Thermal constraints on in vivo optogenetic manipulations

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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.

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Fig. 1: Light delivery suppresses MSN activity in vivo and in acute slices.
Fig. 2: Light-induced heating increases an inwardly rectifying potassium conductance in MSNs.
Fig. 3: Light delivery in the dorsal striatum drives rotational behavior.

Data availability

The data that support the findings of this study are available from the corresponding author upon request.

Code availability

Modeling is based on previously a published code that is freely available1; all other analysis code is available upon reasonable request.


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The authors thank B. Margolin for assistance with genotyping, histology, and microscopy, and members of A.C.K.’s laboratory for comments on the manuscript. This work was funded by NIH R01 NS078435 and RF1 AG047655 (to A.C.K.), a NARSAD Young Investigator Award (to S.F.O.), F32 NS083369 (to S.F.O.) and K99 MH110597 (to S.F.O.), and RR018928 (to the Gladstone Institutes).

Author information

S.F.O., M.H.L., and A.C.K. designed the experiments. S.F.O. and M.H.L. performed the experiments and analyzed the data, and all authors wrote the manuscript.

Correspondence to Anatol C. Kreitzer.

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Supplementary Figure 1 Light suppresses MSN firing rates in vivo.

. A, Recording configuration for in vivo physiology. B, Histogram of peak-trough latencies for single-unit waveforms. Putative MSNs identified by longer latencies colored in black. C, Laser stimulation protocol, interleaved blocks of 50 stimuli for 3 mW and 15 mW with 532 nm light. D, Peri-stimulus aligned mean firing rate for MSNs in response to 532 nm laser light at 3 mW. E,F, Histogram representing group data for light-driven modulation of single unit firing rates at 3 mW or 15 mW. Modulation Index = (Laser On – Laser Off) / (Laser On + Laser Off). G-I, Recording configuration, time-course, and group data for light-driven modulation of MSN firing rates in response to 3 mW at 532 nm. Two-sided signed-rank test (N=2 mice, n=10 cells; P=0.002 and P=0.010). J, Configuration for hippocampal slice recordings. K, Time-course for firing rates in CA1 pyramidal neurons in response to 15 mW at 532 nm. Same data as Fig. 1i (N=6 mice, n=10 cells). L, Time-course for firing rates in DG Granule cells in response to 15 mW at 532 nm. Same data as Fig. 1j (N=5 mice; n=9 cells). M, Configuration for cortical slice recordings. N, Time-course for firing rates in cortical L5 pyramidal neurons in response to 15 mW at 532 nm. Same data as Fig. 1k (N=5 mice; n=15 cells). O, Time-course for firing rates in cortical L5 fast-spiking interneurons. Same data as Fig. 1l (N=8 mice; n=13 cells). All shaded regions and error bars are s.e.m. * P<0.05, ** P<0.01, *** P<0.001.

Supplementary Figure 2 Time-course of light-driven temperature changes.

. A, Full time-course of temperature changes measured using an acutely inserted thermocouple probe in dorsal striatum in response to light delivery at 3 mW or 15 mW (N = 1 mouse). Data reproduced from Fig. 2b. B, Time-course of opsin-independent decrease in MSN firing. Data reproduced from Fig. 1c in response to 532 nm light at 15 mW with time-scale adjusted to highlight contrast with optogenetic manipulation in Panel C. C, Optogenetic silencing of FSIs (green) and disinhibition of neighboring MSNs (blue) occurs on a time-scale much shorter than light-driven temperature changes recorded in Panel B (N=5 PV-2A-cre mice; n=64 MSNs, n=16 FSIs). D, Computer model simulations highlight the sensitivity of temperature changes to light pulse duration. Each trace represents equivalent light delivery (3 sec light over a 30 sec period = 10% duty cycle). Condensing this light into fewer pulses of longer duration each drives increased fluctuations in temperature. Simulation measured at 1 mm from the tip of a 200 μm optical fiber tip using 532 nm light at 15 mW. E, Computer model of predicted temperature changes in response to 1 sec at 15 mW light delivered with commonly used wavelengths. Simulation measured 1 mm from the tip of a 200 μm optical fiber. F. Computer model of predicted temperature change in response to 20 min illumination with 473 nm light through a 200 μm NA 0.39 fiber, 200um below fiber tip. Shaded regions indicated mean +/- s.e.m.

Supplementary Figure 3 Computer model of how changes in light power, duty cycle, and pulse duration affect temperature.

. A, Simulation of light delivery at varying powers using a published computer model1. Temperature measurements predicted at 1 mm from the tip of a 200 μm optical fiber with 532 nm light delivered for a 1 sec continuous pulse. B, Simulation of light delivery with varying duty cycle at 15 mW. Other parameters equivalent to Panel A. C, Simulation of light delivery with varying pulse duration at 15 mW. Other parameters equivalent to Panel A. Note near equivalence of temperature responses in each case as the amount of light is changed by altering the power (Panel A), duty cycle (Panel B), or pulse duration (Panel C).

Supplementary Figure 4 Additional quantification of open field behavior.

. A, Mouse speed aligned to light onset. B, Total number of rotations, clockwise and counterclockwise, across behavioral sessions. Mean (bar) and individual hemispheres (gray circles). C, Total distance traveled across behavioral sessions, organized as described above. D. Average speed during Pre, Light, and Post periods. No significant changes were detected using two-sided signed rank test (3 mW, P=0.73 and P=0.81; 7 mW, P=0.41 and P=0.26; 15 mW, P=0.53 and P=0.08; 15 mW 20 Hz, P=0.73 and P=0.32). E, Mean change in body direction aligned to light onset. Dotted lines represent 95% percentile bounds based on baseline angle change (-20 to 0 seconds) for all sessions. F. Mean change in body direction. Two-sided signed-rank test (3 mW, P=0.06 and P = 0.13; 7 mW, P=0.002 and P=2.6x10-4; 15 mW, P=6.1x10-5 and P=2.4x10-5; 15 mW at 20 Hz, P=0.67 and P=0.03). For all panels in this figure, N=11 mice, n=22 sessions per condition (1 session per hemisphere per light condition). Each data point represents a single session. All error bars and shaded regions represent s.e.m.

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Owen, S.F., Liu, M.H. & Kreitzer, A.C. Thermal constraints on in vivo optogenetic manipulations. Nat Neurosci 22, 1061–1065 (2019) doi:10.1038/s41593-019-0422-3

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