Holographic optogenetic stimulation in AC and experimental procedure.

A: An example brain slice showing cells in AC expressing both GCaMP and opsin (AAV9-hSyn-GC8s-T2A-rsChRmine). B: An example field of view (FOV) where single cell targeting precision was tested and response traces to the holographic stimulation from an example cell. Stimulation was offset from the original position (red circle) to distance-shifted positions in 10 μm increments (gray dashed circles in the x-axis or y-axis of the FOV). Responses were the greatest when the stimulation was performed on the original cell position (red solid line trace). Rapid amplitude decay along the position shift was observed (red dashed line traces). Grey error shades indicate SEM across trials. A right inset errorbar plot shows a grand average amplitude change per stimulation point across all tested cells (n = 15 cells, 3 animals). Error bars indicate SEM across cells. C: An example FOV showing a population of cells (left) and amplitude changes to 5-cell stimulation as a stimulation effect (Δ F/Fstim -Δ F/Fspont., right). Filled squares indicate each cell. White circles indicate stimulation targeted cells. D: (left) Proportion of stimulated cells that showed an increase in fluorescence following photostimulation. Error bars indicate SEM across FOVs. A horizontal dashed line indicates average permutation results (random permutation test on 100 iterations, p < 0.0001). (right) Grand average of the stimulation effect across imaging sessions. Error bars indicate SEM across FOVs. A horizontal dashed line indicates average permutation results (random permutation test on 100 iterations, p < 0.0001). E: Experimental procedure. A total of four consecutive imaging sessions were acquired: 1) A cell selection session to identify neurons selective for 16 kHz pure tones, 2) a baseline imaging session to acquire tone-evoked activity response to either 16 or 54 kHz pure tone, 3) a stimulation session representing five cells of either 16 kHz or 54 kHz responsive cells as target stimulation to examine the effect of stimulation synchronized to tone presentations, and 4) a post-stimulation session to examine network persistence after stimulation-related changes.

Targeted cells and non-target cells show response changes due to stimulation.

A: (top) An example FOV showing the stimulation effect (Δ F/Fstim - Δ F/Fbaseline) of sound responsive cells for 16 kHz target cell stimulation (filled squares). Black circles indicate stimulated target cells. (bottom) Mean response traces of an example target and non-target cell in baseline session (black) and stimulation session (red). Error bars indicate SEM across trials. An example target cell shows an increased response due to the stimulation. An example non-target cell shows a decreased response due to stimulation on the target cells. B: (left) A violin plot of the proportion of stimulated cells that showed increased activity due to stimulation across FOVs. Horizontal solid line indicates mean proportion, empty circle indicates median proportion, and gray filled circles indicate individual FOVs. (right) Mean amplitude changes of target cells for stimulation session and post-stimulation session normalized to the baseline session. Error bars indicate SEM across cells. Dashed horizontal lines on both panels indicate average permutation results (random permutation test on 100 iterations, p < 0.0001). DE: Same as ABC for 54 kHz target cell stimulation.

Non-target co-tuned cells show more decreased response amplitudes due to stimulation when synchronized with their preferred tones.

A: Stimulation effect (Δ F/Fstim-Δ F/Fbaseline) in 16 kHz (blue) and 54 kHz (orange) preferring cells. Both cell groups show decreased amplitude to their preferred frequency regardless of conditions due to acoustic stimulus-specific adaptation. Only co-tuned cells (16 kHz preferring cells for 16 kHz stimulation or 54 kHz preferring cells for 54 kHz stimulation) show a further decrease in response amplitudes due to the stimulation, when the preferred pure tone (PT) frequency was synchronized. Error bars indicate SEM across FOVs (*: p < 0.05). B: Post-stimulation effect (Δ F/Fpost - Δ F/Fstim) 16 kHz (blue) and 54 kHz (orange) preferring cells. No significant response amplitude changes were observed. Error bars indicate SEM across FOVs. C: Sub-categorization of cells based on the frequency selectivity for each target stimulation condition (left: 16 kHz stim, right 54 kHz stim). Cells were first grouped into either 16 kHz preferring cells (blue) or 54 kHz preferring cells (orange). Within each cell group, cells were further subdivided into low, mid, and high frequency selectivity categories based on their 33% quartile ranges. Note that the frequency preference was log-transformed for visualization, but the x-axis labels kept the original frequency selectivity values before transformation. Vertical dashed lines indicate 33% quartile ranges. D: Response amplitude change based on the frequency selectivity category for each cell groups (blue: 16 kHz preferring cells, orange: 54 kHz preferring cells). Significant response amplitude changes relative to the control condition were observed only for high frequency selectivity category when target stimulated cells were co-tuned (*: p < 0.05).

Rebalanced response changes on non-target 16 kHz cells are immediate and widely distributed.

A: Stimulation effect (Δ F/Fstim - Δ F/Fbaseline) in 16 kHz (blue) and 54 kHz (orange) preferring cells per each trial for the 5-cell stimulation condition (left) and the no-cell control condition (right). Decreased amplitudes to preferred frequencies were observed from as early as trial 1 with no significant further changes across trials, regardless of frequencies and conditions (sum-of-squares F-test, all p > 0.05). Solid lines indicate fitted curves and dashed lines indicate 95% confidence intervals. B: Stimulation effect (Δ F/Fstim - Δ F/Fbaseline) of each non-target 16 kHz (blue) or 54 kHz (orange) preferring cells for either 16 kHz (top row) or 54 kHz (bottom row) presentation in relation to the minimum distance to any target cells for the stimulation condition (left) and the control condition (right). For the control condition, we considered top 5 most tone-responsive cells from the baseline session as “target” cells, as there was no stimulation. Non-target cells are widely distributed within the FOV (550 μm2), regardless of cell groups, frequencies, and conditions. Gray lines indicate fitted curves, excluding cells that are closer than 15 μm (vertical green lines; cells < 15 μm marked in lighter shades), and dashed lines indicate 95% confidence intervals.