Figure 3. Deep brain holographic stimulation of individual BLA neurons is specific and robust.

(A) Schematic of head-fixed 2-photon calcium imaging via 920nm laser path combined with a second 1040nm laser path for stimulation. (B) In vivo imaging of GCaMP6f (920nm) or the red-shifted opsin ChRmine (1080nm) expressed in BLA neurons. (C) Schematic depicting experimental setup for testing X/Y specificity of single ROI activation using a spatial light modulator (SLM). Red spirals indicate SLM targets, with each subsequent stimulation moving a fixed distance (+10, +20, +30, +40μm) laterally from the target cell. (D) Mean fluorescence across 5 stimulation trials (10hz, 2s, 6mW power) at each distance, darker colors indicate SLM spiral target being closer to the target cell. (Inset) Mean projection image of neuron recorded from (green) and spiral stimulation targets moving laterally. Order of spiral stimulation target (1–5) was randomized. (E) Mean fluorescence of single BLA neuron across 5 stimulation trials at each SLM ROI plotted as a function of distance from the target neuron. (F) Schematized representation of strategy to assess axial specificity of single-cell SLM stimulation. To stimulate (10hz, 2s, 6mW power) both above and below our target cell, the microscope Z-position was moved at fixed distances (−90 to +90μm in microscope space). At the same time, a separate imaging path with an electrotunable lens (ETL) was used to counteract this microscope movement to ensure imaging of the plane where our target cell was located. (G) Sample position and corresponding objective position for determining the degree of axial magnification caused by the GRIN lens. (H) Trial averaged fluorescence of a single BLA neuron plotted as a function of both time (left Y-axis) and stimulation distance in microscope units (X-axis). (I) Heatmaps showing stimulation aligned fluorescence traces (dotted line indicates onset of SLM stimulation) for 3 distances across 5 repeated stimulation trials (ITI=15s). (J) SLM stimulation evoked fluorescence averaged across all neurons (n=6) plotted as a function of distance corrected for GRIN lens magnification (brain distance in μm). Negative numbers indicate SLM stimulation ROIs above the target neuron, while positive numbers indicate SLM stimulation ROIs below. (K) Simultaneous activation of 6 BLA neurons through a GRIN lens using SLM stimulation (10hz, 2s, 6mW power per ROI; ITI=20–25s). Yellow box indicates first stimulation of the session, green box indicates final stimulation of the session. (L) Aligned SLM stimulation evoked fluorescence from the first stimulation (left; yellow line) and the final (right; green line) stimulation. Traces are shaded according to their color in K.