Addendum: Independent optical excitation of distinct neural populations

Klapoetke, Nathan C.; Murata, Yasunobu; Kim, Sung Soo; Pulver, Stefan R.; Birdsey-Benson, Amanda; Cho, Yong Ku; Morimoto, Tania K.; Chuong, Amy S.; Carpenter, Eric J.; Tian, Zhendong; Wang, Jun; Xie, Yinlong; Yan, Zhixiang; Zhang, Yong; Chow, Brian Y.; Surek, Barbara; Melkonian, Michael; Jayaraman, Vivek; Constantine‐Paton, Martha; Wong, Gane Ka‐Shu; Boyden, Edward S.

doi:10.1038/nmeth0914-972

Cited by 40 publications

(33 citation statements)

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“…In this study, we compared the ion selectivity of our previously published XXL and XXM and found that XXM showed a four-fold increased Na + selectivity over H + together with a two-fold increased K + selectivity over H + , compared to wild type ChR2. Based on this finding, we made further aspartate to histidine and cysteine mutations of PsChR (Platymonas subcordiformis channelrhodopsin) [20] and Chronos [9]. PsChR wild type was already reported to be highly Na + conductive and indeed showed a six-fold increased Na + and K + selectivity over H + compared to wild type ChR2 in our measurements.…”

Section: Introductionmentioning

confidence: 81%

“…Such changes can also be engineered by point mutations. In this study we compared the properties of ChR2, Chronos [9], PsChR [20], and their corresponding mutants of the aspartate in TM4 (DC gate aspartate).…”

Section: Discussionmentioning

confidence: 99%

“…We further synthesized Chronos [9] and PsChR [20] and characterized the corresponding aspartate to cysteine, and the histidine mutations as the aspartate in transmembrane helix 4 (TM4) were conserved in all three channelrhodopsins (Appendix B). Chronos D173C, D173H, and PsChR D139H, D139C, all showed an increased expression level, compared to their wild type (Figure 1a).…”

Section: Mutation Of the Aspartate In Tm4 Influences The Ca 2+ Permeamentioning

confidence: 99%

“…The calcium translocating channelrhodopsin CatCh (a ChR2 leucine to cysteine mutation at position 132, L132C) showed improved Ca 2+ conductivity together with a larger photocurrent and higher light sensitivity [8]. Newly discovered Chronos (Stigeoclonium helveticum channelrhodopsin = ShChR) and Chrimson (CnChR1 from Chlamydomonas noctigama) showed a faster channel closing and a red-shifted action spectrum, respectively [9]. An E90R (glutamate to arginine mutation at position 90) point mutation could extend the cation conductance of ChR2 to additional anion conductance [10].…”

Section: Introductionmentioning

confidence: 99%

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Mutated Channelrhodopsins with Increased Sodium and Calcium Permeability

2019

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(1) Background: After the discovery and application of Chlamydomonas reinhardtii channelrhodopsins, the optogenetic toolbox has been greatly expanded with engineered and newly discovered natural channelrhodopsins. However, channelrhodopsins of higher Ca2+ conductance or more specific ion permeability are in demand. (2) Methods: In this study, we mutated the conserved aspartate of the transmembrane helix 4 (TM4) within Chronos and PsChR and compared them with published ChR2 aspartate mutants. (3) Results: We found that the ChR2 D156H mutant (XXM) showed enhanced Na+ and Ca2+ conductance, which was not noticed before, while the D156C mutation (XXL) influenced the Na+ and Ca2+ conductance only slightly. The aspartate to histidine and cysteine mutations of Chronos and PsChR also influenced their photocurrent, ion permeability, kinetics, and light sensitivity. Most interestingly, PsChR D139H showed a much-improved photocurrent, compared to wild type, and even higher Na+ selectivity to H+ than XXM. PsChR D139H also showed a strongly enhanced Ca2+ conductance, more than two-fold that of the CatCh. (4) Conclusions: We found that mutating the aspartate of the TM4 influences the ion selectivity of channelrhodopsins. With the large photocurrent and enhanced Na+ selectivity and Ca2+ conductance, XXM and PsChR D139H are promising powerful optogenetic tools, especially for Ca2+ manipulation.

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Section: Introductionmentioning

confidence: 81%

Section: Discussionmentioning

confidence: 99%

Section: Mutation Of the Aspartate In Tm4 Influences The Ca 2+ Permeamentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Mutated Channelrhodopsins with Increased Sodium and Calcium Permeability

2019

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“…The following fly lines were used: MBON split-Gal4 lines (28);n 20xUAS-CsChrimson-mVenus (attP18) (36); 10xUAS-Chrimson88-tdTomato3.1 (attp18) (David Anderson lab); UAS-Shibire ts (37); 20xUAS-Shibire ts (38); 20xUAS-gtACR1 (39); UAS-Kir2. 1 (40); DAN split-Gal4 lines (28); w 1118 ; pBPp65ADZpUw (attP40); pBPZpGAL4DBDUw (attP2) (Empty Split) (41); 20xUAS-dsFRT-CsChrimson-mVenus (42) ; LexAop-FLP (43) ; scr-LexA (44).…”

Section: Methodsmentioning

confidence: 99%

Activation of specific mushroom body output neurons inhibits proboscis extension and feeding behavior

Chia¹,

Scott²

2019

Preprint

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15The ability to modify behavior based on prior experience is essential to an animal's 16 survival. For example, animals may become attracted to a previously neutral odor or reject a 17 previously appetitive food source upon learning. In Drosophila, the mushroom bodies (MBs) are 18 critical for olfactory associative learning and conditioned taste aversion, but how the output of 19 the MBs affects specific behavioral responses is unresolved. In conditioned taste aversion, 20 Drosophila shows a specific behavioral change upon learning: proboscis extension to sugar is 21 reduced after a sugar stimulus is paired with an aversive stimulus. While studies have identified 22 MB output neurons (MBONs) that drive approach or avoidance behavior, whether the same 23 MBONs impact proboscis extension behavior is unknown. Here, we tested the role of MB 24 pathways in modulating proboscis extension and identified 10 MBON split-GAL4 lines that upon 25 activation significantly decreased proboscis extension to sugar. Activating several of these lines 26 also decreased sugar consumption, revealing that these MBONs have a general role in modifying 27 feeding behavior beyond proboscis extension. Although the MBONs that decreased proboscis 28 extension and ingestion are different from those that drive avoidance behavior in another context, 29 the diversity of their arborizations demonstrates that a distributed network influences proboscis 30 extension behavior. These studies provide insight into how the MB flexibly alters the response to 31 taste compounds and modifies feeding decisions.32 33 Introduction 34A key role of the brain is to prioritize relevant sensory information to guide behavior.35 Animals exhibit innate behaviors to a variety of sensory stimuli including tastes and odors, and 3 36 the ability to modify those behaviors based on contextual cues and prior experience is essential 37 to an animal's survival. 38In Drosophila, the mushroom body (MB) has long been implicated as a center for 39 learning and memory, and has been studied most extensively in the context of olfactory 40 associative learning (1-4). The dendrites of the principal cells of the MB, Kenyon cells (KCs), 41 receive sparse, random synaptic inputs from olfactory projection neurons (5). The parallel axonal 42 fibers of the KCs form the MB lobes, the output region of the MB. The axonal lobes comprised 43 of ~2000 KC axons are beautifully organized into 15 compartments, defined anatomically by the 44 dendrites of 21 types of MB output neurons (MBONs) (6). These compartments also contain the 45 axon terminals of 20 types of dopaminergic neurons (DANs), which similarly tile the MB lobes.46 The DANs convey signals of reward or punishment for sensory associations (7-14). In Pavlovian 47 terminology for a classical conditioning paradigm, the odor is designated the conditioned 48 stimulus (CS), and the DANs carry the unconditioned stimulus (US). 49In the current model of olfactory associative learning, behavior is determined through the 50 summation of activity in di...

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Algal Photobiology: A Rich Source of Unusual Light Sensitive Proteins for Synthetic Biology and Optogenetics

Kianianmomeni

Hallmann

2016

Methods in Molecular Biology

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The light absorption system in eukaryotic (micro)algae includes highly sensitive photoreceptors, which change their conformation in response to different light qualities on a subsecond time scale and induce physiological and behavioral responses. Some of the light sensitive modules are already in use to engineer and design photoswitchable tools for control of cellular and physiological activities in living organisms with various degrees of complexity. Thus, identification of new light sensitive modules will not only extend the source material for the generation of optogenetic tools but also foster the development of new light-based strategies in cell signaling research. Apart from searching for new proteins with suitable light-sensitive modules, smaller variants of existing light-sensitive modules would be helpful to simplify the construction of hybrid genes and facilitate the generation of mutated and chimerized modules. Advances in genome and transcriptome sequencing as well as functional analysis of photoreceptors and their interaction partners will help to discover new light sensitive modules.

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Addendum: Independent optical excitation of distinct neural populations

Cited by 40 publications

References 1 publication

Mutated Channelrhodopsins with Increased Sodium and Calcium Permeability

Mutated Channelrhodopsins with Increased Sodium and Calcium Permeability

Activation of specific mushroom body output neurons inhibits proboscis extension and feeding behavior

Algal Photobiology: A Rich Source of Unusual Light Sensitive Proteins for Synthetic Biology and Optogenetics

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