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Novel Luciferase–Opsin Combinations for Improved Luminopsins

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Novel Luciferase–Opsin Combinations for Improved Luminopsins Received: 1 May 2017 | Revised: 15 August 2017 | Accepted: 16 August 2017 DOI: 10.1002/jnr.24152 RESEARCH ARTICLE Novel luciferase–opsin combinations for improved luminopsins Sung Young Park1 | Sang-Ho Song2,3 | Brandon Palmateer4,5 | Akash Pal4,5 | Eric D. Petersen4,5 | Gabrielle P. Shall4 | Ryan M. Welchko4 | Keiji Ibata6,7 | Atsushi Miyawaki6 | George J. Augustine1,2,3 | Ute Hochgeschwender4,5 1Center for Functional Connectomics, Korea Institute of Science and Technology, Seoul, Republic of Korea 2Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore 3Institute of Molecular and Cell Biology, Singapore 4Neuroscience Program, Central Michigan University, Mt. Pleasant, Michigan 5College of Medicine, Central Michigan University, Mt. Pleasant, Michigan 6Laboratory for Cell Function Dynamics, Brain Science Institute, Riken, Saitama, Japan 7School of Medicine, Keio University, Tokyo, Japan Correspondence Ute Hochgeschwender, Neuroscience Abstract Program and College of Medicine, Central Previous work has demonstrated that fusion of a luciferase to an opsin, to create a luminescent Michigan University, 1280 SE Campus opsin or luminopsin, provides a genetically encoded means of manipulating neuronal activity via Drive, CMED 2405, Mount Pleasant, both chemogenetic and optogenetic approaches. Here we have expanded and refined the versatility MI 48859. Email: [email protected] of luminopsin tools by fusing an alternative luciferase variant with high light emission, Gaussia lucif- erase mutant GLucM23, to depolarizing and hyperpolarizing channelrhodopsins with increased light Funding Information sensitivity. The combination of GLucM23 with Volvox channelrhodopsin-1 produced LMO4, while This work was supported by the National combining GLucM23 with the anion channelrhodopsin iChloC yielded iLMO4. We found efficient Institutes of Health (R21MH101525; R21EY026427; U01NS099709), the activation of these channelrhodopsins in the presence of the luciferase substrate, as indicated by National Science Foundation (CBET- responses measured in both single neurons and in neuronal populations of mice and rats, as well as 1464686), the W.M. Keck Foundation, the by changes in male rat behavior during amphetamine-induced rotations. We conclude that these Singapore Ministry of Education (2015-T1- new luminopsins will be useful for bimodal opto- and chemogenetic analyses of brain function. 001-069), and the World Class Institute (WCI) program of the National Research Foundation of Korea (NRF), funded by the KEYWORDS Ministry of Science, ICT & Future Planning (WCI 2009-003). 6-OHDA rat, bioluminescence, chemogenetics, GLucM23, iChloC, optogenetics Significance | 1 INTRODUCTION Manipulation of neuronal activity in behaving experimental animals is crucial for elucidating neuronal networks underlying Interrogation of neural circuits at a wide range of spatial and temporal brain function. Both optogenetic and chemogenetic approaches scales is necessary to elucidate the relationship between neural activity continue to be useful for identifying the contributions of and behavior. For this purpose, both optogenetic (Fenno, Yizhar, & genetically defined neuron populations to circuit and behavioral Deisseroth, 2011) and chemogenetic (Sternson & Roth, 2014) probes outputs. Luminopsins, which are fusions of light-emitting lucifer- have proven very valuable because of their ability to manipulate the ases and light-sensing optogenetic elements, provide both acute electrical activity of genetically defined populations of neurons. Previ- optical and chronic chemical control in one molecule. Our new- ous work has developed a tool set that integrates opto- and chemoge- est probes, LMO4 and iLMO4, combine a brighter luciferase netic approaches by fusing a light-emitting luciferase to an optogenetic with activating and silencing opsins for improved bimodal control light-responsive element, resulting in a luminescent opsin, or luminop- of neural activity. sin (LMO) (Berglund, Birkner, Augustine, & Hochgeschwender, 2013; JNeuroRes. 2017;1–12. wileyonlinelibrary.com/journal/jnr VC 2017 Wiley Periodicals, Inc. | 1 2 | PARK ET AL. Berglund et al., 2016; Tung, Gutekunst, & Gross, 2015). In these LMOs, an AAV vector downstream of a human synapsin promoter as bioluminescence produced by oxidation of a diffusible luciferin sub- described previously (Berglund et al., 2016). strate by the luciferase enzyme activates a tethered, nearby opsin. Depending on the biophysical properties of the opsin, the light gener- 2.2 | Cell culture ated by the luciferase can either excite or inhibit target neurons Human embryonic kidney fibroblasts (HEK293, RRID:CVCL_0045; and expressing the LMO. Such integration of opto- and chemogenetic HEK293FT, RRID:CVCL_6911) were grown in Dulbecco’s modified approaches allows manipulation of neural activity over a range of spa- Eagle’s medium supplemented with 10% fetal bovine serum (FBS), 100 tial and temporal scales in the same experimental animal. Specifically, U penicillin, and 0.1 mg streptomycin per milliliter, at 37 8Cand5% LMOs allow rapid optogenetic control of the activity of subsets of neu- CO in a humidified atmosphere. Cells were transfected using Lipofect- rons as well as slower chemogenetic control of the activity of entire 2 amine 2000 (Invitrogen, Waltham, MA) according to the manufacturer’s populations of neurons. protocol. For luminescence experiments 1 3 104 cells were seeded in Initially, wild-type Gaussia luciferase (GLuc) (Verhaegen & Christo- white 96-well plates (BD Falcon, Franklin Lakes, NJ). Cells were used poulos, 2002) was combined with channelrhodopsin 2 (ChR2) (Nagel for luminescence experiments 36 hr after transfection. et al., 2003) to yield the excitatory LMO1 (Berglund et al., 2013), while For in vitro electrophysiology studies, primary hippocampal neu- combination of the red-shifted Renilla luciferase version TagRFP-RLuc rons were cultured from newborn mouse pups (P0–P2) of both sexes (Dragulescu-Andrasi, Chan, De, Massoud, & Gambhir, 2011) with of strain B6129F1 hybrid (RRID:IMSR_TAC:b6129) as described previ- Natronomonas halorhodopsin (NpHR) (Gradinaru, Thompson, & Dei- ously (Berglund et al., 2013). Lipofection was used for transfecting cul- sseroth, 2008) resulted in the inhibitory iLMO1 (Tung et al. 2015). The tured neurons on days in vitro (DIV) 2 to 4. We used 1/10th of the performance of subsequent LMO versions was significantly improved recommended amount of Lipofectamine and otherwise followed the by using luciferase variants with higher light emission, such as the manufacturer’s protocol. Cells were plated in culture medium consisting GLuc variants “slow burn” (sbGLuc) (Welsh, Patel, Manthiram, & of Neurobasal Medium (Invitrogen) containing B-27 (Invitrogen), 2 mM Swartz, 2009) and “superluminescent” (slGLuc) (Kim, Suzuki, Sato, & Glutamax (Invitrogen), and 5% FCS (NB-FCS). The medium was Tao, 2011), as well as the RLuc-based luciferase–fluorescent fusion replaced with culture medium without serum the next day (NB-Plain). protein Nano-lantern (Saito et al., 2012). These probes allowed activa- For multielectrode array (MEA) experiments, primary neurons tion and silencing of neurons via noninvasive peripheral application of were collected from embryonic day 18 rat embryos of both sexes from the luciferase substrate in freely moving mice and rats, resulting in Sprague–Dawley females (RRID:RGD_5508397) obtained directly from observable changes in their behavior (Berglund et al., 2016; Tung et al., a commercial vendor (Harlan) or from BrainBits, LLC. Neurons were 2015). transduced with virus on DIV2. Virus was added to the culture medium While the current LMOs are usable, their performance is not fully (1 ml per well of concentrated stocks). All supplements were purchased optimized. Here we have explored the potential of a new GLuc mutant from Gibco/Invitrogen/Thermo Fisher unless indicated otherwise. with very efficient light generation, GLucM23 (Lindberg, Mizukami, Ibata, Miyawaki, & Kikuchi, 2013), as the light-emitting moiety in 2.3 | Virus LMOs. By fusing GLucM23 to the depolarizing opsin Volvox channelrhodopsin-1 (VChR1), we created a fourth-generation excita- AAVs carrying LMOs were produced by transfecting subconfluent tory LMO, LMO4. Similarly, by fusing GLucM23 to a hyperpolarizing HEK293FT cells per 10-cm culture dish with 24 lg of the helper plas- anion channelrhodopsin, iChloC (Wietek et al., 2015), we created a mid pAd delta F6, 20 lg of the serotype plasmid AAV2/9, and 12 lgof novel inhibitory LMO, iLMO4. Validation of these new constructs, both the LMO3 plasmid using Lipofectamine 2000. After 72 hr, supernatant in vitro and in vivo, revealed that these new LMOs have the highest was harvested from culture plates and virus was purified from cells and known coupling between the luciferase enzyme and the opsin actua- supernatant following the method of Guo et al. (2012), but without the tors. We conclude that these new LMOs are useful additions to the partitioning step in the aqueous 2-phase system (Berglund et al., 2016). LMO tool set for bimodal opto- and chemogenetic experiments. Viral titers were determined by q-PCR for the WPRE element. Prepara- tions with titers ranging from 1 3 1010 to 1 3 1013 vg/ml were used in 2 | MATERIALS AND METHODS this study. 2.1 | Constructs 2.4 | Coelenterazine The generation of LMO plasmids is described in detail in Berglund et al. The luciferin, coelenterazine (CTZ), was purchased from Nanolight (2013). For the new versions of LMOs, the wild-type
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