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Activation of Distinct Channelrhodopsin Variants Engages Different Patterns of Network Activity

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Activation of Distinct Channelrhodopsin Variants Engages Different Patterns of Network Activity New Research Sensory and Motor Systems Activation of Distinct Channelrhodopsin Variants Engages Different Patterns of Network Activity Na Young Jun1 and Jessica A. Cardin2,3 https://doi.org/10.1523/ENEURO.0222-18.2019 1Department of Ophthalmology, Yale University, New Haven, CT 06520, 2Department of Neuroscience, Yale University, New Haven, CT 06520, and 3Kavli Institute for Neuroscience, Yale University, New Haven, CT 06520 Abstract Several recently developed Channelrhodopsin (ChR) variants are characterized by rapid kinetics and reduced desensitization in comparison to the widely used ChR2. However, little is known about how varying opsin properties may regulate their interaction with local network dynamics. We compared evoked cortical activity in mice expressing three ChR variants with distinct temporal profiles under the CamKII promoter: Chronos, Chrimson, and ChR2. We assessed overall neural activation by measuring the amplitude and temporal progres- sion of evoked spiking. Using ␥-range (30–80 Hz) local field potential (LFP) power as an assay for local network engagement, we examined the recruitment of cortical network activity by each tool. All variants caused light-evoked increases in firing in vivo, but each demonstrated different temporal patterning of evoked activity. In addition, the three ChRs had distinct effects on cortical ␥-band activity. Our findings suggest the properties of optogenetic tools can substantially affect their efficacy in vivo, as well their engagement of circuit resonance. Key words: Channelrhodopsin; Chrimson; Chronos; cortex; ␥ oscillations; optogenetics Significance Statement Genetically modified opsins are some of the most widely used experimental tools in modern neuroscience. However, although these tools are well characterized at the single-cell level, little is known about how the varying properties of the opsins affect their interactions with active neural networks in vivo. Here, we present data from experiments using three optogenetic tools with distinct activation/inactivation and kinetic profiles. We find that opsin properties regulate the amplitude and temporal pattern of activity evoked in vivo. Despite all evoking elevated spiking, the three opsins also differentially regulate cortical ␥ oscillations. These data suggest that the kinetic properties of optogenetic tools interact with active neural circuits on several time scales. Optogenetic tool selection should therefore be a key element of experimental design. Introduction rhodopsins (ChRs), Halorhodopsins, and Archaerho- The advent of easily accessible optogenetic tools for dopsins that enable activation and suppression of neural manipulating neural activity has substantially altered ex- activity with millisecond-timescale precision. Within the perimental neuroscience. The current optogenetics toolkit ChR family, many variants have now been made with for neuroscience comprises a large number of Channel- altered activation spectra, photocycle kinetics, and ion Received June 4, 2018; accepted December 1, 2019; First published ship Award; an Alfred P. Sloan Fellowship; a National Alliance for Research on December 10, 2019. Schizophrenia and Depression Young Investigator Award; a grant from the The authors declare no competing financial interests. Ludwig Foundation; and a McKnight Fellowship (J.A.C.). Author contributions: N.Y.J. and J.A.C. designed research; N.Y.J. and J.A.C. Acknowledgements: We thank Dr. M.J. Higley for valuable discussions. We performed research; N.Y.J. and J.A.C. analyzed data; N.Y.J. and J.A.C. wrote thank Dr. E.S. Boyden for generously sharing samples of viral vectors for the paper. Chronos and Chrimson used in initial pilot experiments. We thank Mr. Jong This work was supported by National Institutes of Health Grants R01 Wook Kim for assistance with coding and data analysis. MH102365, R01 EY022951, R01 MH113852, and P30 EY026878; a Smith Correspondence should be addressed to Jessica A. Cardin at Family Award for Excellence in Biomedical Research; a Klingenstein Fellow- [email protected]. January/February 2020, 7(1) ENEURO.0222-18.2019 1–10 New Research 2 of 10 selectivity. The first tool to be widely used in neuroscien- s) under active network conditions in vivo (Adesnik and tific approaches, ChR2, is a nonspecific cation channel Scanziani, 2010; Bortone et al., 2014; Phillips and Hasen- with sensitivity to blue light. ChR2 conferred the ability to staub, 2016; Burgos-Robles et al., 2017). Here, we tested evoke action potentials with high precision and reliability the impact of optogenetic tool properties on evoked ac- across a wide range of cell types (Boyden et al., 2005; tivity patterns in the intact brain. We took advantage of the Cardin et al., 2009; Deisseroth, 2015). However, the utility well-characterized ␥ oscillation rhythm in mouse primary of this tool has been somewhat limited by its relatively visual cortex in vivo (Adesnik and Scanziani, 2010; Niell long offset kinetics and fairly rapid inactivation of photo- and Stryker, 2010; Vinck et al., 2015) as a metric for currents in response to sustained strong light stimulation optogenetic recruitment of local network activity. Using (Boyden et al., 2005; Bamann et al., 2008; Ritter et al., optogenetic activation of excitatory pyramidal cells as a 2008; Schoenenberger et al., 2011). In addition, most paradigm to evoke both spiking and cortical ␥ oscillations, naturally occurring ChRs are sensitive to blue-green light, we compared three ChRs with robust photocurrents but presenting a challenge to the use of multiple tools for distinct kinetic profiles: Chronos, with high-speed on and simultaneous optogenetic control of distinct neural pop- off kinetics (Klapoetke et al., 2014); ChR2, with fast on but ulations. A significant effort in the field has therefore been relatively slow off kinetics (Boyden et al., 2005); and made to develop ChR variants with faster on- and offset Chrimson (Klapoetke et al., 2014), with slow on and off temporal kinetics, less desensitization over time, and red- kinetics. We found that these tools, although expressed in shifted activation spectra. the same cell types in the same brain region and effective Previous work has suggested that the ChRs are highly at eliciting action potentials, evoked distinct patterns of effective tools for probing the cellular interactions under- activity and had different effects on ␥ activity. Together, lying intrinsically generated patterns of brain activity. our data suggest that the kinetic properties of engineered Stimulation of parvalbumin (PV)-expressing interneurons opsin tools affect optogenetic interactions with local cir- in the cortex via ChR2 evokes ␥ oscillations, entrains the cuit activity and should be a key factor in experimental firing of excitatory pyramidal neurons, and regulates sen- design. sory responses (Cardin et al., 2009; Sohal et al., 2009). Similarly, ChR2 stimulation of PVϩ GABAergic long-range Materials and Methods projection neurons in the basal forebrain generates ␥ Animals -range oscillations in frontal cortex circuits (Brown and All animal procedures were performed in accordance McKenna, 2015). Recent work further suggests that ChR2 with the Yale University Institutional Animal Care and Use activation of Somatostatin-expressing interneurons, Committee animal care committee’s regulations. We used which synapse on both PVϩ cells and excitatory neurons, ␥ both female and male C57BL/6J mice ranging from three evokes cortical oscillations in a low range (Veit et al., to five months old. 2017). Sustained depolarization of excitatory sensory cor- tical neurons via ChR2 activation likewise evokes ␥ oscil- Surgical procedures lations, likely by engaging reciprocal interactions with To express ChR2, Chronos, and Chrimson in pyramidal local GABAergic interneurons. (Adesnik and Scanziani, neurons, we injected AAV5-CAMKII-ChR2-GFP (Addgene 2010). In comparison, activation of pyramidal neurons in # 26969), AAV5-CAMKII-CHRONOS-GFP (Addgene # mouse motor cortex via ChRGR, another ChR variant, 58805), or AAV5-CAMKII-CHRIMSON-GFP (Addgene # evokes activity in a broad range of lower-band frequen- 62718), respectively, in the cortex of C57BL/6J mice. For cies (Wen et al., 2010). High-fidelity spiking recruited by the virus injection surgery, 1 ␮l of AAV was injected Chronos, oChiEF, and ReaChR has been used in vitro and through a small burr hole craniotomy in the skull over the in vivo in visual cortex (Chaigneau et al., 2016; Hass and left visual cortex (–3.2 mm posterior, –2.5 mm lateral, –500 Glickfeld, 2016; Ronzitti et al., 2017) and the auditory ␮m deep relative to bregma) using a glass pipette. Injec- midbrain (Guo et al., 2015; Hight et al., 2015), but the tions were made via beveled glass micropipette at a rate impact of such stimulation on the surrounding network of ϳ100 nl/min. After injection, pipettes were left in the remains unclear. brain for ϳ5 min to prevent backflow. Mice were given Despite the substantial increase in available ChR variants four weeks for virus expression before experiments. with diverse kinetic and spectral properties, it remains un- clear how these properties interact with endogenous tem- poral patterns of neural circuit activity like ␥ oscillations in Electrophysiological recordings vivo. Furthermore, the properties of optogenetic tools are Mice were anesthetized with 0.3–0.5% isoflurane in typically validated using short pulses of light (1–100 ms) oxygen and head-fixed by cementing a titanium headpost under quiet network conditions
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