COMPLEX TEMPORAL ACTIVITY PATTERNS REPLAYED WITH FAST 3D ACOUSTO-OPTICAL STIMULATION FOR PARTIAL VISUAL RESTORATION

Two-photon optogenetics enables cell-resolved mapping and patterned manipulation of neuronal circuits, supporting applications from high-throughput connectivity mapping to causal tests of population codes of perception. However, existing strategies for combined 3D readout and 3D manipulation are constrained by fundamental trade-offs between temporal and spatial resolution and the ability to access widely distributed targets within large volumes.
Here we introduce a dual-wavelength acousto-optic (AO) scanning system that overcomes these constraints by enabling microsecond-scale switching between imaging and photostimulation while maintaining diffraction-limited resolution and true 3D random-access targeting throughout the entire imaging volume. This unified optical framework supports rapid functional readout together with precise, distributed manipulation of neuronal networks at single-cell level without sacrificing spatiotemporal performance. Motivated by evidence that perceptual representations are embedded in structured temporal activity patterns rather than simple co-firing, we leverage this capability to replay endogenous dynamics with fine control over timing, power, and 3D targeting at single-cell resolution. Using this approach, we achieve simultaneous recording and stimulation of up to 850 neurons across a 700 µm z-range in mouse primary visual cortex and demonstrate the reactivation of complex activity sequences with up to millisecond-precision. In behaving mice, selective replay of perceptually evoked 3D activity patterns enhanced performance in visual detection and discrimination tasks, providing causal evidence that temporally structured ensemble dynamics contribute to perception. Together, our results establish a general framework for cell-resolved, temporally structured control of neural activity in three dimensions and lay groundwork for future closed-loop optical manipulation of cortical circuits.