptors in the mesoaccumbens and nigrostriatal systems.

Δ9-tetrahydrocannabinol (THC), through its action on cannabinoid type-1 receptor (CB1R), is known to activate dopamine (DA) neurotransmission. Functional evidence of a direct antagonistic interaction between CB1R and DA D2-receptors (D2R) suggests that D2R may be an important target for the modulation of DA neurotransmission by THC. The current study evaluated, in rodents, the effects of chronic exposure to THC (1 mg/kg/day; 21 days) on D2R and D3R availabilities using the D2R-prefering antagonist and the D3R-preferring agonist radiotracers [¹8F]fallypride and [³H]-(+)-PHNO, respectively. At 24 h after the last THC dose, D2R and D3R densities were significantly increased in midbrain. In caudate/putamen (CPu), THC exposure was associated with increased densities of D2R with no change in D2R mRNA expression, whereas in nucleus accumbens (NAcc) both D3R binding and mRNA levels were upregulated. These receptor changes, which were completely reversed in CPu but only partially reversed in NAcc and midbrain at 1 week after THC cessation, correlated with an increased functionality of D2/3R in vivo, based on findings of increased locomotor suppressive effect of a presynaptic dose and enhanced locomotor activation produced by a postsynaptic dose of quinpirole. Concomitantly, the observations of a decreased gene expression of tyrosine hydroxylase in midbrain together with a blunted psychomotor response to amphetamine concurred to indicate a diminished presynaptic DA function following THC. These findings indicate that the early period following THC treatment cessation is associated with altered presynaptic D2/3R controlling DA synthesis and release in midbrain, with the concurrent development of postsynaptic D2/3R supersensitivity in NAcc and CPu. Such D2/3R neuroadaptations may contribute to the reinforcing and habit-forming properties of THC.

Acute and chronic effects of citalopram on 5-HT1A receptor-labeling by [18F]MPPF and -coupling to receptors-G proteins.

Selective serotonin reuptake inhibitors take several weeks to produce their maximal therapeutic antidepressant effect. This delay has been attributed to the gradual desensitization of somatodendritic serotonin 5-HT(1A) autoreceptors. We evaluated adaptive changes of 5-HT(1A) receptors after acute and chronic citalopram challenges in rat. Small animal positron emission tomography trial and quantitative ex vivo autoradiography studies using [(18)F]MPPF were employed, as well as in vitro 8-OH-DPAT-stimulated [(35)S]-GTPgammaS binding assay. Additionally, 5-HT(1A) receptor knock-out mice were used to assess the specificity of [(18)F]MPPF. Acute treatment with citalopram did not alter [(18)F]MPPF binding in dorsal raphe nucleus (DR), frontal cortex, or hippocampus. The absence of [(18)F]MPPF binding in the brain of 5-HT(1A) knock-out mice demonstrates the specificity of MPPF for 5-HT(1A) receptor brain imaging, but the high affinity of [(18)F]MPPF compared to 5-HT suggests that it would only be displaced by dramatic increases in extracellular 5-HT. Chronic citalopram did not modify 5-HT(1A) receptor density in any of the brain regions studied. In addition, this treatment did not modify 8-OH-DPAT-stimulated [(35)S]-GTPgammaS binding in DR, although a significant increase was observed in frontal cortex and hippocampus. [(18)F]MPPF appears to be an efficient radioligand to quantify specifically 5-HT(1A) receptor density in brain imaging. The delayed therapeutic efficacy of citalopram did not appear to be linked to either a downregulation of 5-HT(1A) receptors or to a 5-HT(1A) receptor-G protein decoupling process in serotonergic neurons, but to increased functional sensitivity of postsynaptic 5-HT(1A) receptors.

Chloramphenicol decreases brain glucose utilization and modifies the sleep-wake cycle architecture in rats.

We studied the effects of chloramphenicol on brain glucose utilization and sleep-wake cycles in rat. After slightly anaesthetized animals were injected with [18F]fluoro-2-deoxy-D-glucose, we acquired time-concentration curves from three radiosensitive beta microprobes inserted into the right and left frontal cortices and the cerebellum, and applied a three-compartment model to calculate the cerebral metabolic rates for glucose. The sleep-wake cycle architecture was analysed in anaesthetic-free rats by recording electroencephalographic and electromyographic signals. Although chloramphenicol is a well-established inhibitor of oxidative phosphorylation, no compensatory increase in glucose utilization was detected in frontal cortex. Instead, chloramphenicol induced a significant 23% decrease in the regional cerebral metabolic rate for glucose. Such a metabolic response indicates a potential mismatch between energy supply and neuronal activity induced by chloramphenicol administration. Regarding sleep-wake states, chloramphenicol treatment was followed by a 64% increase in waking, a 20% decrease in slow-wave sleep, and a marked 59% loss in paradoxical sleep. Spectral analysis of the electroencephalogram indicates that chloramphenicol induces long-lasting modifications of delta-band power during slow-wave sleep.