Frédéric Pain

In vivo quantification of localized neuronal activation and inhibition in the rat brain using a dedicated high temporal-resolution β+-sensitive microprobe

Understanding brain disorders, the neural processes implicated in cognitive functions and their alterations in neurodegenerative pathologies, or testing new therapies for these diseases would benefit greatly from combined use of an increasing number of rodent models and neuroimaging methods specifically adapted to the rodent brain. Besides magnetic resonance (MR) imaging and functional MR, positron-emission tomography (PET) remains a unique methodology to study in vivo brain processes. However, current high spatial-resolution tomographs suffer from several technical limitations such as high cost, low sensitivity, and the need of restraining the animal during image acquisition. We have developed a β+-sensitive high temporal-resolution system that overcomes these problems and allows the in vivo quantification of cerebral biochemical processes in rodents. This β-MICROPROBE is an in situ technique involving the insertion of a fine probe into brain tissue in a way very similar to that used for microdialysis and cell electrode recordings. In this respect, it provides information on molecular interactions and pathways, which is complementary to that produced by these technologies as well as other modalities such as MR or fluorescence imaging. This study describes two experiments that provide a proof of concept to substantiate the potential of this technique and demonstrate the feasibility of quantifying brain activation or metabolic depression in individual living rats with 2-[18F]fluoro-2-deoxy-d-glucose and standard compartmental modeling techniques. Furthermore, it was possible to identify correctly the origin of variations in glucose consumption at the hexokinase level, which demonstrate the strength of the method and its adequacy for in vivo quantitative metabolic studies in small animals.

Autofluorescence imaging of NADH and flavoproteins in the rat brain: insights from Monte Carlo simulations.

There has been recently a renewed interest in using Autofluorescence imaging (AF) of NADH and flavoproteins (Fp) to map brain activity in cortical areas. The recording of these cellular signals provides complementary information to intrinsic optical imaging based on hemodynamic changes. However, which of NADH or Fp is the best candidate for AF functional imaging is not established, and the temporal profile of AF signals is not fully understood. To bring new theoretical insights into these questions, Monte Carlo simulations of AF signals were carried out in realistic models of the rat somatosensory cortex and olfactory bulb. We show that AF signals depend on the structural and physiological features of the brain area considered and are sensitive to changes in blood flow and volume induced by sensory activation. In addition, we demonstrate the feasibility of both NADHAF and Fp-AF in the olfactory bulb.

Characterization, comparison, and choice of a commercial double-clad fiber for nonlinear endomicroscopy

Abstract. Several endomicroscope prototypes for nonlinear optical imaging were developed in the last decade for in situ analysis of tissue with cellular resolution by using short infrared light pulses. Fourier-transform-limited pulses at the tissue site are necessary for optimal excitation of faint endogenous signals. However, obtaining these transform-limited short pulses remains a challenge, and previously proposed devices did not achieve an optimal pulse delivery. We present a study of fibered endomicroscope architecture with an efficient femtosecond pulse delivery and a high excitation level at the output of commercially available double-clad fibers (DCFs). The endomicroscope incorporates a module based on a grism line to compensate for linear and nonlinear effects inside the system. Simulations and experimental results are presented and compared to the literature. Experimentally, we obtained short pulses down to 24 fs at the fiber output, what represents to the best of our knowledge the shortest pulse duration ever obtained at the output of a nonlinear endoscopic system without postcompression. The choice of the optimal DCF among four possible commercial components is discussed and evaluated in regard to multiphoton excitation and fluorescence emission.

Extension of the GATE Monte-Carlo simulation package to model bioluminescence and fluorescence imaging

Abstract. The Geant4 Application for Emission Tomography (GATE) is an advanced open-source software dedicated to Monte-Carlo (MC) simulations in medical imaging involving photon transportation (Positron emission tomography, single photon emission computed tomography, computed tomography) and in particle therapy. In this work, we extend the GATE to support simulations of optical imaging, such as bioluminescence or fluorescence imaging, and validate it against the MC for multilayered media standard simulation tool for biomedical optics in simple geometries. A full simulation set-up for molecular optical imaging (bioluminescence and fluorescence) is implemented in GATE, and images of the light distribution emitted from a phantom demonstrate the relevance of using GATE for optical imaging simulations.

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

PurposeMultimodal 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 β-MicroProbe instead of PET for local measurements of radioactivity coupled with MRI.MethodsWe simultaneously recorded local radioactivity due to [18F]MPPF (a 5-HT1A receptor PET radiotracer) binding in the hippocampus with the β-MicroProbe and carried out anatomical MRI in the same anaesthetised rat.ResultsThe comparison of [18F]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 β-MicroProbe reliably measures radioactivity in rat brains under an intense magnetic field of 7 Tesla.ConclusionThe biological validation of a β-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.

Combining the radiosensitive Beta MicroProbe to Nuclear Magnetic Resonance: theoretical approach for in vivo studies in small animals

In vivo small animal imaging with multiple modalities has become an important tool in modern biomedical research. Indeed, combining exploratory techniques allows simultaneous recording of complementary data, which is required to elucidate complex physiopathological mechanisms. In this field, because of strict technical constraints in vivo, an exciting challenge remains in the combination of Nuclear Magnetic Resonance (NMR) and Positron Emission Tomography (PET). Coupling NMR with a radiosensitive Beta MicroProbe offers therefore a very interesting technical alternative. Here, we assessed the feasibility of this new combination by theoretically evaluating the ability of the Beta MicroProbe to monitor radioactivity in a magnet. To that aim, we modelled with Geant4 the effect of an intense magnetic field on the probe field of view and showed that the field should not have an impact on the global efficiency of the probe.

PIXSIC: A Wireless Intracerebral Radiosensitive Probe in Freely Moving Rats.

The aim of this study was to demonstrate the potential of a wireless pixelated β+-sensitive intracerebral probe (PIXSIC) for in vivo positron emission tomographic (PET) radiopharmacology in awake and freely moving rodents. The binding of [11C]raclopride to D2 dopamine receptors was measured in anesthetized and awake rats following injection of the radiotracer. Competitive binding was assessed with a cold raclopride injection 20 minutes later. The device can accurately monitor binding of PET ligands in freely moving rodents with a high spatiotemporal resolution. Reproducible time-activity curves were obtained for pixels throughout the striatum and cerebellum. A significantly lower [11C]raclopride tracer-specific binding was observed in awake animals. These first results pave the way for PET tracer pharmacokinetics measurements in freely moving rodents.

Imaging Odor-Evoked Activities in the Mouse Olfactory Bulb using Optical Reflectance and Autofluorescence Signals

In the brain, sensory stimulation activates distributed populations of neurons among functional modules which participate to the coding of the stimulus. Functional optical imaging techniques are advantageous to visualize the activation of these modules in sensory cortices with high spatial resolution. In this context, endogenous optical signals that arise from molecular mechanisms linked to neuroenergetics are valuable sources of contrast to record spatial maps of sensory stimuli over wide fields in the rodent brain. Here, we present two techniques based on changes of endogenous optical properties of the brain tissue during activation. First the intrinsic optical signals (IOS) are produced by a local alteration in red light reflectance due to: (i) absorption by changes in blood oxygenation level and blood volume (ii) photon scattering. The use of in vivo IOS to record spatial maps started in the mid 1980s with the observation of optical maps of whisker barrels in the rat and the orientation columns in the cat visual cortex1. IOS imaging of the surface of the rodent main olfactory bulb (OB) in response to odorants was later demonstrated by Larry Katzs group2. The second approach relies on flavoprotein autofluorescence signals (FAS) due to changes in the redox state of these mitochondrial metabolic intermediates. More precisely, the technique is based on the green fluorescence due to oxidized state of flavoproteins when the tissue is excited with blue light. Although such signals were probably among the first fluorescent molecules recorded for the study of brain activity by the pioneer studies of Britton Chances and colleagues3, it was not until recently that they have been used for mapping of brain activation in vivo. FAS imaging was first applied to the somatosensory cortex in rodents in response to hindpaw stimulation by Katsuei Shibukis group4. The olfactory system is of central importance for the survival of the vast majority of living species because it allows efficient detection and identification of chemical substances in the environment (food, predators). The OB is the first relay of olfactory information processing in the brain. It receives afferent projections from the olfactory primary sensory neurons that detect volatile odorant molecules. Each sensory neuron expresses only one type of odorant receptor and neurons carrying the same type of receptor send their nerve processes to the same well-defined microregions of ˜100μm3 constituted of discrete neuropil, the olfactory glomerulus (Fig. 1). In the last decade, IOS imaging has fostered the functional exploration of the OB5, 6, 7 which has become one of the most studied sensory structures. The mapping of OB activity with FAS imaging has not been performed yet. Here, we show the successive steps of an efficient protocol for IOS and FAS imaging to map odor-evoked activities in the mouse OB.

The Potential of a Radiosensitive Intracerebral Probe to Monitor 18F-MPPF Binding in Mouse Hippocampus In Vivo

As mouse imaging has become more challenging in preclinical research, efforts have been made to develop dedicated PET systems. Although these systems are currently used for the study of physiopathologic murine models, they present some drawbacks for brain studies, including a low temporal resolution that limits the pharmacokinetic study of radiotracers. The aim of this study was to demonstrate the ability of a radiosensitive intracerebral probe to measure the binding of a radiotracer in the mouse brain in vivo. Methods: The potential of a probe 0.25 mm in diameter for pharmacokinetic studies was assessed. First, Monte Carlo simulations followed by experimental studies were used to evaluate the detection volume and sensitivity of the probe and its adequacy for the size of loci in the mouse brain. Second, ex vivo autoradiography of 5-hydroxytryptamine receptor 1A (5-HT1A) receptors in the mouse brain was performed with the PET radiotracer 2′-methoxyphenyl-(N-2′-pyridinyl)-p-18F-fluorobenzamidoethylpiperazine (18F-MPPF). Finally, the binding kinetics of 18F-MPPF were measured in vivo in both the hippocampus and the cerebellum of mice. Results: Both the simulations and the experimental studies demonstrated the feasibility of using small probes to measure radioactive concentrations in specific regions of the mouse brain. Ex vivo autoradiography showed a heterogeneous distribution of 18F-MPPF consistent with the known distribution of 5-HT1A in the mouse brain. Finally, the time–activity curves obtained in vivo were reproducible and validated the capacity of the new probe to accurately measure 18F-MPPF kinetics in the mouse hippocampus. Conclusion: Our results demonstrate the ability of the tested radiosensitive intracerebral probe to monitor binding of PET radiotracers in anesthetized mice in vivo, with high temporal resolution suited for compartmental modeling.

Experimental assessment of the safety and potential efficacy of high irradiance photostimulation of brain tissues

Optogenetics is widely used in fundamental neuroscience. Its potential clinical translation for brain neuromodulation requires a careful assessment of the safety and efficacy of repeated, sustained optical stimulation of large volumes of brain tissues. This study was performed in rats and not in non-human primates for ethical reasons. We studied the spatial distribution of light, potential damage, and non-physiological effects in vivo, in anesthetized rat brains, on large brain volumes, following repeated high irradiance photo-stimulation. We generated 2D irradiance and temperature increase surface maps based on recordings taken during optical stimulation using irradiance and temporal parameters representative of common optogenetics experiments. Irradiances of 100 to 600 mW/mm2 with 5 ms pulses at 20, 40, and 60 Hz were applied during 90 s. In vivo electrophysiological recordings and post-mortem histological analyses showed that high power light stimulation had no obvious phototoxic effects and did not trigger non-physiological functional activation. This study demonstrates the ability to illuminate cortical layers to a depth of several millimeters using pulsed red light without detrimental thermal damages.Optogenetics is widely used in fundamental neuroscience. Its potential clinical translation for brain neuromodulation requires a careful assessment of the safety and efficacy of repeated, sustained optical stimulation of large volumes of brain tissues. This study was performed in rats and not in non-human primates for ethical reasons. We studied the spatial distribution of light, potential damage, and non-physiological effects in vivo, in anesthetized rat brains, on large brain volumes, following repeated high irradiance photo-stimulation. We generated 2D irradiance and temperature increase surface maps based on recordings taken during optical stimulation using irradiance and temporal parameters representative of common optogenetics experiments. Irradiances of 100 to 600 mW/mm2 with 5 ms pulses at 20, 40, and 60 Hz were applied during 90 s. In vivo electrophysiological recordings and post-mortem histological analyses showed that high power light stimulation had no obvious phototoxic effects and did not trigger non-physiological functional activation. This study demonstrates the ability to illuminate cortical layers to a depth of several millimeters using pulsed red light without detrimental thermal damages.