Holographic Dissection of Neural Circuits – NeurHolog

Light is the tool of the 21st century. The combination of light microscopy with functional reporters, caged compounds and, more recently, optogenetics offers the possibility to control activation and inhibition of neuronal activity and monitor functional responses in a non-invasive manner enabling the analysis of well-defined neuronal population within intact neuronal circuits and systems. This ongoing revolution has motivated the development of new optical methods for light stimulation. Among them, a new class of optical techniques based on the use of wave front shaping through liquid crystal matrices has been recently proposed as a powerful tool to dynamically generate precise, optically confined excitation patterns in single and two-photon excitation. This was achieved by combining different approaches for lateral light shaping including digital holography and the generalized phase contrast method. In two-photon excitation, axial confinement has been achieved by combining lateral light shaping with the technique of temporal focusing. Aim of this project is to improve and apply these emerging techniques to studying the structure-function relationships in active neural networks. The biological objects at stake are two vital minimal rhythmic neuronal ensembles (oscillators) composed of about 500 genetically identified and optically accessible neurons, operating in mouse embryos as a forerunning version of the vital respiratory rhythm generator (RRG). One of these functional modules is the preBötzinger Complex (preBötC) oscillator, the best characterized component of the RRG, entraining respiration4. The other one is the embryonic parafacial (e-pF) oscillator that couples to the preBötC and has progressively shared the front stage of respiratory neurophysiology with the latter, in part owing to its crucial role in CO2 chemosensitivity, and its dysfunction likely causal of the Central Congenital Hypoventilation Syndrome (CCHS). Still unknown are the respective neuronal connectivity that supports their function. We want to test a structural hypothesis of the oscillators (adapted to the necessary robustness and flexibility of the respiratory rhythm) whereby their function may depend on the activity of a very small number of their composing neurons. For this, we will use wave front shaping for 1P and 2P patterned light excitation.This will enable for the first time, to reversibly and selectively stimulate scalable numbers of channelrhodopsin-2 (ChR2)-expressing oscillators’ cells and image the responding cells to derive the “links” connecting the “nodes” defining the structure of the oscillators’ network. Apart from their biological interest, the e-pF, a 100µm thick structure comprising 3-4 superposed cell layers on the ventral surface of the brain, and the preBötC a collection of cell scattered over the 450µm thick rhythmic “preBötC slice”, constitute graded optical challenges perfectly matching the technological solutions proposed in the program. To reach deep cells with high spatial precision, we plan to use two-photon patterned excitation with amplified laser pulses.Moreover, the combination of wave front shaping with advanced optical schemes for remote focusing (using variable group velocity dispersion or an adjustable mirror in the remote space) will permit patterned excitation at a fixed excitation plane while concurrently image evoked calcium responses iteratively in multiple distinct imaging planes. This may give access to a larger number of responding cells, opening ways to the 3D reconstruction of the oscillators’ connected neuronal ensembles.If successful, a useful way to inspect the topology of neural networks will become available to the scientific community.This program should also shed light on the still elusive aspect of neural network development linking cellular identity, dictated by genetic developmental programs, to the emergence of network assembly and function.