Spectral Unmixing: Analysis of Performance in the Olfactory Bulb In Vivo

Background: The generation of transgenic mice expressing combinations of fluorescent proteins has greatly aided the reporting of activity and identification of specific neuronal populations. Methods capable of separating multiple overlapping fluorescence emission spectra, deep in the living brain, with high sensitivity and temporal resolution are therefore required. Here, we investigate to what extent spectral unmixing addresses these issues. Methodology/Principal Findings: Using fluorescence resonance energy transfer (FRET)-based reporters, and two-photon laser scanning microscopy with synchronous multichannel detection, we report that spectral unmixing consistently improved FRET signal amplitude, both in vitro and in vivo. Our approach allows us to detect odor-evoked FRET transients 180-250 mm deep in the brain, the first demonstration of in vivo spectral imaging and unmixing of FRET signals at depths greater than a few tens of micrometer. Furthermore, we determine the reporter efficiency threshold for which FRET detection is improved by spectral unmixing. Conclusions/Significance: Our method allows the detection of small spectral variations in depth in the living brain, which is essential for imaging efficiently transgenic animals expressing combination of multiple fluorescent proteins

Etude du couplage neurovasculaire et neurométabolique in vivo dans le bulbe olfactif des rongeurs par une approche associant microscopie biphotonique, enregistrements oxymétriques et électrophysiologiques (rôle de l'adaptation périphérique dans le codage des odeurs in vivo)

Dans différentes structures cérébrales, l activation neuronale déclenche une augmentation du métabolisme énergétique et du flux sanguin dans la région activée. Mon sujet de thèse a consisté en l étude du rôle de l activation neuronale postsynaptique dans le couplage neurométabolique et neurovasculaire, in vivo, au niveau des glomérules du bulbe olfactif des rongeurs, en réponse à une stimulation olfactive. En combinant des mesures de la vitesse des érythrocytes en microscopie biphotonique, à des enregistrements de l activité calcique postsynaptique chez la souris G-CaMP2 ou à des enregistrements électrophysiologiques extracellulaires et oxymétriques chez le rat, et en réalisant des applications pharmacologiques, nous avons montré que le déclenchement des réponses vasculaires et la consommation d oxygène dans les glomérules olfactifs activés résultent principalement de l activation dendritique postsynaptique par le glutamate. En combinant des enregistrements de l activité calcique des terminaisons axonales des neurones sensoriels olfactifs en microscopie biphotonique à des enregistrements de l activité postsynaptique en potentiel de champ, et à des applications pharmacologiques, nous avons aussi montré la contribution majeure de l adaptation périphérique dans l adaptation glomérulaire au niveau du bulbe olfactif chez le rat. Nous proposons que l adaptation périphérique joue un rôle majeur dans le codage des odeurs à haute concentration au niveau des glomérules.PARIS-BIUSJ-Thèses (751052125) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Multiple forms of activity-dependent intrinsic plasticity in layer V cortical neurones in vivo

Synaptic plasticity is classically considered as the neuronal substrate for learning and memory. However, activity-dependent changes in neuronal intrinsic excitability have been reported in several learning-related brain regions, suggesting that intrinsic plasticity could also participate to information storage. Compared to synaptic plasticity, there has been little exploration of the properties of induction and expression of intrinsic plasticity in an intact brain. Here, by the means of in vivo intracellular recordings in the rat we have examined how the intrinsic excitability of layer V motor cortex pyramidal neurones is altered following brief periods of repeated firing. Changes in membrane excitability were assessed by modifications in the discharge frequency versus injected current (F–I) curves. Most (∼64%) conditioned neurones exhibited a long-lasting intrinsic plasticity, which was expressed either by selective changes in the current threshold or in the slope of the F–I curve, or by concomitant changes in both parameters. These modifications in the neuronal input–output relationship led to a global increase or decrease in intrinsic excitability. Passive electrical membrane properties were unaffected by the intracellular conditioning, indicating that intrinsic plasticity resulted from modifications of voltage-gated ion channels. These results demonstrate that neocortical pyramidal neurones can express in vivo a bidirectional use-dependent intrinsic plasticity, modifying their sensitivity to weak inputs and/or the gain of their input–output function. These multiple forms of experience-dependent intrinsic changes, which expand the computational abilities of individual neurones, could shape new network dynamics and thus might participate in the formation of mnemonic motor engrams

Local Ca(2+) detection and modulation of synaptic release by astrocytes.

Astrocytes communicate with synapses by means of intracellular calcium ([Ca(2+)](i)) elevations, but local calcium dynamics in astrocytic processes have never been thoroughly investigated. By taking advantage of high-resolution two-photon microscopy, we identify the characteristics of local astrocyte calcium activity in the adult mouse hippocampus. Astrocytic processes showed intense activity, triggered by physiological transmission at neighboring synapses. They encoded synchronous synaptic events generated by sparse action potentials into robust regional (∼12 μm) [Ca(2+)](i) elevations. Unexpectedly, they also sensed spontaneous synaptic events, producing highly confined (∼4 μm), fast (millisecond-scale) miniature Ca(2+) responses. This Ca(2+) activity in astrocytic processes is generated through GTP- and inositol-1,4,5-trisphosphate-dependent signaling and is relevant for basal synaptic function. Thus, buffering astrocyte [Ca(2+)](i) or blocking a receptor mediating local astrocyte Ca(2+) signals decreased synaptic transmission reliability in minimal stimulation experiments. These data provide direct evidence that astrocytes are integrated in local synaptic functioning in adult brain

Limited Resection Versus Pancreaticoduodenectomy for Duodenal Gastrointestinal Stromal Tumors? Enucleation Interferes in the Debate: A European Multicenter Retrospective Cohort Study

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