Stéphane Dieudonné Dr at IBENS, Ecole Normale Supérieure, CNRS UMR 8197, INSERM U1024, Paris, FranceStéphane DieudonnéDr at IBENS, Ecole Normale Supérieure, CNRS UMR 8197, INSERM U1024, Paris, France
A sequential strategy for multiphoton actuation and recording of neuronal membrane voltage in awake animals, by Stéphane Dieudonné
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Technologies for recording and manipulating neuronal membrane potential in vivo in defined neuronal populations with high fidelity will be essential to understand how information is represented, processed, and propagated in the brain. Genetically encoded voltage indicators (GEVIs) and optogenetic actuators are especially promising as they can be expressed in defined cell types and are compatible with long-term chronic imaging in vivo. Optical recording and actuation of cellular voltage in vivo, however, suffers from limitations of both speed and sensitivity inherent in current indicators and imaging modalities.
In vivo 2P excitation of membrane proteins has been limited by three main factors: the low number of membrane proteins within a 2P focal volume, the low frame rate of standard 2P imaging and the possible imaging artefacts linked to brain motion in awake behaving mice. To address these three issues, we have developed light patterning strategies based on the acousto-optic technology. These strategies combine fast scanning (resonant rate), fast random-access (100 kHz- 1 MHz), holographic shaping of the focal volume and beam multiplexing.
We demonstrate 10 kHz random-access voltage recordings from ensembles of neurons in awake behaving mice. Using ASAP3, a newly developed fast GEVI, we report single spike detection with sub-millisecond precision and subthreshold membrane potential recordings with cellular resolution deep in the cortex and hippocampus of awake mice. We show activation of the optogenetic actuator ChR2 at reduced laser power and with equivalent XY and Z resolution. Finally, we present new developments for 3D imaging and stimulation and demonstrate GCaMP6 calcium recording of neurons at different laminar positions within a 400x400x400 µm volume of mouse sensory cortex at 1 kHz sampling speed.
Dr Dieudonné short biography:
Stéphane Dieudonné is a cellular and systems neurobiologist with expertise in both electrophysiology and imaging of neuronal activity. During his PhD thesis under the supervision of Philippe Ascher (Paris, France), he studied glycinergic transmission and produced the first patch-clamp recordings of Golgi interneurons and Lugaro interneurons in the cerebellum. Following a short postdoc in the laboratory of K. Delaney (SFU, Vancouver, Canada), where he learned calcium imaging, he was recruited as a permanent researcher by INSERM in 2001. Anticipating the neurophotonics revolution, Stéphane Dieudonné has been an important contributor to the development of Random-Access Multiphoton microscopy, as a strategy for fast optical recording and actuation of neuronal activity. He is involved in several international training courses in optics for the neurosciences and acts as scientific director of the imaging facility at the Biology Institute of the Ecole Normale Supérieure (IBENS). Since 2005, he has been heading a research team at IBENS, investigating the function of inhibitory neurons in neuronal homeostasis and computation. The group has identified functional organizing principles of mixed GABA/glycine inhibitory transmission at the molecular and microcircuit levels and described long-range inhibitory pathways involved in motor control. The team is now combining molecular and cellular expertise in optogenetics with the implementation of Random-Access imaging and actuation technology in the behaving animal to answer fundamental questions on the role of cerebellar microcircuits in motor learning and motor control.
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