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. 2019 Jul;22(7):1061-1065.
doi: 10.1038/s41593-019-0422-3. Epub 2019 Jun 17.

Thermal Constraints on in Vivo Optogenetic Manipulations

Free PMC article

Thermal Constraints on in Vivo Optogenetic Manipulations

Scott F Owen et al. Nat Neurosci. .
Free PMC article


A key assumption of optogenetics is that light only affects opsin-expressing neurons. However, illumination invariably heats tissue, and many physiological processes are temperature-sensitive. Commonly used illumination protocols increased the temperature by 0.2-2 °C and suppressed spiking in multiple brain regions. In the striatum, light delivery activated an inwardly rectifying potassium conductance and biased rotational behavior. Thus, careful consideration of light-delivery parameters is required, as even modest intracranial heating can confound interpretation of optogenetic experiments.

Conflict of interest statement


The authors have no competing interests to disclose.


Figure 1.
Figure 1.. Light delivery suppresses MSN activity in vivo and in acute slices.
A,B, Recording and electrode configuration for acute, in vivo, head-fixed recordings from awake mice. C,D Mean peri-stimulus aligned firing rate of MSNs in response to 15 mW of 532 nm light and corresponding population average of modulation index. Two-sided signed rank test to compare each distribution against zero. (N=3 mice, n=99 MSNs; P=6.57×10−7 for 3 mW, P=6.84×10−18 for 15 mW). E, Configuration for acute slice whole-cell recordings. F, Exemplar current-clamp recording with spiking elicited by current injection. Light delivered at 15 mW, 532 nm through an optical fiber. G,H, Mean suppression of spiking by light delivery for light on (green) or light off (black) traces. Two-sided signed-rank test. (N=2 mice, n=11 cells; P=0.002 and P=0.010). I-L, Firing rates for CA1 pyramidal neurons (N=6 mice, n=10 cells; P=0.375 and P=0.432), DG Granule cells (N=5 mice, n=9 cells; P=0.012 and P=0.098), cortical L5 pyramidal neurons (N=5 mice, n=15 cells; P=0.026 and P=0.008), and cortical L5 fast-spiking interneurons (N=8 mice, n=13 cells; P=0.001 and P=0.001) in response to light delivery at 15 mW, 532 nm. All tests are two-sided signed-rank tests. * P<0.05, ** P<0.01, *** P<0.001. All error bars and shaded regions represent s.e.m.
Figure 2.
Figure 2.. Light-induced heating increases an inwardly-rectifying potassium conductance in MSNs.
A, Configuration for in vivo head-fixed temperature measurements and physiology. B, Temperature increase in vivo fit to a single exponential (dashed line) (N=1 mouse). C, Time-course of decrease in mean MSN firing rate fit to a single exponential (dashed line). Same data as Figure 1C (N=3 mice, n=99 MSNs). D, Configuration for whole-cell recordings or temperature measurements in acute slices. E, Exemplar temperature change and F, average whole-cell current in exemplar MSN held at −50 mV with potassium-based internal. Light delivery at 3 mW or 15 mW, 532 nm. G, Group data for light-evoked current in MSNs (N=2 mice, n=9 cells for 3mW; N=2 mice, n=10 cells for 15 mW; two-sided rank sum test, P=4.33×10−5). H, Configuration for whole-cell recordings with temperature modulation. I, Temperature changes and J, exemplar whole-cell currents in wild-type MSNs, recorded in voltage-clamp with potassium-based internal. K, Linear relationship between temperature change and current (N=2 mice, n=7 cells for −2.82 °C; N=2 mice, n=7 cells for +0.89 °C; N=2 mice, n=10 cells for +1.92 °C). L, Modeled temperature changes predicted for this recording configuration. M, Recording configuration, N, exemplar traces, and O, group data for voltage sensitivity of light-activated currents (N=2 mice; n=8 cells). P, Exemplar light-activated current recorded at −50 mV with cesium-based internal. Light off (black), light on (green). (N=2 mice, n=10 cells). Q, Exemplar light-activated current recorded at −50 mV with potassium-based internal before (green) and after (purple) bath application of 250 μM BaCl2. R, Group data for BaCl2 sensitivity of light-activated conductance (N=2 mice, n=7 cells sham; N=2 mice, n=6 cells BaCl2, two-sided rank sum test, P=0.001). Error bars and shaded regions are s.e.m. ** P<0.01, *** P<0.001.
Figure 3:
Figure 3:. Light delivery in dorsal striatum drives rotational behavior.
A. Video frame illustrating body position tracking. B, Modeled temperature change for continuous or pulsed light delivery at 532 nm. Duty cycle was 20% for pulsed light. C, Light delivery protocol for open field behavioral tests. D, Mean rotational bias in response to light delivery. Two-sided signed-rank test (N=11 mice, 2 hemispheres per mouse, n=22 sessions per condition; 3 mW, P=0.37 and P=0.66; 7 mW, P=2.7×10−3 and P=3.9×10−4; 15 mW, P=2.3×10−4 and P=1.1×10−5; 15 mW at 20 Hz, P=0.94 and P=0.06). ** P<0.01, *** P<0.001. Error bars and shaded regions are s.e.m.

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