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.

Design of a serotonin 4 receptor radiotracer with decreased lipophilicity for single photon emission computed tomography.

With the aim to develop a suitable radiotracer for the brain imaging of the serotonin 4 receptor subtype (5-HT4R) using single photon emission computed tomography (SPECT), we synthesized and evaluated a library of di- and triazaphenanthridines with lipophilicity values which were in the range expected to favour brain penetration, and which demonstrated specific binding to the target of interest. Adding additional nitrogen atoms to previously described phenanthridine ligands exhibiting a high unspecific binding, we were able to design a radioiodinated compound [(125)I]14. This compound exhibited a binding affinity value of 0.094 nM toward human 5-HT4R and a high selectivity over other serotonin receptor subtypes (5-HTR). In vivo SPECT imaging studies and competition experiments demonstrated that the decreased lipophilicity (in comparison with our previously reported compounds 4 and 5) allowed a more specific labelling of the 5-HT4R brain-containing regions.

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.

Improved leptin sensitivity as a potential candidate responsible for the spontaneous food restriction of the Lou/C rat.

The Lou/C rat, an inbred strain of Wistar origin, was described as a model of resistance to age- and diet-induced obesity. Although such a resistance involves many metabolic parameters described in our previous studies, Lou/C rats also exhibit a spontaneous food restriction due to decreased food consumption during the nocturnal period. We then attempted to delineate the leptin sensitivity and mechanisms implicated in this strain, using different protocols of acute central and peripheral leptin administration. A first analysis of the meal patterns revealed that Lou/C rats eat smaller meals, without any change in meal number compared to age-matched Wistar animals. Although the expression of the recognized leptin transporters (leptin receptors and megalin) measured in the choroid plexus was normal in Lou/C rats, the decreased triglyceridemia observed in these animals is compatible with an increased leptin transport across the blood brain barrier. Improved hypothalamic leptin signaling in Lou/C rats was also suggested by the higher pSTAT3/STAT3 (signal transducer and activator of transcription 3) ratio observed following acute peripheral leptin administration, as well as by the lower hypothalamic mRNA expression of the suppressor of cytokine signaling 3 (SOCS3), known to downregulate leptin signaling. To conclude, spontaneous hypophagia of Lou/C rats appears to be related to improved leptin sensitivity. The main mechanism underlying such a phenomenon consists in improved leptin signaling through the Ob-Rb leptin receptor isoform, which seems to consequently lead to overexpression of brain-derived neurotrophic factor (BDNF) and thyrotropin-releasing hormone (TRH).

Synthesis and structure-affinity relationships of selective high-affinity 5-HT(4) receptor antagonists: application to the design of new potential single photon emission computed tomography tracers.

The work described herein aims at finding new potential ligands for the brain imaging of 5-HT(4) receptors (5-HT(4)Rs) using single-photon emission computed tomography (SPECT). Starting from the nonsubstituted phenanthridine compound 4a, exhibiting a K(i) value of 51 nM on the 5-HT(4)R, we explored the structure-affinity in this series. We found that substitution in position 4 of the tricycle with a fluorine atom gave the best result. Introduction of an additional nitrogen atom inside the tricyclic framework led to an increase of both the affinity and selectivity for 5-HT(4)R, suggesting the design of the antagonist 4v, exhibiting a high affinity of 0.04 nM. Several iodinated analogues were then synthesized as potential SPECT tracers. The iodinated compound 11d was able to displace the reference radioiodinated 5-HT(4)R antagonist (1-butylpiperidin-4-yl)methyl-8-amino-7-iodo[(123)I]-2,3-dihydrobenzo[b][1,4]dioxine-5-carboxylate {[(123)I]1, [(123)I]SB 207710} both in vitro and in vivo in brain. Compound 11d was radiolabeled with [(125)I]iodine, providing a potential SPECT candidate for brain imaging of 5-HT(4)R.

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

Clusterin in neurological disorders: molecular perspectives and clinical relevance.

Firstly discovered in rete testis fluid, clusterin is a glycoprotein present in most of the other biological fluids. Several isoforms of clusterin are encoded from a single gene located on chromosome 8 in human species. Among the different isoforms, the secreted form of clusterin is expressed by a variety of tissues, including the nervous system under normal conditions. This form is presumed to play an anti-apoptotic role and seems to be a major determinant in cell survival and neuroplasticity after stroke. In animal models of this pathology, both neuronal and astroglial subpopulations express high levels of clusterin early after the ischemic damage. Recent lines of evidence point also to its possible involvement in neurodegenerative disorders. It is thought that in Alzheimer's disease the association between amyloidogenic peptides and clusterin contributes to limit Aß species misfolding and facilitates their clearance from the extracellular space. Thus, intercellular and intracellular factors that modulate local clusterin expression in the nervous system may represent potent targets for neurodegenerative disease therapies. In this review we provide a critical overview of the most recent data on the involvement of clusterin in neurodegenerative diseases with special reference to their putative clinical relevance.

Synthesis and characterization of a iodine-125-labeled pyrrolo[1,2-a]thieno[3,2-e]pyrazine and evaluation as a potential 5-HT4R SPECT tracer.

In the aim to find new radiotracers for the in vivo imaging of 5-HT(4) receptors by ultra-high resolution quantitative SPECT, we have developed the synthesis of a radioiodinated 5-HT(4) ligand using an iododestannylation procedure. The [(125)I]-ligand was obtained in a high radiochemical yield. Preliminary autoradiographic and ex vivo studies failed to show a specific labeling of 5-HT(4) receptors.

Brain serotonergic circuitries.

Brain serotonergic circuitries interact with other neurotransmitter systems on a multitude of different molecular levels. In humans, as in other mammalian species, serotonin (5-HT) plays a modulatory role in almost every physiological function. Furthermore, serotonergic dysfunction is thought to be implicated in several psychiatric and neurodegenerative disorders. We describe the neuroanatomy and neurochemistry of brain serotonergic circuitries. The contribution of emergent in vivo imaging methods to the regional localization of binding site receptors and certain aspects of their functional connectivity in correlation to behavior is also discussed. 5-HT cell bodies, mainly localized in the raphe nuclei, send axons to almost every brain region. It is argued that the specificity of the local chemocommunication between 5-HT and other neuronal elements mainly depends on mechanisms regulating the extracellular concentration of 5-HT the diversity of high-affinity membrane receptors, and their specific transduction modalities.

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.

Extracellular clusterin promotes neuronal network complexity in vitro.

Clusterin (apolipoprotein J), a highly conserved amphiphatic glycoprotein and chaperone, has been implicated in a wide range of physiological and pathological processes. As a secreted protein, clusterin has been shown to act extracellularly where it is involved in lipid transportation and clearance of cellular debris. Intracellularly, clusterin may regulate signal transduction and is upregulated after cell stress. After neural injury, clusterin may be involved in nerve cell survival and postinjury neuroplasticity. In this study, we investigated the role of extracellular clusterin on neuronal network complexity in vitro. Quantitative analysis of clustrin-treated neuronal cultures showed significantly higher network complexity. These findings suggest that in addition to previously demonstrated neuroprotective roles, clusterin may also be involved in neuronal process formation, elongation, and plasticity.

Clusterin increases post-ischemic damages in organotypic hippocampal slice cultures.

Clusterin or apolipoprotein J is a heterodimeric glycoprotein which is known to be increased during tissue involution in response to hormonal changes or injury and under circumstances leading to apoptosis. Previous studies in wild-type (WT) and clusterin-null (Clu-/-) mice indicated a protective role of clusterin over-expression in astrocytes lasting up to 90 days post-ischemia. However, in in vitro and in vivo models of neonatal hypoxia-ischemia, clusterin exacerbates necrotic cell death. We developed recombinant forms of clusterin and examined their effect on propidium iodide uptake, neuronal and synaptic markers as well as electrophysiological recordings in hippocampal slice cultures from Clu-/- and WT mice subjected to oxygen-glucose deprivation (OGD). WT mice displayed a marked up-regulation of clusterin associated with electrophysiological deficits and dramatic increase of propidium iodide uptake 5 days post-OGD. Immunocytochemical and western blot analyses revealed a substantial decrease of neuronal nuclei and synaptophysin immunoreactivity that predominated in WT mice. These findings contrasted with the relative post-OGD resistance of Clu-/- mice. The addition of biologically active recombinant forms of human clusterin for 24 h post-OGD led to the abolishment of the ischemic tolerance in Clu-/- slices. This deleterious effect of clusterin was reverted by the concomitant administration of the NMDA receptor antagonist, d-2-amino-5-phosphonopentanoate. The present data indicate that in an in vitro model of ischemia characterized by the predominance of NMDA-mediated cell death, clusterin exerts a negative effect on the structural integrity and functionality of hippocampal neurons.

In vivo quantification of 5-HT1A-[18F]MPPF interactions in rats using the YAP-(S)PET scanner and a beta-microprobe.

We performed full modeling analysis of 5-HT(1A)-[(18)F]MPPF interactions using the beta-microprobe (beta P) and a YAP-(S)PET scanner. Sixteen Wistar rats were used for beta P (n=5) and YAP-(S)PET (n=5) acquisitions and metabolite studies (n=6). Time-concentration curves were obtained in the hippocampus, raphe dorsalis, frontal cortex and cerebellum, using three injections of [(18)F]MPPF at different specific activities. B'(max) values were estimated from a two (2T-5k)- and three (3T-7k)-tissue-compartment model with beta P and YAP-(S)PET time-concentration curves. The simplified reference tissue model (SRTM) was used to estimate binding potential (BP(SRTM)) values from data obtained with the first injection and the cerebellum as the reference region. Overall, the 3T-7k model provided a better fit than the 2T-5k model, as evaluated from AIC criteria in all experiments. The rank order of receptor density (B'max) values was as follows: hippocampus>raphe approximately frontal cortex>cerebellum. Non-negligible specific binding was observed in the cerebellum (B'max (beta P)=1.5+/-0.9 pmol/ml). Significant correlations (p<0.001) between B'max and BP(SRTM) values were evident with both beta P (r=0.895) and YAP-(S)PET (r=0.695). The YAP-(S)PET system underestimated the [18F]MPPF binding levels in brain due to limited resolution (i.e. partial volume), but led to similar conclusions.

Anorexia induced by activation of serotonin 5-HT4 receptors is mediated by increases in CART in the nucleus accumbens.

Anorexia nervosa is a growing concern in mental health, often inducing death. The potential neuronal deficits that may underlie abnormal inhibitions of food intake, however, remain largely unexplored. We hypothesized that anorexia may involve altered signaling events within the nucleus accumbens (NAc), a brain structure involved in reward. We show here that direct stimulation of serotonin (5-hydroxytryptamine, 5-HT) 4 receptors (5-HT(4)R) in the NAc reduces the physiological drive to eat and increases CART (cocaine- and amphetamine-regulated transcript) mRNA levels in fed and food-deprived mice. It further shows that injecting 5-HT(4)R antagonist or siRNA-mediated 5-HT(4)R knockdown into the NAc induced hyperphagia only in fed mice. This hyperphagia was not associated with changes in CART mRNA expression in the NAc in fed and food-deprived mice. Results include that 5-HT(4)R control CART mRNA expression into the NAc via a cAMP/PKA signaling pathway. Considering that CART may interfere with food- and drug-related rewards, we tested whether the appetite suppressant properties of 3,4-N-methylenedioxymethamphetamine (MDMA, ecstasy) involve the 5-HT(4)R. Using 5-HT(4)R knockout mice, we demonstrate that 5-HT(4)R are required for the anorectic effect of MDMA as well as for the MDMA-induced enhancement of CART mRNA expression in the NAc. Directly injecting CART peptide or CART siRNA into the NAc reduces or increases food consumption, respectively. Finally, stimulating 5-HT(4)R- and MDMA-induced anorexia were both reduced by injecting CART siRNA into the NAc. Collectively, these results demonstrate that 5-HT(4)R-mediated up-regulation of CART in the NAc triggers the appetite-suppressant effects of ecstasy.

Metabolic compartmentalization in the human cortex and hippocampus: evidence for a cell- and region-specific localization of lactate dehydrogenase 5 and pyruvate dehydrogenase.

BACKGROUND: For a long time now, glucose has been thought to be the main, if not the sole substrate for brain energy metabolism. Recent data nevertheless suggest that other molecules, such as monocarboxylates (lactate and pyruvate mainly) could be suitable substrates. Although monocarboxylates poorly cross the blood brain barrier (BBB), such substrates could replace glucose if produced locally. The two key enzymatiques systems required for the production of these monocarboxylates are lactate dehydrogenase (LDH; EC1.1.1.27) that catalyses the interconversion of lactate and pyruvate and the pyruvate dehydrogenase complex that irreversibly funnels pyruvate towards the mitochondrial TCA and oxydative phosphorylation. RESULTS: In this article, we show, with monoclonal antibodies applied to post-mortem human brain tissues, that the typically glycolytic isoenzyme of lactate dehydrogenase (LDH-5; also called LDHA or LDHM) is selectively present in astrocytes, and not in neurons, whereas pyruvate dehydrogenase (PDH) is mainly detected in neurons and barely in astrocytes. At the regional level, the distribution of the LDH-5 immunoreactive astrocytes is laminar and corresponds to regions of maximal 2-deoxyglucose uptake in the occipital cortex and hippocampus. In hippocampus, we observed that the distribution of the oxidative enzyme PDH was enriched in the neurons of the stratum pyramidale and stratum granulosum of CA1 through CA4, whereas the glycolytic enzyme LDH-5 was enriched in astrocytes of the stratum moleculare, the alveus and the white matter, revealing not only cellular, but also regional, selective distributions. The fact that LDH-5 immunoreactivity was high in astrocytes and occurred in regions where the highest uptake of 2-deoxyglucose was observed suggests that glucose uptake followed by lactate production may principally occur in these regions. CONCLUSION: These observations reveal a metabolic segregation, not only at the cellular but also at the regional level, that support the notion of metabolic compartmentalization between astrocytes and neurons, whereby lactate produced by astrocytes could be oxidized by neurons.

Distribution of neurokinin A-like and serotonin immunoreactivities within the vertical lobe complex in Sepia officinalis.

Immunohistochemistry, using antibodies raised against mammalian neurokinin A (NKA) and serotonin (5-HT), was applied in double-staining experiments to map these molecules within the vertical lobe complex (inferior frontal, superior frontal, post-frontal, vertical, subvertical and precommissural lobes). NKA-like and 5-HT immunoreactivities were detected in all the lobes of the vertical lobe complex but were never colocalized in cell bodies or fibres. Except for the cell layers of the superior frontal lobe, both types of labelled cell bodies were observed in all the lobes. Both types of immunoreactive fibres were detected in all the neuropils and interestingly revealed clear subdivisions within some lobes, e.g., 5-HT-IR fibres were more abundant in the peripheral part of the vertical lobe whereas NKA-IR ones were widely observed in both the peripheral and central parts. In cephalopods, the vertical lobe complex is involved in learning and memory; thus, our results strongly suggest that one or more NKA-like and 5-HT molecules may function as neurochemical messengers in these cognitive processes.

The NADPH oxidase NOX4 drives cardiac differentiation: Role in regulating cardiac transcription factors and MAP kinase activation.

Reactive oxygen species (ROS) generated by the NOX family of NADPH oxidases have been described to act as second messengers regulating cell growth and differentiation. However, such a function has hitherto not been convincingly demonstrated. We investigated the role of NOX-derived ROS in cardiac differentiation using mouse embryonic stem cells. ROS scavengers prevented the appearance of spontaneously beating cardiac cells within embryoid bodies. Down-regulation of NOX4, the major NOX isoform present during early stages of differentiation, suppressed cardiogenesis. This was rescued by a pulse of low concentrations of hydrogen peroxide 4 d before spontaneous beating appears. Mechanisms of ROS-dependent signaling included p38 mitogen-activated protein kinase (MAPK) activation and nuclear translocation of the cardiac transcription factor myocyte enhancer factor 2C (MEF2C). Our results provide first molecular evidence that the NOX family of NADPH oxidases regulate vertebrate developmental processes.

A comparison of in vivo and in vitro neuroimaging of 5-HT 1A receptor binding sites in the cat brain.

To validate the cat as a suitable model for positron emission tomography imaging (PET) and to gain further knowledge on the anatomical distribution of the serotonin-1A receptor (5-HT 1A) in the feline brain, we used PET with [18F]MPPF and in vitro autoradiography with [3H]MPPF, [3H]8-OH-DPAT and [3H]paroxetine. PET radioactivity curves with [18F]MPPF were very reproducible in anaesthetized cats, with the highest radioactivity uptakes recorded in the hippocampus, cingulate cortex, septum, infralimbic cortex and raphe nucleus, whereas the lowest were found in the cerebellum. [3H]8-OH-DPAT binding displayed a comparable, albeit lower, regional distribution than with [3H]MPPF. Autoradiography also revealed the presence of 5-HT 1A receptor binding sites in the cortex and in the interpeduncular nucleus, due to its greater sensitivity and spatial resolution compared with PET imaging. The cat constitutes an interesting experimental model for PET imaging, as many physiological concepts have been well established with this animal. Our study also shows the advantages of combining complementary neuroimaging techniques such as in vivo PET imaging and in vitro autoradiography to visualize the distribution of the 5-HT 1A receptors.