High-throughputin vivosynaptic connectivity mapping of neuronal micro-circuits using two-photon holographic optogenetics and compressive sensing

Abstract

SummaryUnderstanding the intricate synaptic connectivity in living neural circuits is crucial for unraveling the relationship between network structure and function, as well as its evolution during development, learning, and recovery from injury. However, current methodologies for identifying connected neuronsin vivosuffer from limitations, particularly with regards to their throughput. In this study, we introduce a groundbreaking framework forin vivoconnectivity mapping that combines two-photon holographic optogenetics for activating single or multiple potential presynaptic neurons, whole-cell recording of postsynaptic responses, and a compressive sensing strategy for efficiently retrieving individual postsynaptic neurons’ responses when multiple potential presynaptic neurons are simultaneously activated. The approach was validated in the layer 2/3 of the visual cortex in anesthetized mice, enabling rapid probing of up to 100 cells in approximately 5 minutes. By identifying tens of synaptic pairs, including their connection strength, kinetics, and spatial distribution, this method showcases its potential to significantly advance circuit reconstruction in large neuronal networks with minimal invasiveness. Moreover, through simultaneous multi-cell stimulation and compressive sensing, we demonstrate up to a three-fold reduction in the number of required measurements to infer connectivity with limited loss in accuracy, thereby enabling high-throughput connectivity mappingin vivo. These results pave the way for a more efficient and rapid investigation of neuronal circuits, leading to deeper insights into brain function and plasticity.

Multicolor two-photon light-patterning microscope exploiting the spatio-temporal properties of a fiber bundle

“…These numbers of achievable targets are compatible with a wide range of applications. They can be applied, e.g., to study how the activation of a portion or the entirety of a specific cell assembly, or even just a single hub neuron [ 15 ], can impact the circuit dynamics or to perform high-throughput intra and inter-layer connectivity mapping [ 16 – 19 ]. For applications where, higher total power or a greater number of achievable targets are required [ 11 ], it becomes imperative to conduct a thorough assessment of signal losses, axial resolution, and attainable output power for evaluating the benefit of using this approach compared to more traditional configurations.…”

“…Pioneering experiments using similar methods have started to show that precise optogenetics photostimulation of selected neurons in the mouse brain can influence animal behavior and affect perception [ 11 – 14 ] or influence neuronal population dynamics [ 15 ]. Sequential optogenetic activation of one or few cells at cellular resolution also enabled in vitro and in vivo high throughput connectivity mapping [ 16 – 19 ].…”

Compressive sensing of functional connectivity maps from patterned optogenetic stimulation of neuronal ensembles

“…Model parameters were informed by literature where possible with the goal of recapitulating properties of in vivo experiments. 64 The number of observed neurons in the model was motivated by the average number of neurons typically visible in a 2p optogenetic experiment. 17 , 28 , 58 Parameters of the neuron model were randomized over the biophysical range to demonstrate that mapping is robust to variability in neuron parameters.…”

“…In the context of using a method for a mapping experiment, patching onto cells declared to be connected is a likely follow-up step. 64 Whether or not that connection exists would be a better metric to use, as results can be more easily confirmed or rejected.…”

“…In practice, optically evoked presynaptic spiking and synaptic reliability can be a function of many variables such as intrinsic cell properties, 72 opsin expression level, time since last spike, 61 and up/down brain states. 73 , 64 …”