PIXSIC, a pixelated ß⁺-sensitive probe for radiopharmacological investigations in rat brain: binding studies with [¹8F]MPPF.

PURPOSE: The aim of this work was to demonstrate the pharmacokinetic potential of a wireless pixelated ß(+)-sensitive probe (PIXSIC). PROCEDURES: The binding of 2'-methoxyphenyl-(N-2'-pyridinyl)-p-[(18)F]fluoro-benzamidoethylpiperazine ([(18)F]MPPF), a 5-HT1A serotonin receptor radiopharmaceutical, was measured in anesthetized rats and compared to microPET data. The effects of a 5-HT1A antagonist injection on in vivo [(18)F]MPPF binding were monitored by PIXSIC. RESULTS: PIXSIC allowed differentiating the radioactive kinetics according to the location of its pixels in the hippocampus, cortex, corpus callosum, and cerebellum. The device accurately detected the changes in [(18)F]MPPF binding, after 5-HT1A antagonist blockade. The time-activity curves were reproducible and consistent with kinetics obtained simultaneously with a microPET camera. CONCLUSIONS: These results demonstrate the ability of the PIXSIC device to record reliably the binding of PET ligands, with a high spatiotemporal resolution in anesthetized rodents. These first in vivo results are a key stage on the path to its implementation in awake freely moving animals.

Functional ultrasound imaging reveals different odor-evoked patterns of vascular activity in the main olfactory bulb and the anterior piriform cortex.

Topographic representation of the outside world is a key feature of sensory systems, but so far it has been difficult to define how the activity pattern of the olfactory information is distributed at successive stages in the olfactory system. We studied odor-evoked activation patterns in the main olfactory bulb and the anterior piriform cortex of rats using functional ultrasound (fUS) imaging. fUS imaging is based on the use of ultrafast ultrasound scanners and detects variations in the local blood volume during brain activation. It makes deep brain imaging of ventral structures, such as the piriform cortex, possible. Stimulation with two different odors (hexanal and pentylacetate) induced the activation of odor-specific zones that were spatially segregated in the main olfactory bulb. Interestingly, the same odorants triggered the activation of the entire anterior piriform cortex, in all layers, with no distinguishable odor-specific areas detected in the power Doppler images. These fUS imaging results confirm the spatial distribution of odor-evoked activity in the main olfactory bulb, and furthermore, they reveal the absence of such a distribution in the anterior piriform cortex at the macroscopic scale in vivo.

A wireless beta-microprobe based on pixelated silicon for in vivo brain studies in freely moving rats.

The investigation of neurophysiological mechanisms underlying the functional specificity of brain regions requires the development of technologies that are well adjusted to in vivo studies in small animals. An exciting challenge remains the combination of brain imaging and behavioural studies, which associates molecular processes of neuronal communications to their related actions. A pixelated intracerebral probe (PIXSIC) presents a novel strategy using a submillimetric probe for beta(+) radiotracer detection based on a pixelated silicon diode that can be stereotaxically implanted in the brain region of interest. This fully autonomous detection system permits time-resolved high sensitivity measurements of radiotracers with additional imaging features in freely moving rats. An application-specific integrated circuit (ASIC) allows for parallel signal processing of each pixel and enables the wireless operation. All components of the detector were tested and characterized. The beta(+) sensitivity of the system was determined with the probe dipped into radiotracer solutions. Monte Carlo simulations served to validate the experimental values and assess the contribution of gamma noise. Preliminary implantation tests on anaesthetized rats proved PIXSIC's functionality in brain tissue. High spatial resolution allows for the visualization of radiotracer concentration in different brain regions with high temporal resolution.

A method based on Monte Carlo simulations and voxelized anatomical atlases to evaluate and correct uncertainties on radiotracer accumulation quantitation in beta microprobe studies in the rat brain.

The beta-microprobe is a simple and versatile technique complementary to small animal positron emission tomography (PET). It relies on local measurements of the concentration of positron-labeled molecules. So far, it has been successfully used in anesthetized rats for pharmacokinetics experiments and for the study of brain energetic metabolism. However, the ability of the technique to provide accurate quantitative measurements using (18)F, (11)C and (15)O tracers is likely to suffer from the contribution of 511 keV gamma rays background to the signal and from the contribution of positrons from brain loci surrounding the locus of interest. The aim of the present paper is to provide a method of evaluating several parameters, which are supposed to affect the quantification of recordings performed in vivo with this methodology. We have developed realistic voxelized phantoms of the rat whole body and brain, and used them as input geometries for Monte Carlo simulations of previous beta-microprobe reports. In the context of realistic experiments (binding of (11)C-Raclopride to D2 dopaminergic receptors in the striatum; local glucose metabolic rate measurement with (18)F-FDG and H(2)O(15) blood flow measurements in the somatosensory cortex), we have calculated the detection efficiencies and corresponding contribution of 511 keV gammas from peripheral organs accumulation. We confirmed that the 511 keV gammas background does not impair quantification. To evaluate the contribution of positrons from adjacent structures, we have developed beta-Assistant, a program based on a rat brain voxelized atlas and matrices of local detection efficiencies calculated by Monte Carlo simulations for several probe geometries. This program was used to calculate the 'apparent sensitivity' of the probe for each brain structure included in the detection volume. For a given localization of a probe within the brain, this allows us to quantify the different sources of beta signal. Finally, since stereotaxic accuracy is crucial for quantification in most microprobe studies, the influence of stereotaxic positioning error was studied for several realistic experiments in favorable and unfavorable experimental situations (binding of (11)C-Raclopride to D2 dopaminergic receptors in the striatum; binding of (18)F-MPPF to 5HT1A receptors in the dorsal raphe nucleus).

Simultaneous in vivo magnetic resonance imaging and radioactive measurements with the beta-MicroProbe.

PURPOSE: Multimodal instrumentation is a new technical approach allowing simultaneous and complementary in vivo recordings of complementary biological parameters. To elucidate further the physiopathological mechanisms in intact small animal models, especially for brain studies, a challenging issue is the actual coupling of magnetic resonance imaging (MRI) techniques with positron emission tomography (PET): it has been shown that running the technology for radioactive imaging in a magnet alters the spatiotemporal performance of both modalities. Thus, we propose an alternative coupling of techniques that uses the beta-MicroProbe instead of PET for local measurements of radioactivity coupled with MRI. METHODS: We simultaneously recorded local radioactivity due to [(18)F]MPPF (a 5-HT(1A) receptor PET radiotracer) binding in the hippocampus with the beta-MicroProbe and carried out anatomical MRI in the same anaesthetised rat. RESULTS: The comparison of [(18)F]MPPF kinetics obtained from animals in a magnet with kinetics from a control group outside the magnet allowed us to determine the stability of tracer biokinetic measurements over time in the magnet. We were thus able to show that the beta-MicroProbe reliably measures radioactivity in rat brains under an intense magnetic field of 7 Tesla. CONCLUSION: The biological validation of a beta-MicroProbe/MRI dual system reported here opens up a wide range of future multimodal approaches for functional and pharmacological measurements by the probe combined with various magnetic resonance technologies, including anatomical MRI, functional MRI and MR spectroscopy.

The potential of the beta-Microprobe, an intracerebral radiosensitive probe, to monitor the [(18)F]MPPF binding in the rat dorsal raphe nucleus.

The aim of this study was to demonstrate the ability of a recently developed beta(+)-range sensitive intracerebral probe (beta-Microprobe) to measure the binding kinetics of [(18)F]MPPF, a well-documented 5-HT(1A) serotoninergic receptor ligand, in the dorsal raphe nucleus (DRN) of the anaesthetised rat. This midbrain nucleus presents a high concentration of 5-HT(1A) receptors known to be implicated in the effects of antidepressants. The difficulty confronting this study lay in the fact that the dimensions of the DRN are smaller than the detection volume of the beta-Microprobe. In the first part of the study, we studied the feasibility of this measurement from a theoretical point of view by autoradiography and a Monte Carlo simulation. We determined the optimal beta-Microprobe location close to the DRN and verified that this configuration allowed accurate determination of [(18)F]MPPF specific binding in the nucleus. In the second part of our study, we measured the in vivo time-concentration curves of [(18)F]MPPF binding in the DRN in comparison with the cerebellum. The specificity of [(18)F]MPPF binding in the DRN was confirmed by its displacement after non-labelled 5-HT(1A)antagonist injection (MPPF or WAY-100635). Moreover, we verified the feasibility of using beta-Microprobe monitoring and simultaneous validation by microdialysis to study the effect of an increase in extracellular serotonin, induced by fenfluramine injection, on [(18)F]MPPF binding in the DRN. Our theoretical simulations, confirmed by our experimental results, demonstrate the ability of this new device to monitor in vivo the binding of [(18)F]MPPF in the DRN of anaesthetised rodents.

Radio-imaging for quantitative autoradiography in biology.

We present here an overview of new in vitro and ex vivo radio-imaging systems developed to overcome the limitations of films and emulsions currently used in histological autoradiography experiments. The shortcomings of films for quantitative studies are first introduced. Principles and performances of each family of imagers are discussed and illustrated in various biological contexts. Finally, perspectives of development including nonradioactive labeling techniques are briefly presented.

A new high resolution radioimager for the quantitative analysis of radiolabelled molecules in tissue section.

We present a high-speed, high-resolution imager of beta particles. It is devoted to be used in autoradiography experiments such as receptor binding or in situ hybridization experiments, either instead of, or in complement with autoradiographic film and emulsions. It allows the user to locate and perform quantitative analyses of (3H, 14C, 35S, 33P, 32P, 125I) labelled molecules with a 15 microm spatial resolution on a 0.9 x 1.3 cm2 sensitive area. Combining recent techniques (specific scintillator thin sheets and intensified charge-coupled device (CCD)) this imager offers a wide dynamic range and real-time acquisition.

Radioimagers as an alternative to film autoradiography for in situ quantitative analysis of 125I-ligand receptor binding and pharmacological studies.

Three radioimagers, the mu-imager, the beta-imager and the phosphorimager, were tested as alternatives to quantitative autoradiography on film, for receptor imaging and pharmacological in situ quantitative analysis. Two iodinated ligands 125I-interleukin-1 alpha and 125I-gonadotropin releasing hormone agonist were used for receptor characterization in mouse brain and pituitary sections. Due to the high number of the agonist receptors in rat pituitary gland, this tissue was used to compare measurements obtained from digital autoradiograms with classical gamma detector determination. This permits the evaluation of radioimager efficiency and absolute quantification. Radioimagers represent an improvement in terms of time of image acquisition. All the radioimagers are more sensitive than film for the detection of low levels of radioactivity. The spatial resolution provided by the mu-imager compares favourably with that obtained on film autoradiograms while digital autoradiograms from the phosphorimager and beta-imager did not show precise definition under our experimental conditions. Superimposition of histological structures from the stained sections with radiolabelled areas in the autoradiograms remains, at this time, the unique advantage of film. In conclusion, radioimagers represent an alternative to autoradiography on film or emulsion for in situ quantitative studies on tissue sections. They combine precise imaging for in situ binding studies with easy and direct access to counts in cpm. The improvement in radioimaging technology has, therefore, brought in situ analysis of iodinated ligand binding to the level of accuracy that is obtained with classical detectors of radioactivity.

A novel rat tyrosine hydroxylase mRNA species generated by alternative splicing.

Tyrosine hydroxylase (TH) catalyzes the first and rate-limiting step in the biosynthesis of catecholamines. Among the various mechanisms implicated in the regulation of TH activity, alternative splicing of TH primary transcript has been described as a characteristic of higher primates and Drosophila. We investigated whether there is such a regulatory mechanism in the rat. Reverse transcriptase-PCR experiments were performed with RNA from PC12 cells. A new TH mRNA species was evidenced, resulting from the use of an alternative donor site in exon 2. RNase protection assays and in situ hybridization experiments detected this mRNA species in the adrenal medulla but not in the main catecholaminergic nuclei of the CNS. The corresponding putative protein lacks 33 amino acids in the N-terminal regulatory domain. A recombinant protein was produced in E. coli. Its in vitro specific activity was similar to that of the previously identified TH protein.

Rat interleukin-1 beta binding sites in rat hypothalamus and pituitary gland.

In this study, radiolabeled recombinant rat interleukin-1 beta (r125I-IL-1 beta) was used to localize and characterize IL-1 beta binding in rat hypothalamus and pituitary gland by quantitative autoradiography. The ability of this ligand to bind to type I IL-1 receptor was first tested on murine lymphoma cells (EL-4). In the rat-tissue sections, high densities of specific r125I-IL-1 beta binding sites were localized in the anterior as well as the posterior pituitary and in the choroid plexus. A fine labeling was observed in meninges and third ventricle walls while no binding was detected in the hypothalamic nuclei. Saturation experiments, in the anterior and posterior pituitary, revealed one specific binding site with an affinity constant (Kd) of 0.5 nM. Competition experiments were achieved using either rat IL-1 beta (rIL-1 beta) or human IL-1s (hIL-1 alpha, hIL-1 beta and IL-1 receptor antagonist: hIL-1a). Affinity constants (Ki) were drastically different according to the ligand used, while Ki values were found similar in anterior and posterior pituitary. Competition with rIL-1 beta revealed one binding affinity (Ki of 0.1 nM range). In contrast, competition with hIL-1 beta revealed two binding affinities: a high (Ki: 0.1 pM range) and a low one (Ki: 1 nM range). Competition with hIL-1ra was obtained for high concentrations only (Ki: 10-100 nM range), whereas human IL-1 alpha (hIL-1 alpha) was unable to compete at 1-100 nM.(ABSTRACT TRUNCATED AT 250 WORDS)

HRRI: a high resolution radioimager for fast, direct quantification in in situ hybridization experiments.

We present a high-speed, high-resolution beta imager. It has been developed to be used in in situ hybridization experiments, either instead of or in complement with autoradiographic film and emulsions that are currently used for these experiments. It allows the user to locate and perform quantitative analyses of (3H-, 14C-, 35S-, 32P-, 125I-) labeled molecules with a 15-microns spatial resolution on a 1.2 cm2 area. We have combined recent techniques (specific scintillator thin sheets and intensified charge-coupled device [CCD]) so that this imager offers a wide dynamic range and real-time acquisition. Several biological applications will be discussed.

"In situ" characterization of GnRH receptors: use of two radioimagers and comparison with quantitative autoradiography.

New radioimagers, the HRRI (high resolution radioimager) and the Phosphorimager (phosphor screen : PS), apt to display more ample linear dose-response scale than radio-sensitive films, were tested in comparison with quantitative autoradiography (QA). GnRH receptor saturation experiments were achieved on tissue sections (rat pituitary, rat brain, human ovary) with a iodinate GnRH agonist (125I-[D-Ala6,Des-Gly10]-LH-RH Ethylamide) for determination of affinity constant (Kd). In rat pituitary, comparable results were obtained with the 3 methods (Kd: 0.4 to 0.6 nM). Discrepancies occurred in the hippocampus and in the granulosa cell layer of the preovulatory follicle, due to low resolutive (PS) or short linear dose-response (films) performances. In the hippocampus GnRH receptor affinity was under-estimated with PS (Kd: 2.3 vs 0.5 and 0.6 nM for QA and HRRI respectively). In the follicular granulosa cell layer it was over-estimated by QA (0.5 vs 50 nM for the HRRI), while PS did not allow resolution of this thin cell layer. In conclusion, the HRRI is a very powerful tool for the quantification of in situ radioligand binding (binding sites study and in situ hybridization) in very discrete areas.