Research Article: New Research | Sensory and Motor Systems Activation of distinct Channelrhodopsin variants engages different patterns of network activity https://doi.org/10.1523/ENEURO.0222-18.2019 Cite as: eNeuro 2019; 10.1523/ENEURO.0222-18.2019 Received: 4 June 2018 Revised: 25 October 2019 Accepted: 1 December 2019 This Early Release article has been peer-reviewed and accepted, but has not been through the composition and copyediting processes. The final version may differ slightly in style or formatting and will contain links to any extended data. Alerts: Sign up at www.eneuro.org/alerts to receive customized email alerts when the fully formatted version of this article is published. Copyright © 2019 Jun and Cardin This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license, which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed. 1 Manuscript Title: Activation of distinct Channelrhodopsin variants engages 2 different patterns of network activity. 3 4 Abbreviated title: Variable network impact of optogenetic tools. 5 6 Authors: Na Young Jun1 and Jessica A. Cardin2,3 7 8 1Department of Ophthalmology, Yale University, New Haven CT 06520 9 10 2Department of Neuroscience, Yale University, New Haven CT 06520 11 12 3Kavli Institute for Neuroscience, Yale University, New Haven CT 06520 13 14 Author contributions: N.Y.J. and J.A.C. designed the study. N.Y.J. performed 15 experiments and analyzed the data. N.Y.J. and J.A.C. wrote the manuscript. 16 17 Correspondence should be addressed to: 18 Jessica A. Cardin 19 333 Cedar St., PO Box 208001 20 New Haven, CT 06511 21 [email protected] 22 23 Figures: 6 Abstract: 152 words 24 Tables: 0 Significance Statement: 121 words 25 Multimedia: 0 Introduction: 713 words 26 Discussion: 1134 words 27 28 Acknowledgments 29 The authors thank Dr. M.J. Higley for valuable discussions. We thank Dr. E.S. Boyden 30 for generously sharing samples of viral vectors for Chronos and Chrimson used in initial 31 pilot experiments. We thank Mr. Jong Wook Kim for assistance with coding and data 32 analysis. 33 34 Funding 35 This work was supported by NIH R01 MH102365, NIH R01 EY022951, NIH R01 36 MH113852, NIH P30 EY026878, a Smith Family Award for Excellence in Biomedical 37 Research, a Klingenstein Fellowship Award, an Alfred P. Sloan Fellowship, a NARSAD 38 Young Investigator Award, a grant from the Ludwig Foundation, and a McKnight 39 Fellowship to JAC. 40 41 Competing interests 42 The authors declare no competing interests 43 44 45 1 46 Abstract 47 48 Several recently developed Channelrhodopsin (ChR) variants are characterized 49 by rapid kinetics and reduced desensitization in comparison to the widely used 50 Channelrhodopsin-2 (ChR2). However, little is known about how varying opsin 51 properties may regulate their interaction with local network dynamics. We compared 52 evoked cortical activity in mice expressing three ChR variants with distinct temporal 53 profiles under the CamKII promoter: Chronos, Chrimson, and ChR2. We assessed 54 overall neural activation by measuring the amplitude and temporal progression of 55 evoked spiking. Using gamma-range (30-80Hz) LFP power as an assay for local network 56 engagement, we examined the recruitment of cortical network activity by each tool. All 57 variants caused light-evoked increases in firing in vivo, but each demonstrated different 58 temporal patterning of evoked activity. In addition, the three ChRs had distinct effects on 59 cortical gamma-band activity. Our findings suggest the properties of optogenetic tools 60 can substantially affect their efficacy in vivo, as well their engagement of circuit 61 resonance. 62 63 64 Significance statement 65 66 Genetically modified opsins are some of the most widely used experimental tools 67 in modern neuroscience. However, although these tools are well characterized at the 68 single-cell level, little is known about how the varying properties of the opsins affect their 69 interactions with active neural networks in vivo. Here we present data from experiments 70 using three optogenetic tools with distinct activation/inactivation and kinetic profiles. We 71 find that opsin properties regulate the amplitude and temporal pattern of activity evoked 72 in vivo. Despite all evoking elevated spiking, the three opsins also differentially regulate 73 cortical gamma oscillations. These data suggest that the kinetic properties of 74 optogenetic tools interact with active neural circuits on several time scales. Optogenetic 75 tool selection should therefore be a key element of experimental design. 76 77 78 79 2 80 Introduction 81 82 The advent of easily accessible optogenetic tools for manipulating neural activity 83 has substantially altered experimental neuroscience. The current optogenetics toolkit for 84 neuroscience comprises a large number of Channelrhodopsins (ChRs), Halorhodopsins, 85 and Archaerhodopsins that enable activation and suppression of neural activity with 86 millisecond-timescale precision. Within the Channelrhodopsin family, many variants 87 have now been made with altered activation spectra, photocycle kinetics, and ion 88 selectivity. The first tool to be widely used in neuroscientific approaches, 89 Channelrhodopsin-2 (ChR2), is a nonspecific cation channel with sensitivity to blue light. 90 ChR2 conferred the ability to evoke action potentials with high precision and reliability 91 across a wide range of cell types (Boyden et al., 2005; Cardin et al., 2009; Deisseroth, 92 2015). However, the utility of this tool has been somewhat limited by its relatively long 93 offset kinetics and fairly rapid inactivation of photocurrents in response to sustained 94 strong light stimulation (Boyden et al., 2005; Bamann et al., 2008; Ritter et al., 2008; 95 Schoenenberger et al., 2011). In addition, most naturally occurring Channelrhodopsins 96 are sensitive to blue-green light, presenting a challenge to the use of multiple tools for 97 simultaneous optogenetic control of distinct neural populations. A significant effort in the 98 field has therefore been made to develop Channelrhodopsin variants with faster on- and 99 offset temporal kinetics, less desensitization over time, and red-shifted activation spectra. 100 Previous work has suggested that the Channelrhodopsins are highly effective 101 tools for probing the cellular interactions underlying intrinsically generated patterns of 102 brain activity. Stimulation of Parvalbumin (PV)-expressing interneurons in the cortex via 103 ChR2 evokes gamma oscillations, entrains the firing of excitatory pyramidal neurons, 104 and regulates sensory responses (Cardin et al., 2009; Sohal et al., 2009). Similarly, 105 ChR2 stimulation of PV+ GABAergic long-range projection neurons in the basal forebrain 106 generates gamma-range oscillations in frontal cortex circuits (Brown and McKenna, 107 2015). Recent work further suggests that ChR2 activation of Somatostatin-expressing 108 interneurons, which synapse on both PV+ cells and excitatory neurons, evokes cortical 109 oscillations in a low gamma range (Veit et al., 2017). Sustained depolarization of 110 excitatory sensory cortical neurons via ChR2 activation likewise evokes gamma 111 oscillations, likely by engaging reciprocal interactions with local GABAergic interneurons. 112 (Adesnik and Scanziani, 2010). In comparison, activation of pyramidal neurons in 113 mouse motor cortex via ChRGR, another ChR variant, evokes activity in a broad range 3 114 of lower-band frequencies (Wen et al., 2010). High-fidelity spiking recruited by Chronos, 115 oChiEF, and ReaChR has been used in vitro and in vivo in visual cortex (Chaigneau et 116 al., 2016; Hass and Glickfeld, 2016; Ronzitti et al., 2017) and the auditory midbrain (Guo 117 et al., 2015; Hight et al., 2015), but the impact of such stimulation on the surrounding 118 network remains unclear. 119 Despite the substantial increase in available ChR variants with diverse kinetic 120 and spectral properties, it remains unclear how these properties interact with 121 endogenous temporal patterns of neural circuit activity like gamma oscillations in vivo. 122 Furthermore, the properties of optogenetic tools are typically validated using short 123 pulses of light (1 to 100ms) under quiet network conditions in vitro, but these tools are 124 widely used for sustained neural activation (100s of ms to s) under active network 125 conditions in vivo (Adesnik and Scanziani, 2010; Bortone et al., 2014; Phillips and 126 Hasenstaub, 2016; Burgos-Robles et al., 2017). Here we tested the impact of 127 optogenetic tool properties on evoked activity patterns in the intact brain. We took 128 advantage of the well-characterized gamma oscillation rhythm in mouse primary visual 129 cortex in vivo (Adesnik and Scanziani, 2010; Niell and Stryker, 2010; Vinck et al., 2015) 130 as a metric for optogenetic recruitment of local network activity. Using optogenetic 131 activation of excitatory pyramidal cells as a paradigm to evoke both spiking and cortical 132 gamma oscillations, we compared three Channelrhodopsins with robust photocurrents 133 but distinct kinetic profiles: Chronos, with high-speed on and off kinetics (Klapoetke et al., 134 2014); ChR2, with fast on but relatively slow off kinetics (Boyden et al., 2005); and 135 Chrimson (Klapoetke et al., 2014), with slow on and off kinetics. We found that these 136 tools, although expressed in the same cell types in the same