Bidirectional Optogenetic Control of Inhibitory Neurons in Freely-Moving Mice

Abstract

Objective: Optogenetic manipulations of excitable cells enable activating or silencing specific types of neurons. By expressing two types of exogenous proteins, a single neuron can be depolarized using light of one wavelength and hyperpolarized with another. However, routing two distinct wavelengths into the same brain locality typically requires bulky optics that cannot be implanted on the head of a freely-moving animal. Methods: We developed a lens-free approach for constructing dual-color headmounted, fiber-based optical units: any two wavelengths can be combined. Results: Here, each unit was comprised of one 450 nm and one 638 nm laser diode, yielding light power of 0.4 mW and 8 mW at the end of a 50 micrometer multimode fiber. To create a multi-color/multi-site optoelectronic device, a four-shank silicon probe mounted on a microdrive was equipped with two dual-color and two single-color units, for a total weight under 3 g. Devices were implanted in mice expressing the blue-light sensitive cation channel ChR2 and the red-light sensitive chloride pump Jaws in parvalbumin-immunoreactive (PV) inhibitory neurons. The combination of dual-color units with recording electrodes was free from electromagnetic interference, and device heating was under 7 °C even after prolonged operation. Conclusion: Using these devices, the same cortical PV cell could be activated and silenced. This was achieved for multiple cells both in neocortex and hippocampus of freely-moving mice. Significance: This technology can be used for controlling spatially intermingled neurons that have distinct genetic profiles, and for controlling spike timing of cortical neurons during cognitive tasks.

Colocalized, Bidirectional Optogenetic Modulations in Freely Behaving Mice with a Wireless Dual-Color Optoelectronic Probe

“…Results summarized for multiple cells show that the basal spike firing rate (13 Hz) increases to 28 Hz during red illumination and decreases to 2 Hz during blue illumination (Figures S11f and S11g). These results showcase the capability for in vivo bidirectional modulations and are in good accordance with the previous report based on a different opsin combination (ChR2 and Jaws) 36 .…”

“…We also design a customized, miniaturized flexible circuit module to wirelessly operate the dual-color micro-LED probe. Compared to previous reports 22, 36 on tethered fiber or laser coupled devices for dual-color stimulations, the wireless operation here enables the study of complex behaviors of freely moving animals more conveniently. Controlled by radio frequency (RF) communication at 2.4 GHz, the battery-powered circuit (weight ~1.9 g) independently addresses the red and the blue micro-LEDs, by adjusting their pulse frequencies, durations, and injection currents for versatile neural modulations in vivo (Figure 1h, and details in the Methods and Figures S5–S9).…”

“…Certain efforts have been attempted, including explorations based on experiments by expressing excitatory and inhibitory opsins in separate animals 28,29 or distinct cells in different neural regions 17,30 . There are few reports on dualcolor optogenetic activation and inhibition by co-expressing different opsins in the same cells [31][32][33] , but the efficacy and utility of such bidirectional modulations could be limited by the potential spectral overlay of the two opsins and the lack of miniaturized light delivery systems. Therefore, we envision that wirelessly operated light sources with polychromatic emissions, which collaborate with co-expressed opsins, can be implemented for untethered use in freely moving animals and enable previously inaccessible applications.…”

“…Certain efforts have been attempted, including explorations based on experiments by expressing excitatory and inhibitory opsins in separate animals 30, 31 or distinct cells in different neural regions 17, 32 . There are also reports on dual-color optogenetic activation and inhibition by co-expressing different opsins in the same cells 33–36 . Recent efforts also remarkably demonstrate bidirectional optogenetic modulations with implantable waveguides coupled to extra-cranial dual-color laser sources and their combination with in vivo electrophysiological recordings 23, 36, 37 , but the wireless operation has not been achieved.…”

“…There are also reports on dual-color optogenetic activation and inhibition by co-expressing different opsins in the same cells 33–36 . Recent efforts also remarkably demonstrate bidirectional optogenetic modulations with implantable waveguides coupled to extra-cranial dual-color laser sources and their combination with in vivo electrophysiological recordings 23, 36, 37 , but the wireless operation has not been achieved. Therefore, we envision that wirelessly operated light sources with polychromatic emissions, which collaborate with co-expressed opsins, can be implemented for untethered use in freely moving animals and enable previously inaccessible applications.…”