All-optical electrophysiology in behaving animals with optimized two-photon excitation

The NeuroSchool PhD Program of Aix-Marseille University (France) has launched its annual calls for PhD contracts for students with a master's degree in a non-French university and for  international co-supervised PhDs.

This project is one of the proposed projects. Not all proposed projects will be funded, check our website for details.

State of the art: Today, two-photon excitation (TPE) is routinely used to study function and connectivity in neural circuits by combining genetically encoded fluorescent calcium or voltage (GEVI) indicators with photosensitive actuators (e.g., channelrhodopsins), the goal being to photostimulate a large number of cells while being temporally accurate. Two main approaches are available today: one scans a user-selected 3D pattern of focal points in spirals the size of a cell body; the other generates a chosen 3D pattern of holograms of similar size as the spirals. The latter is temporally accurate but curbs TPE efficiency, the heating threshold thus drastically limiting the number of excitable cells. Conversely, spiral-scanning is TPE-efficient but lacks temporal accuracy: cells respond when a threshold number of opsins is activated and their spatial distribution varies among cells.

The objective of this PhD project is to characterize and optimize, first in slices and then in vivo (mice, marmosets) a novel photostimulation (PS) method that combines the advantages of the two above strategies - without suffering from their shortcomings. The project’s first part consists in applying to two-photon stimulation “dynamic holograms”, a recent optical method developed for in-vivo two-photon GEVI recordings (“2D-ULoVE”, Villette & al., 2019). The objective here is to characterize, in slices under patch-clamp control (INT), the dependency of the stimulation’s efficacy and temporal precision on the ULoVE parameters. The project’s second part consists in extending 2D-ULoVE, which covers only one cell at a time, to “3D-ULoVE”, i.e., to many selected spatial positions (cells) simultaneously, by using a spatial light modulator, in mice. To assess the novel technique’s neuroscientific impact, we will use 3D-ULoVE to evoke hippocampal neural activity patterns in behaving mice, using experimental protocols developed at INMED.  Finally, the technique will be applied in (behaving) marmosets (INT).

Methods: First, patch-clamp will be used to characterize the cells’ response as a function of the dynamic hologram’s parameters used for stimulation (type of ULoVE pattern, laser power…), in slices. Next, to achieve population-level TPE PS, we will implement 3D-ULoVE on a commercial two-photon microscope equipped with spiral-scan PS, thus allowing direct comparison between the two methods, and apply it first to slices, then to behaving mice and finally to marmosets.

Complementarity: Today, this specific microscope exists within the CIRCUITPHOTONICS facility (joint INMED-INT-Inst. Fresnel Equipex+ 2020) at INMED and will be replicated at INT within ~1 year (ERC F. Cazettes). J-C Platel (collaborator) from R. Cossart’s team that already practices PS, will contribute also their experience in PS, fostering the collaboration between the two institutes.

 Expected results: The patch-clamp characterization will yield, for various opsins, the key information on the maximal power usable for imaging without unwantedly inducing PS. This will allow using the fluorescence from cells co-expressing a GECI and a fast opsin as readout of their response to 3D-ULoVE stimulation, expected to (~3x) outperform spiral scan both in timing accuracy and addressable number of neurons at given laser power. Last, the protocol developed at INMED for benchmarking the new 3D-ULoVE against existing methods shall become available to the community as a standard reading/writing procedure.

Feasibility (#APAFIS): INT: 51819-2024101716408210_v6, 48723-202403251009126_v3, 16235-2017122017031124_v7. INMED: 30716-2021032215171216_v8, microscope is available for project and training assured

Within Aix Marseille Université, NeuroMarseille brings together 8 research laboratories and NeuroSchool, a graduate school in neuroscience, to increase the attractiveness of the university, international collaborations, interdisciplinarity, links with the clinical and industrial worlds and the integration of students into professional life. 

Launched in July 2018, NeuroSchool unifies and harmonizes the training of the third year of the Bachelor of Life Sciences (Neuroscience track), the Master's and the PhD in Neuroscience. 

• Master's degree from a non-French university in neuroscience or related field
• Fluent in English

The ideal candidate should have a background in cellular neuroscience, biophotonics or engineering. Hands-on experience in optical microscopy techniques (TPE, optogenetics…) or patch-clamp recordings is a plus.