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Optogenetics is a recent breakthrough in neuroscience, and one of the most promising applications is the treatment of retinal degenerative diseases. Multiple clinical trials are currently ongoing, less than a decade after the first attempt at visual restoration using optogenetics. Optogenetic therapy has great value in providing hope for visual restoration in late-stage retinal degeneration, regardless of the genotype. This alternative gene therapy consists of multiple elements including the choice of target retinal cells, optogenetic tools, and gene delivery systems. Currently, there are various options for each element, all of which have been developed as a product of technological success. In particular, the performance of optogenetic tools in terms of light and wavelength sensitivity have been improved by engineering microbial opsins and applying human opsins. To provide better post-treatment vision, the optimal choice of optogenetic tools and effective gene delivery to retinal cells is necessary. In this review, we provide an overview of the advancements in optogenetic therapy for visual restoration, focusing on available options for optogenetic tools and gene delivery methods.

All optical approaches to control and read out the electrical activity in a cardiac syncytium can improve our understanding of cardiac electrophysiology. Here, we demonstrate optogenetic stimulation of cardiomyocytes with high spatial precision using light foci generated with a ferroelectric spatial light modulator. Computer generated holograms binarized by bidirectional error diffusion create multiple foci with more even intensity distribution compared with thresholding approach. We evoke the electrical activity of cardiac HL1 cells expressing the channelrhodopsin-2 variant, ChR2(H134R) using single and multiple light foci and at the same time visualize the action potential using a calcium sensitive indicator called Cal-630. We show that localized regions in the cardiac monolayer can be stimulated enabling us to initiate signal propagation from a precise location. Furthermore, we demonstrate that probing the cardiac cells with multiple light foci enhances the excitability of the cardiac network. This approach opens new applications in manipulating and visualizing the electrical activity in a cardiac syncytium.

The cerebellum is emerging as a powerful regulator of cognitive and affective processing and memory in both humans and animals and has been implicated in affective disorders. How the cerebellum supports affective function remains poorly understood. The short-latency (just a few milliseconds) functional connections that were identified between the cerebellum and amygdala-a structure crucial for the processing of emotion and valence-more than four decades ago raise the exciting, yet untested, possibility that a cerebellum-amygdala pathway communicates information important for emotion. The major hurdle in rigorously testing this possibility is the lack of knowledge about the anatomy and functional connectivity of this pathway. Our initial anatomical tracing studies in mice excluded the existence of a direct monosynaptic connection between the cerebellum and amygdala. Using transneuronal tracing techniques, we have identified a novel disynaptic circuit between the cerebellar output nuclei and the basolateral amygdala. This circuit recruits the understudied intralaminar thalamus as a node. Using ex vivo optophysiology and super-resolution microscopy, we provide the first evidence for the functionality of the pathway, thus offering a missing mechanistic link between the cerebellum and amygdala. This discovery provides a connectivity blueprint between the cerebellum and a key structure of the limbic system. As such, it is the requisite first step toward obtaining new knowledge about cerebellar function in emotion, thus fundamentally advancing understanding of the neurobiology of emotion, which is perturbed in mental and autism spectrum disorders.

Optogenetics is a recent breakthrough in neuroscience, and one of the most promising applications is the treatment of retinal degenerative diseases. Multiple clinical trials are currently ongoing, less than a decade after the first attempt at visual restoration using optogenetics. Optogenetic therapy has great value in providing hope for visual restoration in late-stage retinal degeneration, regardless of the genotype. This alternative gene therapy consists of multiple elements including the choice of target retinal cells, optogenetic tools, and gene delivery systems. Currently, there are various options for each element, all of which have been developed as a product of technological success. In particular, the performance of optogenetic tools in terms of light and wavelength sensitivity have been improved by engineering microbial opsins and applying human opsins. To provide better post-treatment vision, the optimal choice of optogenetic tools and effective gene delivery to retinal cells is necessary. In this review, we provide an overview of the advancements in optogenetic therapy for visual restoration, focusing on available options for optogenetic tools and gene delivery methods.

Kanalrhodopsine (ChRs), lichtgesteuerte Ionenkanäle, vermitteln phototaktische Reaktionen in beweglichen Algen und sind als optogenetische Werkzeuge zur Manipulation der Zellaktivität mittels Lichts weit verbreitet. Viele Kationen- und Anionen-leitende ChRs (CCRs und ACRs) wurden aus kultivierbaren Chlorophyten- und Cryptophytenarten identifiziert. Die meisten mikrobiellen Organismen kann jedoch nicht kultiviert werden, was zu einem unvollständigen Bild der ChR-Vielfalt führt. Die Metagenomik öffnet die Tür für Erkenntnisse über die Verteilung von ChRs in unkultivierten Organismen. Diese Arbeit beschreibt die biophysikalische Charakterisierung von zwei Gruppen metagenomisch identifizierter ChRs. Die MerMAIDs (Metagenomically discovered marine, anion-conducting, and intensely desensitizing ChRs) sind eine neue ChR-Familie und zeigen nahezu komplette Photostrom-Inaktivierung unter Dauerlicht. Die Photoströme lassen sich durch einen Photozyklus erklären, der zur Akkumulation eines langlebigen und nicht-leitenden Photointermediats führt. Ein konserviertes Cystein ist für dieses Phänomen entscheidend, da seine Substitution zu einer stark reduzierten Inaktivierung führt. Die Prasinophyten ChRs, die große carboxyterminale Domänen aufweisen, wurden in großen, marinen Viren identifiziert, die sie von ihren beweglichen und einzelligen Grünalgen-Wirten durch lateralen Gentransfer übernommen haben. Heterolog exprimiert, sind die viralen ChRs nur nach Ergänzung von Transportsequenzen und carboxyterminaler Kürzung funktional. Die Grünalgen- und viralen ChRs sind Anionen-leitend mit nicht-inaktivierenden Photoströmen, wenn sie in Säugetierzellen exprimiert werden, obwohl die viralen Vertreter weniger leitfähig und zytotoxisch sind. Nichtsdestotrotz repräsentiert diese ChR-Gruppe die ersten Grünalgen- und Virus-ACRs. Diese Arbeit zeigt eine breite Verteilung der ACRs unter marinen mikrobiellen Organismen und die Bedeutung der Funktionsmetagenomik bei der Entdeckung neuer ChRs., Channelrhodopsins (ChRs) are light-gated ion channels mediating phototactic responses in motile algae and widely used as optogenetic tools to manipulate cellular activity using light. Many cation- and anion-conducting ChRs (CCRs and ACRs) have been identified from culturable chlorophyte and cryptophyte species. However, most microbial organisms cannot be cultured, resulting in an incomplete view of the diversity of ChRs. Metagenomics opens the door to gather insights on the distribution of ChRs in uncultured organisms. Here, the biophysical characterization of two groups of metagenomically identified ChRs is described. The MerMAIDs (Metagenomically discovered marine, anion-conducting, and intensely desensitizing ChRs) represent a new ChR family with near-complete photocurrent desensitization under continuous illumination. The photocurrents can be explained by a single photocycle leading to the accumulation of a long-lived and non-conducting photointermediate. A conserved cysteine is critical for this phenomenon, as its substitution results in a strongly reduced desensitization. The prasinophyte ChRs, harboring large carboxy-terminal extensions, were identified in marine giant viruses that acquired them from their motile and unicellular green algal hosts via lateral gene transfer. Expressed in cell culture, the viral ChRs are only functional upon the addition of trafficking sequences and carboxy-terminal truncation. The green algal and viral ChRs are anion-conducting and display non-desensitizing photocurrents when expressed in mammalian cells, though the viral representatives are less conductive and cytotoxic. Nonetheless, this group of ChRs represents the first green algal and viral ACRs. This thesis highlights a broad distribution of ACRs among marine microbial organisms and the importance of functional metagenomics in discovering new ChRs.

Kanalrhodopsine (ChRs), lichtgesteuerte Ionenkanäle, vermitteln phototaktische Reaktionen in beweglichen Algen und sind als optogenetische Werkzeuge zur Manipulation der Zellaktivität mittels Lichts weit verbreitet. Viele Kationen- und Anionen-leitende ChRs (CCRs und ACRs) wurden aus kultivierbaren Chlorophyten- und Cryptophytenarten identifiziert. Die meisten mikrobiellen Organismen kann jedoch nicht kultiviert werden, was zu einem unvollständigen Bild der ChR-Vielfalt führt. Die Metagenomik öffnet die Tür für Erkenntnisse über die Verteilung von ChRs in unkultivierten Organismen. Diese Arbeit beschreibt die biophysikalische Charakterisierung von zwei Gruppen metagenomisch identifizierter ChRs. Die MerMAIDs (Metagenomically discovered marine, anion-conducting, and intensely desensitizing ChRs) sind eine neue ChR-Familie und zeigen nahezu komplette Photostrom-Inaktivierung unter Dauerlicht. Die Photoströme lassen sich durch einen Photozyklus erklären, der zur Akkumulation eines langlebigen und nicht-leitenden Photointermediats führt. Ein konserviertes Cystein ist für dieses Phänomen entscheidend, da seine Substitution zu einer stark reduzierten Inaktivierung führt. Die Prasinophyten ChRs, die große carboxyterminale Domänen aufweisen, wurden in großen, marinen Viren identifiziert, die sie von ihren beweglichen und einzelligen Grünalgen-Wirten durch lateralen Gentransfer übernommen haben. Heterolog exprimiert, sind die viralen ChRs nur nach Ergänzung von Transportsequenzen und carboxyterminaler Kürzung funktional. Die Grünalgen- und viralen ChRs sind Anionen-leitend mit nicht-inaktivierenden Photoströmen, wenn sie in Säugetierzellen exprimiert werden, obwohl die viralen Vertreter weniger leitfähig und zytotoxisch sind. Nichtsdestotrotz repräsentiert diese ChR-Gruppe die ersten Grünalgen- und Virus-ACRs. Diese Arbeit zeigt eine breite Verteilung der ACRs unter marinen mikrobiellen Organismen und die Bedeutung der Funktionsmetagenomik bei der Entdeckung neuer ChRs., Channelrhodopsins (ChRs) are light-gated ion channels mediating phototactic responses in motile algae and widely used as optogenetic tools to manipulate cellular activity using light. Many cation- and anion-conducting ChRs (CCRs and ACRs) have been identified from culturable chlorophyte and cryptophyte species. However, most microbial organisms cannot be cultured, resulting in an incomplete view of the diversity of ChRs. Metagenomics opens the door to gather insights on the distribution of ChRs in uncultured organisms. Here, the biophysical characterization of two groups of metagenomically identified ChRs is described. The MerMAIDs (Metagenomically discovered marine, anion-conducting, and intensely desensitizing ChRs) represent a new ChR family with near-complete photocurrent desensitization under continuous illumination. The photocurrents can be explained by a single photocycle leading to the accumulation of a long-lived and non-conducting photointermediate. A conserved cysteine is critical for this phenomenon, as its substitution results in a strongly reduced desensitization. The prasinophyte ChRs, harboring large carboxy-terminal extensions, were identified in marine giant viruses that acquired them from their motile and unicellular green algal hosts via lateral gene transfer. Expressed in cell culture, the viral ChRs are only functional upon the addition of trafficking sequences and carboxy-terminal truncation. The green algal and viral ChRs are anion-conducting and display non-desensitizing photocurrents when expressed in mammalian cells, though the viral representatives are less conductive and cytotoxic. Nonetheless, this group of ChRs represents the first green algal and viral ACRs. This thesis highlights a broad distribution of ACRs among marine microbial organisms and the importance of functional metagenomics in discovering new ChRs.

Kanalrhodopsine (ChRs), lichtgesteuerte Ionenkanäle, vermitteln phototaktische Reaktionen in beweglichen Algen und sind als optogenetische Werkzeuge zur Manipulation der Zellaktivität mittels Lichts weit verbreitet. Viele Kationen- und Anionen-leitende ChRs (CCRs und ACRs) wurden aus kultivierbaren Chlorophyten- und Cryptophytenarten identifiziert. Die meisten mikrobiellen Organismen kann jedoch nicht kultiviert werden, was zu einem unvollständigen Bild der ChR-Vielfalt führt. Die Metagenomik öffnet die Tür für Erkenntnisse über die Verteilung von ChRs in unkultivierten Organismen. Diese Arbeit beschreibt die biophysikalische Charakterisierung von zwei Gruppen metagenomisch identifizierter ChRs. Die MerMAIDs (Metagenomically discovered marine, anion-conducting, and intensely desensitizing ChRs) sind eine neue ChR-Familie und zeigen nahezu komplette Photostrom-Inaktivierung unter Dauerlicht. Die Photoströme lassen sich durch einen Photozyklus erklären, der zur Akkumulation eines langlebigen und nicht-leitenden Photointermediats führt. Ein konserviertes Cystein ist für dieses Phänomen entscheidend, da seine Substitution zu einer stark reduzierten Inaktivierung führt. Die Prasinophyten ChRs, die große carboxyterminale Domänen aufweisen, wurden in großen, marinen Viren identifiziert, die sie von ihren beweglichen und einzelligen Grünalgen-Wirten durch lateralen Gentransfer übernommen haben. Heterolog exprimiert, sind die viralen ChRs nur nach Ergänzung von Transportsequenzen und carboxyterminaler Kürzung funktional. Die Grünalgen- und viralen ChRs sind Anionen-leitend mit nicht-inaktivierenden Photoströmen, wenn sie in Säugetierzellen exprimiert werden, obwohl die viralen Vertreter weniger leitfähig und zytotoxisch sind. Nichtsdestotrotz repräsentiert diese ChR-Gruppe die ersten Grünalgen- und Virus-ACRs. Diese Arbeit zeigt eine breite Verteilung der ACRs unter marinen mikrobiellen Organismen und die Bedeutung der Funktionsmetagenomik bei der Entdeckung neuer ChRs., Channelrhodopsins (ChRs) are light-gated ion channels mediating phototactic responses in motile algae and widely used as optogenetic tools to manipulate cellular activity using light. Many cation- and anion-conducting ChRs (CCRs and ACRs) have been identified from culturable chlorophyte and cryptophyte species. However, most microbial organisms cannot be cultured, resulting in an incomplete view of the diversity of ChRs. Metagenomics opens the door to gather insights on the distribution of ChRs in uncultured organisms. Here, the biophysical characterization of two groups of metagenomically identified ChRs is described. The MerMAIDs (Metagenomically discovered marine, anion-conducting, and intensely desensitizing ChRs) represent a new ChR family with near-complete photocurrent desensitization under continuous illumination. The photocurrents can be explained by a single photocycle leading to the accumulation of a long-lived and non-conducting photointermediate. A conserved cysteine is critical for this phenomenon, as its substitution results in a strongly reduced desensitization. The prasinophyte ChRs, harboring large carboxy-terminal extensions, were identified in marine giant viruses that acquired them from their motile and unicellular green algal hosts via lateral gene transfer. Expressed in cell culture, the viral ChRs are only functional upon the addition of trafficking sequences and carboxy-terminal truncation. The green algal and viral ChRs are anion-conducting and display non-desensitizing photocurrents when expressed in mammalian cells, though the viral representatives are less conductive and cytotoxic. Nonetheless, this group of ChRs represents the first green algal and viral ACRs. This thesis highlights a broad distribution of ACRs among marine microbial organisms and the importance of functional metagenomics in discovering new ChRs.

Kanalrhodopsine (ChRs), lichtgesteuerte Ionenkanäle, vermitteln phototaktische Reaktionen in beweglichen Algen und sind als optogenetische Werkzeuge zur Manipulation der Zellaktivität mittels Lichts weit verbreitet. Viele Kationen- und Anionen-leitende ChRs (CCRs und ACRs) wurden aus kultivierbaren Chlorophyten- und Cryptophytenarten identifiziert. Die meisten mikrobiellen Organismen kann jedoch nicht kultiviert werden, was zu einem unvollständigen Bild der ChR-Vielfalt führt. Die Metagenomik öffnet die Tür für Erkenntnisse über die Verteilung von ChRs in unkultivierten Organismen. Diese Arbeit beschreibt die biophysikalische Charakterisierung von zwei Gruppen metagenomisch identifizierter ChRs. Die MerMAIDs (Metagenomically discovered marine, anion-conducting, and intensely desensitizing ChRs) sind eine neue ChR-Familie und zeigen nahezu komplette Photostrom-Inaktivierung unter Dauerlicht. Die Photoströme lassen sich durch einen Photozyklus erklären, der zur Akkumulation eines langlebigen und nicht-leitenden Photointermediats führt. Ein konserviertes Cystein ist für dieses Phänomen entscheidend, da seine Substitution zu einer stark reduzierten Inaktivierung führt. Die Prasinophyten ChRs, die große carboxyterminale Domänen aufweisen, wurden in großen, marinen Viren identifiziert, die sie von ihren beweglichen und einzelligen Grünalgen-Wirten durch lateralen Gentransfer übernommen haben. Heterolog exprimiert, sind die viralen ChRs nur nach Ergänzung von Transportsequenzen und carboxyterminaler Kürzung funktional. Die Grünalgen- und viralen ChRs sind Anionen-leitend mit nicht-inaktivierenden Photoströmen, wenn sie in Säugetierzellen exprimiert werden, obwohl die viralen Vertreter weniger leitfähig und zytotoxisch sind. Nichtsdestotrotz repräsentiert diese ChR-Gruppe die ersten Grünalgen- und Virus-ACRs. Diese Arbeit zeigt eine breite Verteilung der ACRs unter marinen mikrobiellen Organismen und die Bedeutung der Funktionsmetagenomik bei der Entdeckung neuer ChRs., Channelrhodopsins (ChRs) are light-gated ion channels mediating phototactic responses in motile algae and widely used as optogenetic tools to manipulate cellular activity using light. Many cation- and anion-conducting ChRs (CCRs and ACRs) have been identified from culturable chlorophyte and cryptophyte species. However, most microbial organisms cannot be cultured, resulting in an incomplete view of the diversity of ChRs. Metagenomics opens the door to gather insights on the distribution of ChRs in uncultured organisms. Here, the biophysical characterization of two groups of metagenomically identified ChRs is described. The MerMAIDs (Metagenomically discovered marine, anion-conducting, and intensely desensitizing ChRs) represent a new ChR family with near-complete photocurrent desensitization under continuous illumination. The photocurrents can be explained by a single photocycle leading to the accumulation of a long-lived and non-conducting photointermediate. A conserved cysteine is critical for this phenomenon, as its substitution results in a strongly reduced desensitization. The prasinophyte ChRs, harboring large carboxy-terminal extensions, were identified in marine giant viruses that acquired them from their motile and unicellular green algal hosts via lateral gene transfer. Expressed in cell culture, the viral ChRs are only functional upon the addition of trafficking sequences and carboxy-terminal truncation. The green algal and viral ChRs are anion-conducting and display non-desensitizing photocurrents when expressed in mammalian cells, though the viral representatives are less conductive and cytotoxic. Nonetheless, this group of ChRs represents the first green algal and viral ACRs. This thesis highlights a broad distribution of ACRs among marine microbial organisms and the importance of functional metagenomics in discovering new ChRs.

Kanalrhodopsine (ChRs) sind lichtaktivierte Ionenkanäle motiler Algen. Heterolog exprimiert erlauben sie es, Ionenflüsse durch Licht zu steuern. Bevorzugt geleitet werden von den meisten ChRs Protonen. Ausprägung und Wirkung lichtaktivierter Protonenflüsse sowie der molekulare Mechanismus protonenselektiver ChRs werden in vorliegender Arbeit untersucht und zur Entwicklung neuer optogenetischer Werkzeuge genutzt. Eine besonders hohe Protonenselektivität zeigten die grün- und rotlicht-aktivierten Kanäle CsChR und Chrimson aus den Algen Chloromonas subdivisa und Chlamydomonas noctigama. Im spektroskopisch detailliert untersuchten CrChR2 aus Chlamydomonas reinhardtii änderte sich die Protonenselektivität nach Anregung mit einem ns-Laserblitz sogar innerhalb eines Aktivierungszyklus und war insbesondere nach Öffnung des Kanals sowie in Folge der Lichtadaptation hoch. Als unentbehrlich für eine effiziente Protonenleitung erwiesen sich in allen drei Kanälen konservierte, titrierbare Reste entlang der Pore, deren individuelle Bedeutung für die Protonenleitung sich je nach Protein wesentlich unterschied. Entsprechend genügte in Chrimson der Austausch einzelner Glutaminsäuren des extrazellulären Halbkanals, dieses in einen grün- oder rotlichtaktivierten Natriumkanal zu transformieren. Aminosäuresubstitutionen der unmittelbaren Retinalumgebung verschoben hingegen das Aktionsmaximum von Chrimson röter als 600 nm und damit röter als in allen bisher beschriebenen ChRs. In Chrimson versperrt hierbei ein zusätzliches äußeres Tor den extrazellulär Halbkanal, während die Retinalbindetasche in Struktur und funktionaler Bedeutung der einzelnen Reste wesentlich jener der Protonenpumpe Bacteriorhodopsin ähnelt. Als Zwei-Komponenten-Optogenetik wurden schließlich protonen-, kationen- und anionenleitende ChRs unterschiedlicher Farbsensitivität fusioniert sowie lichtgetriebene Protonenpumpen mit protonenaktivierten Ionenkanälen kombiniert und neue optogenetische Perspektiven eröffnet., Channelrhodopsins (ChRs) are light-gated ion channels from green algae. Expressed in host cells they are used to control ion fluxes by light and are widely applied in Neurosciences. Although generally classified as either cation or anion channels, most ChRs preferentially conduct protons. This thesis compares proton conductance of different ChRs, examines the molecular mechanism of proton selective ChRs and explores the usage of light regulated proton fluxes in two-component-optogenetics. Proton selectivity varied strongly among different ChRs and was most pronounced for the green- and red-light activated channels CsChR and Chrimson from the algae Chloromonas subdivisa and Chlamydomonas noctigama, that conducted predominantly protons even at high pH. In CrChR2 from Chlamydomonas reinhardtii proton selectivity also changed during a single activation cycle and was especially high directly after channel opening and later on following light adaptation. In all three channels efficient proton conductance depended on conserved titratable residues along the pore with different contribution of the individual side chains in each protein. The substitution of single glutamic acids in the extracellular half pore converted Chrimson into a green or red-light activated sodium channel. A single point mutation close to the retinal chromophore shifted peak absorption of Chrimson beyond 600 nm - further red than all other cation conducting ChRs. Whereas the retinal binding pocket of Chrimson resembles the proton pump Bacteriorhodpsin, the overall pore structure corresponds to other ChRs, but features an additional outer gate, that occludes the extracellular half pore and is important for both, proton selectivity and red light absorption. Finally different Two-Component-Optogenetic approaches combined proton and anion selective ChRs of distinct colour as well as light-driven proton pumps and proton-activated ion channels with major prospect for future optogenetic applications.

Die Modifikation von Genen ist in den molekularen Biowissenschaften ein fundamentales Werkzeug, um die Funktion von Genen zu studieren (Reverse Genetik). Diese Arbeit hat erfolgreich Zinkfinger- und CRISPR/Cas9-Nukleasen für die Verwendung in C. reinhardtii etabliert, um Gene im Kerngenom gezielt auszuschalten und präzise zu verändern. Basierend auf vorausgegangener Arbeit mit Zinkfingernukleasen (ZFN) konnte die Transformationseffizienz um das 300-fache verbessert werden, was die Inaktivierung von Genen auch in motilen Wildtyp-Zellen ermöglichte. Damit war es möglich, die Gene für das Kanalrhodopsin-1 (ChR1), Kanalrhodopsin-2 (ChR2) und das Chlamyopsin-1/2-Gen (COP1/2) einzeln und gemeinsam auszuschalten. Eine Analyse der Phototaxis in diesen Stämmen ergab, dass die Phototaxis durch Inaktivierung von ChR1 stärker beeinträchtigt ist als durch Inaktivierung von ChR2. Um das CRISPR/Cas9-System zu verwenden, wurden die Transformationsbedingungen so angepasst und optimiert, dass der Cas9-gRNA-Komplex als in vitro hergestelltes Ribonukleoprotein in die Zellen transformiert wurde. Um die Bedingungen für präzise Genmodifikationen zu messen und zu verbessern, wurde das SNRK2.2-Gen als Reportergen für eine „Blau-Grün Test“ etabliert. Kleine Insertionen von bis zu 30 bp konnten mit kurzen Oligonukleotiden eingefügt werden, während größere Reportergene (mVenus, SNAP-Tag) mithilfe eines Donor-Plasmids generiert wurden. In dieser Arbeit konnten mehr als 20 nicht-selektierbare Gene – darunter 10 der 15 potenziellen Photorezeptorgene – mit einer durchschnittlichen Mutationsrate von 12,1 % inaktiviert werden. Insgesamt zeigt diese Arbeit in umfassender Weise, wie Gen-Inaktivierungen und Modifikationen mithilfe von ZFNs und des CRISPR/Cas9-Systems in der Grünalge C. reinhardtii durchgeführt werden können. Außerdem bietet die Sammlung der zehn Photorezeptor-Knockouts eine aussichtsreiche Grundlage, um die Vielfalt der Photorezeptoren in C. reinhardtii zu erforschen., Gene editing is a fundamental tool in molecular biosciences in order to study the function of genes (reverse genetics). This study established zinc-finger and CRISPR/Cas9 nucleases for gene editing to target and inactivate the photoreceptor genes in C. reinhardtii. In continuation of previous work with designer zinc-finger nucleases (ZFN), the transformation efficiency could be improved 300-fold, which enabled the inactivation of genes in motile wild type cells. This made it possible to disrupt the Channelrhodopsin-1 (ChR1), Channelrhodopsin-2 (ChR2) and Chlamyopsin-1/2 (COP1/2) genes individually and in parallel. Phototaxis experiments in these strains revealed that the inactivation of ChR1 had a greater effect on phototaxis than the inactivation of ChR2. To apply the CRISPR/Cas9 system, the transformation conditions were adapted and optimized so that the Cas9-gRNA complex was successfully electroporated into the cells as an in vitro synthesized ribonucleoprotein. This approach enabled gene inactivations with CRISPR/Cas9 in C. reinhardtii. In order to measure and improve the conditions for precise gene modifications, the SNRK2.2 gene was established as a reporter gene for a ‘Blue-Green test’. Small insertions of up to 30 bp were inserted using short oligonucleotides, while larger reporter genes (mVenus, SNAP-tag) were integrated using donor plasmids. Throughout this study, more than 20 non-selectable genes were disrupted, including 10 of the photoreceptor genes, with an average mutation rate of 12,1 %. Overall, this work shows in a comprehensive way how gene inactivations and modifications can be performed in green alga C. reinhardtii using ZFNs or CRISPR/Cas9. In addition, the collection of the ten photoreceptor knockouts provides a promising source to investigate the diversity of photoreceptor genes in C. reinhardtii.

Kanalrhodopsine (ChRs) sind lichtaktivierte Ionenkanäle motiler Algen. Heterolog exprimiert erlauben sie es, Ionenflüsse durch Licht zu steuern. Bevorzugt geleitet werden von den meisten ChRs Protonen. Ausprägung und Wirkung lichtaktivierter Protonenflüsse sowie der molekulare Mechanismus protonenselektiver ChRs werden in vorliegender Arbeit untersucht und zur Entwicklung neuer optogenetischer Werkzeuge genutzt. Eine besonders hohe Protonenselektivität zeigten die grün- und rotlicht-aktivierten Kanäle CsChR und Chrimson aus den Algen Chloromonas subdivisa und Chlamydomonas noctigama. Im spektroskopisch detailliert untersuchten CrChR2 aus Chlamydomonas reinhardtii änderte sich die Protonenselektivität nach Anregung mit einem ns-Laserblitz sogar innerhalb eines Aktivierungszyklus und war insbesondere nach Öffnung des Kanals sowie in Folge der Lichtadaptation hoch. Als unentbehrlich für eine effiziente Protonenleitung erwiesen sich in allen drei Kanälen konservierte, titrierbare Reste entlang der Pore, deren individuelle Bedeutung für die Protonenleitung sich je nach Protein wesentlich unterschied. Entsprechend genügte in Chrimson der Austausch einzelner Glutaminsäuren des extrazellulären Halbkanals, dieses in einen grün- oder rotlichtaktivierten Natriumkanal zu transformieren. Aminosäuresubstitutionen der unmittelbaren Retinalumgebung verschoben hingegen das Aktionsmaximum von Chrimson röter als 600 nm und damit röter als in allen bisher beschriebenen ChRs. In Chrimson versperrt hierbei ein zusätzliches äußeres Tor den extrazellulär Halbkanal, während die Retinalbindetasche in Struktur und funktionaler Bedeutung der einzelnen Reste wesentlich jener der Protonenpumpe Bacteriorhodopsin ähnelt. Als Zwei-Komponenten-Optogenetik wurden schließlich protonen-, kationen- und anionenleitende ChRs unterschiedlicher Farbsensitivität fusioniert sowie lichtgetriebene Protonenpumpen mit protonenaktivierten Ionenkanälen kombiniert und neue optogenetische Perspektiven eröffnet., Channelrhodopsins (ChRs) are light-gated ion channels from green algae. Expressed in host cells they are used to control ion fluxes by light and are widely applied in Neurosciences. Although generally classified as either cation or anion channels, most ChRs preferentially conduct protons. This thesis compares proton conductance of different ChRs, examines the molecular mechanism of proton selective ChRs and explores the usage of light regulated proton fluxes in two-component-optogenetics. Proton selectivity varied strongly among different ChRs and was most pronounced for the green- and red-light activated channels CsChR and Chrimson from the algae Chloromonas subdivisa and Chlamydomonas noctigama, that conducted predominantly protons even at high pH. In CrChR2 from Chlamydomonas reinhardtii proton selectivity also changed during a single activation cycle and was especially high directly after channel opening and later on following light adaptation. In all three channels efficient proton conductance depended on conserved titratable residues along the pore with different contribution of the individual side chains in each protein. The substitution of single glutamic acids in the extracellular half pore converted Chrimson into a green or red-light activated sodium channel. A single point mutation close to the retinal chromophore shifted peak absorption of Chrimson beyond 600 nm - further red than all other cation conducting ChRs. Whereas the retinal binding pocket of Chrimson resembles the proton pump Bacteriorhodpsin, the overall pore structure corresponds to other ChRs, but features an additional outer gate, that occludes the extracellular half pore and is important for both, proton selectivity and red light absorption. Finally different Two-Component-Optogenetic approaches combined proton and anion selective ChRs of distinct colour as well as light-driven proton pumps and proton-activated ion channels with major prospect for future optogenetic applications.

Kanalrhodopsine (ChRs) sind lichtaktivierte Ionenkanäle motiler Algen. Heterolog exprimiert erlauben sie es, Ionenflüsse durch Licht zu steuern. Bevorzugt geleitet werden von den meisten ChRs Protonen. Ausprägung und Wirkung lichtaktivierter Protonenflüsse sowie der molekulare Mechanismus protonenselektiver ChRs werden in vorliegender Arbeit untersucht und zur Entwicklung neuer optogenetischer Werkzeuge genutzt. Eine besonders hohe Protonenselektivität zeigten die grün- und rotlicht-aktivierten Kanäle CsChR und Chrimson aus den Algen Chloromonas subdivisa und Chlamydomonas noctigama. Im spektroskopisch detailliert untersuchten CrChR2 aus Chlamydomonas reinhardtii änderte sich die Protonenselektivität nach Anregung mit einem ns-Laserblitz sogar innerhalb eines Aktivierungszyklus und war insbesondere nach Öffnung des Kanals sowie in Folge der Lichtadaptation hoch. Als unentbehrlich für eine effiziente Protonenleitung erwiesen sich in allen drei Kanälen konservierte, titrierbare Reste entlang der Pore, deren individuelle Bedeutung für die Protonenleitung sich je nach Protein wesentlich unterschied. Entsprechend genügte in Chrimson der Austausch einzelner Glutaminsäuren des extrazellulären Halbkanals, dieses in einen grün- oder rotlichtaktivierten Natriumkanal zu transformieren. Aminosäuresubstitutionen der unmittelbaren Retinalumgebung verschoben hingegen das Aktionsmaximum von Chrimson röter als 600 nm und damit röter als in allen bisher beschriebenen ChRs. In Chrimson versperrt hierbei ein zusätzliches äußeres Tor den extrazellulär Halbkanal, während die Retinalbindetasche in Struktur und funktionaler Bedeutung der einzelnen Reste wesentlich jener der Protonenpumpe Bacteriorhodopsin ähnelt. Als Zwei-Komponenten-Optogenetik wurden schließlich protonen-, kationen- und anionenleitende ChRs unterschiedlicher Farbsensitivität fusioniert sowie lichtgetriebene Protonenpumpen mit protonenaktivierten Ionenkanälen kombiniert und neue optogenetische Perspektiven eröffnet., Channelrhodopsins (ChRs) are light-gated ion channels from green algae. Expressed in host cells they are used to control ion fluxes by light and are widely applied in Neurosciences. Although generally classified as either cation or anion channels, most ChRs preferentially conduct protons. This thesis compares proton conductance of different ChRs, examines the molecular mechanism of proton selective ChRs and explores the usage of light regulated proton fluxes in two-component-optogenetics. Proton selectivity varied strongly among different ChRs and was most pronounced for the green- and red-light activated channels CsChR and Chrimson from the algae Chloromonas subdivisa and Chlamydomonas noctigama, that conducted predominantly protons even at high pH. In CrChR2 from Chlamydomonas reinhardtii proton selectivity also changed during a single activation cycle and was especially high directly after channel opening and later on following light adaptation. In all three channels efficient proton conductance depended on conserved titratable residues along the pore with different contribution of the individual side chains in each protein. The substitution of single glutamic acids in the extracellular half pore converted Chrimson into a green or red-light activated sodium channel. A single point mutation close to the retinal chromophore shifted peak absorption of Chrimson beyond 600 nm - further red than all other cation conducting ChRs. Whereas the retinal binding pocket of Chrimson resembles the proton pump Bacteriorhodpsin, the overall pore structure corresponds to other ChRs, but features an additional outer gate, that occludes the extracellular half pore and is important for both, proton selectivity and red light absorption. Finally different Two-Component-Optogenetic approaches combined proton and anion selective ChRs of distinct colour as well as light-driven proton pumps and proton-activated ion channels with major prospect for future optogenetic applications.

Kanalrhodopsine (ChRs) sind lichtaktivierte Ionenkanäle motiler Algen. Heterolog exprimiert erlauben sie es, Ionenflüsse durch Licht zu steuern. Bevorzugt geleitet werden von den meisten ChRs Protonen. Ausprägung und Wirkung lichtaktivierter Protonenflüsse sowie der molekulare Mechanismus protonenselektiver ChRs werden in vorliegender Arbeit untersucht und zur Entwicklung neuer optogenetischer Werkzeuge genutzt. Eine besonders hohe Protonenselektivität zeigten die grün- und rotlicht-aktivierten Kanäle CsChR und Chrimson aus den Algen Chloromonas subdivisa und Chlamydomonas noctigama. Im spektroskopisch detailliert untersuchten CrChR2 aus Chlamydomonas reinhardtii änderte sich die Protonenselektivität nach Anregung mit einem ns-Laserblitz sogar innerhalb eines Aktivierungszyklus und war insbesondere nach Öffnung des Kanals sowie in Folge der Lichtadaptation hoch. Als unentbehrlich für eine effiziente Protonenleitung erwiesen sich in allen drei Kanälen konservierte, titrierbare Reste entlang der Pore, deren individuelle Bedeutung für die Protonenleitung sich je nach Protein wesentlich unterschied. Entsprechend genügte in Chrimson der Austausch einzelner Glutaminsäuren des extrazellulären Halbkanals, dieses in einen grün- oder rotlichtaktivierten Natriumkanal zu transformieren. Aminosäuresubstitutionen der unmittelbaren Retinalumgebung verschoben hingegen das Aktionsmaximum von Chrimson röter als 600 nm und damit röter als in allen bisher beschriebenen ChRs. In Chrimson versperrt hierbei ein zusätzliches äußeres Tor den extrazellulär Halbkanal, während die Retinalbindetasche in Struktur und funktionaler Bedeutung der einzelnen Reste wesentlich jener der Protonenpumpe Bacteriorhodopsin ähnelt. Als Zwei-Komponenten-Optogenetik wurden schließlich protonen-, kationen- und anionenleitende ChRs unterschiedlicher Farbsensitivität fusioniert sowie lichtgetriebene Protonenpumpen mit protonenaktivierten Ionenkanälen kombiniert und neue optogenetische Perspektiven eröffnet., Channelrhodopsins (ChRs) are light-gated ion channels from green algae. Expressed in host cells they are used to control ion fluxes by light and are widely applied in Neurosciences. Although generally classified as either cation or anion channels, most ChRs preferentially conduct protons. This thesis compares proton conductance of different ChRs, examines the molecular mechanism of proton selective ChRs and explores the usage of light regulated proton fluxes in two-component-optogenetics. Proton selectivity varied strongly among different ChRs and was most pronounced for the green- and red-light activated channels CsChR and Chrimson from the algae Chloromonas subdivisa and Chlamydomonas noctigama, that conducted predominantly protons even at high pH. In CrChR2 from Chlamydomonas reinhardtii proton selectivity also changed during a single activation cycle and was especially high directly after channel opening and later on following light adaptation. In all three channels efficient proton conductance depended on conserved titratable residues along the pore with different contribution of the individual side chains in each protein. The substitution of single glutamic acids in the extracellular half pore converted Chrimson into a green or red-light activated sodium channel. A single point mutation close to the retinal chromophore shifted peak absorption of Chrimson beyond 600 nm - further red than all other cation conducting ChRs. Whereas the retinal binding pocket of Chrimson resembles the proton pump Bacteriorhodpsin, the overall pore structure corresponds to other ChRs, but features an additional outer gate, that occludes the extracellular half pore and is important for both, proton selectivity and red light absorption. Finally different Two-Component-Optogenetic approaches combined proton and anion selective ChRs of distinct colour as well as light-driven proton pumps and proton-activated ion channels with major prospect for future optogenetic applications.

Die Modifikation von Genen ist in den molekularen Biowissenschaften ein fundamentales Werkzeug, um die Funktion von Genen zu studieren (Reverse Genetik). Diese Arbeit hat erfolgreich Zinkfinger- und CRISPR/Cas9-Nukleasen für die Verwendung in C. reinhardtii etabliert, um Gene im Kerngenom gezielt auszuschalten und präzise zu verändern. Basierend auf vorausgegangener Arbeit mit Zinkfingernukleasen (ZFN) konnte die Transformationseffizienz um das 300-fache verbessert werden, was die Inaktivierung von Genen auch in motilen Wildtyp-Zellen ermöglichte. Damit war es möglich, die Gene für das Kanalrhodopsin-1 (ChR1), Kanalrhodopsin-2 (ChR2) und das Chlamyopsin-1/2-Gen (COP1/2) einzeln und gemeinsam auszuschalten. Eine Analyse der Phototaxis in diesen Stämmen ergab, dass die Phototaxis durch Inaktivierung von ChR1 stärker beeinträchtigt ist als durch Inaktivierung von ChR2. Um das CRISPR/Cas9-System zu verwenden, wurden die Transformationsbedingungen so angepasst und optimiert, dass der Cas9-gRNA-Komplex als in vitro hergestelltes Ribonukleoprotein in die Zellen transformiert wurde. Um die Bedingungen für präzise Genmodifikationen zu messen und zu verbessern, wurde das SNRK2.2-Gen als Reportergen für eine „Blau-Grün Test“ etabliert. Kleine Insertionen von bis zu 30 bp konnten mit kurzen Oligonukleotiden eingefügt werden, während größere Reportergene (mVenus, SNAP-Tag) mithilfe eines Donor-Plasmids generiert wurden. In dieser Arbeit konnten mehr als 20 nicht-selektierbare Gene – darunter 10 der 15 potenziellen Photorezeptorgene – mit einer durchschnittlichen Mutationsrate von 12,1 % inaktiviert werden. Insgesamt zeigt diese Arbeit in umfassender Weise, wie Gen-Inaktivierungen und Modifikationen mithilfe von ZFNs und des CRISPR/Cas9-Systems in der Grünalge C. reinhardtii durchgeführt werden können. Außerdem bietet die Sammlung der zehn Photorezeptor-Knockouts eine aussichtsreiche Grundlage, um die Vielfalt der Photorezeptoren in C. reinhardtii zu erforschen., Gene editing is a fundamental tool in molecular biosciences in order to study the function of genes (reverse genetics). This study established zinc-finger and CRISPR/Cas9 nucleases for gene editing to target and inactivate the photoreceptor genes in C. reinhardtii. In continuation of previous work with designer zinc-finger nucleases (ZFN), the transformation efficiency could be improved 300-fold, which enabled the inactivation of genes in motile wild type cells. This made it possible to disrupt the Channelrhodopsin-1 (ChR1), Channelrhodopsin-2 (ChR2) and Chlamyopsin-1/2 (COP1/2) genes individually and in parallel. Phototaxis experiments in these strains revealed that the inactivation of ChR1 had a greater effect on phototaxis than the inactivation of ChR2. To apply the CRISPR/Cas9 system, the transformation conditions were adapted and optimized so that the Cas9-gRNA complex was successfully electroporated into the cells as an in vitro synthesized ribonucleoprotein. This approach enabled gene inactivations with CRISPR/Cas9 in C. reinhardtii. In order to measure and improve the conditions for precise gene modifications, the SNRK2.2 gene was established as a reporter gene for a ‘Blue-Green test’. Small insertions of up to 30 bp were inserted using short oligonucleotides, while larger reporter genes (mVenus, SNAP-tag) were integrated using donor plasmids. Throughout this study, more than 20 non-selectable genes were disrupted, including 10 of the photoreceptor genes, with an average mutation rate of 12,1 %. Overall, this work shows in a comprehensive way how gene inactivations and modifications can be performed in green alga C. reinhardtii using ZFNs or CRISPR/Cas9. In addition, the collection of the ten photoreceptor knockouts provides a promising source to investigate the diversity of photoreceptor genes in C. reinhardtii.

Channelrhodopsin2 (ChR2) optogenetic excitation is widely used to study neurons, astrocytes, and circuits. Using complementary approaches in situ and in vivo, we found that ChR2 stimulation leads to significant transient elevation of extracellular potassium ions by ∼5 mM. Such elevations were detected in ChR2-expressing mice, following local in vivo expression of ChR2(H134R) with adeno-associated viruses (AAVs), in different brain areas and when ChR2 was expressed in neurons or astrocytes. In particular, ChR2-mediated excitation of striatal astrocytes was sufficient to increase medium spiny neuron (MSN) excitability and immediate early gene expression. The effects on MSN excitability were recapitulated in silico with a computational MSN model and detected in vivo as increased action potential firing in awake, behaving mice. We show that transient, physiologically consequential increases in extracellular potassium ions accompany ChR2 optogenetic excitation. This coincidental effect may be important to consider during astrocyte studies employing ChR2 to interrogate neural circuits and animal behavior.

Wireless biomedical electronic implants are rapidly being developed to treat a variety of medical conditions. Current technologies include the pacemaker to treat arrhythmias, the cochlear implant to overcome hearing impairment and the deep brain stimulator to treat Parkinson’s disease. Researchers are aiming to create implants that are miniaturised, battery-free, and minimally invasive. This is to ensure that devices are simpler to implant, to avoid surgical battery replacement and to minimise the risk of infection. To meet these demands, future biomedical electronic implants need to be miniaturised and capable of wireless power and data transfer. This thesis explores and extends the capabilities of three different wireless power transfer technologies for biomedical electronic implants: inductive, capacitive and radiative power transfer. This thesis adopts a systems approach to extend the capabilities of wireless power transfer systems. Wireless inductive power transfer has received thorough attention in the literature and involves the use of time-varying magnetic fields to transmit power through biological tissue. Typically, inductive power transfer involves a single transmitter and single receiver. This thesis demonstrates many receiving devices can be operated from a single transmitter - without adding complicated electronics to each receiving device. Moreover, by tuning the receiving coil on each device carefully the transmitter can power individual devices, or multiple devices simultaneously, extending the capabilities of inductive power transfer systems. Optogenetics, a technique used to transfect cells to make them light sensitive, is used to provide biological validation of the multichannel inductive receiving topology. Human embryonic kidney cells are transfected to be sensitive to blue light and then a twin channel inductive receiver with a blue and yellow light is modulated to demonstrate a cell response and no cell response respectively. Inductive coupli

Channelrhodopsin2 (ChR2) optogenetic excitation is widely used to study neurons, astrocytes, and circuits. Using complementary approaches in situ and in vivo, we found that ChR2 stimulation leads to significant transient elevation of extracellular potassium ions by ∼5 mM. Such elevations were detected in ChR2-expressing mice, following local in vivo expression of ChR2(H134R) with adeno-associated viruses (AAVs), in different brain areas and when ChR2 was expressed in neurons or astrocytes. In particular, ChR2-mediated excitation of striatal astrocytes was sufficient to increase medium spiny neuron (MSN) excitability and immediate early gene expression. The effects on MSN excitability were recapitulated in silico with a computational MSN model and detected in vivo as increased action potential firing in awake, behaving mice. We show that transient, physiologically consequential increases in extracellular potassium ions accompany ChR2 optogenetic excitation. This coincidental effect may be important to consider during astrocyte studies employing ChR2 to interrogate neural circuits and animal behavior.

Painful stimuli arising within visceral organs are detected by peripheral nerve endings of spinal afferents, whose cell bodies are located in dorsal root ganglia (DRG). Recent technical advances have made it possible to reliably expose and inject single DRG with neuronal tracers or viruses in vivo. This has facilitated, for the first time, unequivocal identification of different types of spinal afferent endings in visceral organs. These technical advances paved the way for a very exciting series of in vivo experiments where individual DRG are injected to facilitate opsin expression (e.g. Archaerhodopsin). Organ-specific expression of opsins in sensory neurons may be achieved by retrograde viral transduction. This means activity of target specific populations of sensory neurons, within single DRG, can be modulated by optogenetic photo stimulation. Using this approach we implanted micro light-emitting diodes (micro-LEDs) adjacent to DRG of interest, thereby allowing focal DRG-specific control of visceral and/or somatic afferents in conscious mice. This is vastly different from broad photo-illumination of peripheral nerve endings, which are dispersed over much larger surface areas across an entire visceral organ; and embedded deep within multiple anatomical layers. Focal DRG photo-stimulation also avoids the potential that wide-field illumination of the periphery could inadvertently activate other closely apposed organs, or co-activate different classes of axons in the same organ (e.g. enteric and spinal afferent endings in the gut). It is now possible to selectively control nociceptive and/or non-nociceptive pathways to specific visceral organs in vivo, using wireless optogenetics and micro-LEDs implanted adjacent to DRG, for targeted photo-stimulation.

Seit mehr als 10 Jahren kann biologische Aktivität durch eine Vielzahl photosensorischer Proteine beeinflusst werden. In diesem als Optogenetik bezeichneten Forschungsgebiet, werden Kationen leitende Kanalrhodopsine (CCRs) als lichtinduzierte neuronale Aktivatoren eingesetzt. Diese Arbeit soll zur Vervollständigung von optogenetischen Werkzeugen durch die Entwicklung Anionen leitender Kanalrhodopsine (ACRs) dienen, um die bestehenden Nachteile mikrobieller lichtgetriebener Ionenpumpen zu überwinden, die bislang zur neuronale Inhibition genutzt wurden. Der Austausch von E90 in C. reinhardtii Kanalrhodopsin 2 (CrChR2) durch positiv geladene Aminosäuren führte zu Entwicklung Chlorid leitender ChRs (ChloCs), die jedoch eine Restkationen-permeabilität aufwiesen. Durch Substitution zweier weiterer negativen Ladungen innerhalb des Ionenpermeationsweges, konnte die Kationenleitung vollständig aufgehoben werden. Parallel wurde durch A. Berndt et al. ein inhibitorisches C1C2 (iC1C2), basierend auf der CrChR1/2 Chimäre entwickelt. Wie auch bei den ChloCs, zeigte iC1C2 verbesserungswürdige biophysikalische Eigenschaften. Mutagenesestudien des Ionenpermeationsweges führten zur Entwicklung der verbesserten Nachfolgervariante iC++. Um ausgehend von weiteren CCRs neuartige ACRs zu entwickeln (eACRs), wurden die zuvor angewandten Mutagenesestrategien auf weitere CCRs übertragen. Zwei neue eACRs, Phobos und Aurora, mit jeweils blau- und rotverschobenen Aktionsspektrum konnten generiert werden. Bistabile eACRs wurden erzeugt, die ein lichtgesteuertes Schalten zwischen offenen und geschlossenen Zuständen ermöglichen. Schlussendlich wurde ein natürlich vorkommendes ACR (nACR) aus Proteomonas sulcata (PsACR1) identifiziert und charakterisiert. Die Maximalaktivität von PsACR1 zählt mit 540 nm zu den am stärksten rotverschobenen unter den nACRs. Elektrophysiologische und spektroskopische Untersuchungen ergaben, dass sich der Photozyklus von PsACR1 signifikant von jenen der CCRs unterscheid, For more than 10 years, photosensory proteins have developed as powerful tools to manipulate biological activity. In this research field termed optogenetics, cation-conducting channelrhodopsins (CCRs) mainly are utilized as light-induced neural activators. This study aimed at a complementation of the optogenetic tool box by engineering anion-conducting channelrhodopsins (ACRs) to overcome the existing drawbacks of microbial light-driven ion pumps utilized for neural inhibition so far. Replacement of E90 in the cation-conducting C. reinhardtii channelrhodopsin 2 (CrChR2) with positively charged residues reversed the ion selectivity and yielded chloride-conducting ChRs (ChloCs). Applied in neuronal cell culture, ChloCs showed residual cation permeability occasionally leading to excitation instead of the desired inhibition. Further charge elimination within the ion permeation pathway completely abolished cation conduction. In parallel, an inhibitory C1C2 (iC1C2) was developed by A. Berndt et al. based on a CrChR1/2 chimera. Though, iC1C2 displayed unsatisfactory biophysical properties as well. Further mutational modifications of the ion permeation pathway led to the development of the improved successor variant iC++. A systematic transfer of both conversion strategies to other CCRs was conducted to create engineered ACRs (eACRs) with distinct biophysical properties. Two novel eACRs, Phobos and Aurora, with blue- and red-shifted action were obtained. Additionally, step-function mutations greatly enhanced the operational light sensitivity and enabled temporally precise toggling between open and closed states using two different light colors. Finally, a natural ACR (nACR) originating from Proteomonas sulcata (PsACR1) was identified and characterized. With a maximum activation at 540 nm it is one of most red-shifted nACRs. Single turnover electrophysiological measurements and spectroscopic investigations revealed an unusual photocycle compared to that of CCRs.

Kanalrhodopsine (ChRs) sind lichtgesteuerte Ionenkanäle, welche nach Absorption eines Photons durch den Retinal-Cofaktor einen passiven Ionentransport über die Zellmembran katalysieren. Im Zuge von optogenetischen Anwendungen wird diese Reaktion für die Beeinflussung der Ionenhomöostase von verschiedenen Zelltypen und Geweben ausgenutzt. Zu Beginn dieser Arbeit wurden lichtinduzierte Strukturänderungen und Protontransferschritte in einem breiten Zeitbereich (Nanosekunden bis Minuten) in dem grün-absorbierenden ChR ReaChR mithilfe von stationärer und transienter UV-vis- und Fourier-Transform-Infrarot-Spektroskopie (FTIR) untersucht. Auf Basis der experimentellen Daten wurde ein komplexes Photozyklus-Modell konzipiert. Anschließend wurde die IR-aktive, nichtkanonische Aminosäure p-Azido-L-phenylalanin (azF) mittels Stopp-Codon-Suppression ortsspezifisch an mehreren Positionen innerhalb der vermuteten ionenleitenden Kanalpore in ReaChR inkorporiert und mit FTIR untersucht. azF ist sensitiv gegenüber Polaritätsänderungen und absorbiert in einem hochfrequenten Bereich (~2100 cm-1). Aufgrund der großen spektralen Separation zu endogenen Proteinschwingungen (< 1800 cm-1) können globale Konformations- und lokale Hydratisierungsänderungen simultan detektiert werden. Die erhobenen Daten leisten einen wichtigen Beitrag zum Verständnis der Bildung einer temporären Wasserpore in ChRs und demonstrieren zum ersten Mal den erfolgreichen in-vivo-Einbau einer artifiziellen Aminosäure in mikrobielle Rhodopsine und dessen schwingungsspektroskopische Analyse. Die Methode bietet aufgrund ihrer hohen Ortsauflösung ein großes Potential für die Studie von Mikroumgebungen innerhalb komplexer Proteinensemble., Channelrhodopsins (ChRs) are light-gated ion channels. Upon absorption of a photon, the retinal chromophore isomerizes and drives conformational changes within the protein, which lead to a passive ion transport across the cell membrane. This capability is used for optogenetic applications to manipulate ionic homeostasis of different cell types and entire organisms. Within the work, light-induced structural changes and proton transfer steps were studied in the green-absorbing ChR ReaChR in great detail by steady-state and transient UV-vis and Fourier transform infrared spectroscopy (FTIR). The data were merged into a complex photocycle model. Next, the IR-active, unnatural amino acid p-azido-L-phenylalanine (azF) was site-specifically introduced at several sites of the putative ion pore of ReaChR by stop codon suppression. azF is sensitive to polarity changes and absorbs in a clear spectral window lacking endogenous protein vibrations. Thus, FTIR measurements of labeled mutants report for global conformational changes (< 1800 cm-1) and local hydration changes (~2100 cm-1) simultaneously. The presented findings reveal crucial insights regarding formation of a transient water pore in ChRs and demonstrate the first report of the successful in-vivo incorporation of an artificial amino acid into a microbial rhodopsin and its subsequent spectroscopic investigation. Additionally, the so far unprecedented spatial resolution renders this methodology superior over conventional FTIR methods to study microenvironments within complex protein ensembles.

Kanalrhodopsine (ChRs) sind lichtgesteuerte Ionenkanäle, welche nach Absorption eines Photons durch den Retinal-Cofaktor einen passiven Ionentransport über die Zellmembran katalysieren. Im Zuge von optogenetischen Anwendungen wird diese Reaktion für die Beeinflussung der Ionenhomöostase von verschiedenen Zelltypen und Geweben ausgenutzt. Zu Beginn dieser Arbeit wurden lichtinduzierte Strukturänderungen und Protontransferschritte in einem breiten Zeitbereich (Nanosekunden bis Minuten) in dem grün-absorbierenden ChR ReaChR mithilfe von stationärer und transienter UV-vis- und Fourier-Transform-Infrarot-Spektroskopie (FTIR) untersucht. Auf Basis der experimentellen Daten wurde ein komplexes Photozyklus-Modell konzipiert. Anschließend wurde die IR-aktive, nichtkanonische Aminosäure p-Azido-L-phenylalanin (azF) mittels Stopp-Codon-Suppression ortsspezifisch an mehreren Positionen innerhalb der vermuteten ionenleitenden Kanalpore in ReaChR inkorporiert und mit FTIR untersucht. azF ist sensitiv gegenüber Polaritätsänderungen und absorbiert in einem hochfrequenten Bereich (~2100 cm-1). Aufgrund der großen spektralen Separation zu endogenen Proteinschwingungen (< 1800 cm-1) können globale Konformations- und lokale Hydratisierungsänderungen simultan detektiert werden. Die erhobenen Daten leisten einen wichtigen Beitrag zum Verständnis der Bildung einer temporären Wasserpore in ChRs und demonstrieren zum ersten Mal den erfolgreichen in-vivo-Einbau einer artifiziellen Aminosäure in mikrobielle Rhodopsine und dessen schwingungsspektroskopische Analyse. Die Methode bietet aufgrund ihrer hohen Ortsauflösung ein großes Potential für die Studie von Mikroumgebungen innerhalb komplexer Proteinensemble., Channelrhodopsins (ChRs) are light-gated ion channels. Upon absorption of a photon, the retinal chromophore isomerizes and drives conformational changes within the protein, which lead to a passive ion transport across the cell membrane. This capability is used for optogenetic applications to manipulate ionic homeostasis of different cell types and entire organisms. Within the work, light-induced structural changes and proton transfer steps were studied in the green-absorbing ChR ReaChR in great detail by steady-state and transient UV-vis and Fourier transform infrared spectroscopy (FTIR). The data were merged into a complex photocycle model. Next, the IR-active, unnatural amino acid p-azido-L-phenylalanine (azF) was site-specifically introduced at several sites of the putative ion pore of ReaChR by stop codon suppression. azF is sensitive to polarity changes and absorbs in a clear spectral window lacking endogenous protein vibrations. Thus, FTIR measurements of labeled mutants report for global conformational changes (< 1800 cm-1) and local hydration changes (~2100 cm-1) simultaneously. The presented findings reveal crucial insights regarding formation of a transient water pore in ChRs and demonstrate the first report of the successful in-vivo incorporation of an artificial amino acid into a microbial rhodopsin and its subsequent spectroscopic investigation. Additionally, the so far unprecedented spatial resolution renders this methodology superior over conventional FTIR methods to study microenvironments within complex protein ensembles.

Seit mehr als 10 Jahren kann biologische Aktivität durch eine Vielzahl photosensorischer Proteine beeinflusst werden. In diesem als Optogenetik bezeichneten Forschungsgebiet, werden Kationen leitende Kanalrhodopsine (CCRs) als lichtinduzierte neuronale Aktivatoren eingesetzt. Diese Arbeit soll zur Vervollständigung von optogenetischen Werkzeugen durch die Entwicklung Anionen leitender Kanalrhodopsine (ACRs) dienen, um die bestehenden Nachteile mikrobieller lichtgetriebener Ionenpumpen zu überwinden, die bislang zur neuronale Inhibition genutzt wurden. Der Austausch von E90 in C. reinhardtii Kanalrhodopsin 2 (CrChR2) durch positiv geladene Aminosäuren führte zu Entwicklung Chlorid leitender ChRs (ChloCs), die jedoch eine Restkationen-permeabilität aufwiesen. Durch Substitution zweier weiterer negativen Ladungen innerhalb des Ionenpermeationsweges, konnte die Kationenleitung vollständig aufgehoben werden. Parallel wurde durch A. Berndt et al. ein inhibitorisches C1C2 (iC1C2), basierend auf der CrChR1/2 Chimäre entwickelt. Wie auch bei den ChloCs, zeigte iC1C2 verbesserungswürdige biophysikalische Eigenschaften. Mutagenesestudien des Ionenpermeationsweges führten zur Entwicklung der verbesserten Nachfolgervariante iC++. Um ausgehend von weiteren CCRs neuartige ACRs zu entwickeln (eACRs), wurden die zuvor angewandten Mutagenesestrategien auf weitere CCRs übertragen. Zwei neue eACRs, Phobos und Aurora, mit jeweils blau- und rotverschobenen Aktionsspektrum konnten generiert werden. Bistabile eACRs wurden erzeugt, die ein lichtgesteuertes Schalten zwischen offenen und geschlossenen Zuständen ermöglichen. Schlussendlich wurde ein natürlich vorkommendes ACR (nACR) aus Proteomonas sulcata (PsACR1) identifiziert und charakterisiert. Die Maximalaktivität von PsACR1 zählt mit 540 nm zu den am stärksten rotverschobenen unter den nACRs. Elektrophysiologische und spektroskopische Untersuchungen ergaben, dass sich der Photozyklus von PsACR1 signifikant von jenen der CCRs unterscheid, For more than 10 years, photosensory proteins have developed as powerful tools to manipulate biological activity. In this research field termed optogenetics, cation-conducting channelrhodopsins (CCRs) mainly are utilized as light-induced neural activators. This study aimed at a complementation of the optogenetic tool box by engineering anion-conducting channelrhodopsins (ACRs) to overcome the existing drawbacks of microbial light-driven ion pumps utilized for neural inhibition so far. Replacement of E90 in the cation-conducting C. reinhardtii channelrhodopsin 2 (CrChR2) with positively charged residues reversed the ion selectivity and yielded chloride-conducting ChRs (ChloCs). Applied in neuronal cell culture, ChloCs showed residual cation permeability occasionally leading to excitation instead of the desired inhibition. Further charge elimination within the ion permeation pathway completely abolished cation conduction. In parallel, an inhibitory C1C2 (iC1C2) was developed by A. Berndt et al. based on a CrChR1/2 chimera. Though, iC1C2 displayed unsatisfactory biophysical properties as well. Further mutational modifications of the ion permeation pathway led to the development of the improved successor variant iC++. A systematic transfer of both conversion strategies to other CCRs was conducted to create engineered ACRs (eACRs) with distinct biophysical properties. Two novel eACRs, Phobos and Aurora, with blue- and red-shifted action were obtained. Additionally, step-function mutations greatly enhanced the operational light sensitivity and enabled temporally precise toggling between open and closed states using two different light colors. Finally, a natural ACR (nACR) originating from Proteomonas sulcata (PsACR1) was identified and characterized. With a maximum activation at 540 nm it is one of most red-shifted nACRs. Single turnover electrophysiological measurements and spectroscopic investigations revealed an unusual photocycle compared to that of CCRs.

Seit mehr als 10 Jahren kann biologische Aktivität durch eine Vielzahl photosensorischer Proteine beeinflusst werden. In diesem als Optogenetik bezeichneten Forschungsgebiet, werden Kationen leitende Kanalrhodopsine (CCRs) als lichtinduzierte neuronale Aktivatoren eingesetzt. Diese Arbeit soll zur Vervollständigung von optogenetischen Werkzeugen durch die Entwicklung Anionen leitender Kanalrhodopsine (ACRs) dienen, um die bestehenden Nachteile mikrobieller lichtgetriebener Ionenpumpen zu überwinden, die bislang zur neuronale Inhibition genutzt wurden. Der Austausch von E90 in C. reinhardtii Kanalrhodopsin 2 (CrChR2) durch positiv geladene Aminosäuren führte zu Entwicklung Chlorid leitender ChRs (ChloCs), die jedoch eine Restkationen-permeabilität aufwiesen. Durch Substitution zweier weiterer negativen Ladungen innerhalb des Ionenpermeationsweges, konnte die Kationenleitung vollständig aufgehoben werden. Parallel wurde durch A. Berndt et al. ein inhibitorisches C1C2 (iC1C2), basierend auf der CrChR1/2 Chimäre entwickelt. Wie auch bei den ChloCs, zeigte iC1C2 verbesserungswürdige biophysikalische Eigenschaften. Mutagenesestudien des Ionenpermeationsweges führten zur Entwicklung der verbesserten Nachfolgervariante iC++. Um ausgehend von weiteren CCRs neuartige ACRs zu entwickeln (eACRs), wurden die zuvor angewandten Mutagenesestrategien auf weitere CCRs übertragen. Zwei neue eACRs, Phobos und Aurora, mit jeweils blau- und rotverschobenen Aktionsspektrum konnten generiert werden. Bistabile eACRs wurden erzeugt, die ein lichtgesteuertes Schalten zwischen offenen und geschlossenen Zuständen ermöglichen. Schlussendlich wurde ein natürlich vorkommendes ACR (nACR) aus Proteomonas sulcata (PsACR1) identifiziert und charakterisiert. Die Maximalaktivität von PsACR1 zählt mit 540 nm zu den am stärksten rotverschobenen unter den nACRs. Elektrophysiologische und spektroskopische Untersuchungen ergaben, dass sich der Photozyklus von PsACR1 signifikant von jenen der CCRs unterscheid, For more than 10 years, photosensory proteins have developed as powerful tools to manipulate biological activity. In this research field termed optogenetics, cation-conducting channelrhodopsins (CCRs) mainly are utilized as light-induced neural activators. This study aimed at a complementation of the optogenetic tool box by engineering anion-conducting channelrhodopsins (ACRs) to overcome the existing drawbacks of microbial light-driven ion pumps utilized for neural inhibition so far. Replacement of E90 in the cation-conducting C. reinhardtii channelrhodopsin 2 (CrChR2) with positively charged residues reversed the ion selectivity and yielded chloride-conducting ChRs (ChloCs). Applied in neuronal cell culture, ChloCs showed residual cation permeability occasionally leading to excitation instead of the desired inhibition. Further charge elimination within the ion permeation pathway completely abolished cation conduction. In parallel, an inhibitory C1C2 (iC1C2) was developed by A. Berndt et al. based on a CrChR1/2 chimera. Though, iC1C2 displayed unsatisfactory biophysical properties as well. Further mutational modifications of the ion permeation pathway led to the development of the improved successor variant iC++. A systematic transfer of both conversion strategies to other CCRs was conducted to create engineered ACRs (eACRs) with distinct biophysical properties. Two novel eACRs, Phobos and Aurora, with blue- and red-shifted action were obtained. Additionally, step-function mutations greatly enhanced the operational light sensitivity and enabled temporally precise toggling between open and closed states using two different light colors. Finally, a natural ACR (nACR) originating from Proteomonas sulcata (PsACR1) was identified and characterized. With a maximum activation at 540 nm it is one of most red-shifted nACRs. Single turnover electrophysiological measurements and spectroscopic investigations revealed an unusual photocycle compared to that of CCRs.

Channelrhodopsins, such as the algal phototaxis receptor Platymonas subcordiformis channelrhodopsin-2 (PsChR2), are light-gated cation channels used as optogenetic tools for photocontrol of membrane potential in living cells. Channelrhodopsin (ChR)-mediated photocurrent responses are complex and poorly understood, exhibiting alterations in peak current amplitude, extents and kinetics of inactivation, and kinetics of the recovery of the prestimulus dark current that are sensitive to duration and frequency of photostimuli. From the analysis of time-resolved optical absorption data, presented in the accompanying article, we derived a two-cycle model that describes the photocycles of PsChR2. Here, we applied the model to evaluate the transient currents produced by PsChR2 expressed in HEK293 cells under both fast laser excitation and step-like continuous illumination. Interpretation of the photocurrents in terms of the photocycle kinetics indicates that the O states in both cycles are responsible for the channel current and fit the current transients under the different illumination regimes. The peak and plateau currents in response to a single light step, a train of light pulses, and a light step superimposed on a continuous light background observed for ChR2 proteins are explained in terms of contributions from the two parallel photocycles. The analysis shows that the peak current desensitization and recovery phenomena are inherent properties of the photocycles. The light dependence of desensitization is reproduced and explained by the time evolution of the concentration transients in response to step-like illumination. Our data show that photocycle kinetic parameters are sufficient to explain the complex dependence of photocurrent responses to photostimuli.

Channelrhodopsins, such as the algal phototaxis receptor Platymonas subcordiformis channelrhodopsin-2 (PsChR2), are light-gated cation channels used as optogenetic tools for photocontrol of membrane potential in living cells. Channelrhodopsin (ChR)-mediated photocurrent responses are complex and poorly understood, exhibiting alterations in peak current amplitude, extents and kinetics of inactivation, and kinetics of the recovery of the prestimulus dark current that are sensitive to duration and frequency of photostimuli. From the analysis of time-resolved optical absorption data, presented in the accompanying article, we derived a two-cycle model that describes the photocycles of PsChR2. Here, we applied the model to evaluate the transient currents produced by PsChR2 expressed in HEK293 cells under both fast laser excitation and step-like continuous illumination. Interpretation of the photocurrents in terms of the photocycle kinetics indicates that the O states in both cycles are responsible for the channel current and fit the current transients under the different illumination regimes. The peak and plateau currents in response to a single light step, a train of light pulses, and a light step superimposed on a continuous light background observed for ChR2 proteins are explained in terms of contributions from the two parallel photocycles. The analysis shows that the peak current desensitization and recovery phenomena are inherent properties of the photocycles. The light dependence of desensitization is reproduced and explained by the time evolution of the concentration transients in response to step-like illumination. Our data show that photocycle kinetic parameters are sufficient to explain the complex dependence of photocurrent responses to photostimuli.

Two-photon excitation (2PE) overcomes many challenges in fluorescence microscopy. Compared to confocal microscopy, 2PE microscopy improves depth penetration, owing to the longer excitation wavelength required and to the ability to collect scattered emission photons as a useful signal. It also minimizes photodamage because lower energy photons are used and because fluorescence is confined to the geometrical focus of the laser spot. 2PE is therefore ideal for high-resolution, deep-tissue, time-lapse imaging of dynamic processes in cell biology. Here, we provide examples of important applications of 2PE for in vivo imaging of neuronal structure and signals; we also describe how it can be combined with optogenetics or photolysis of caged molecules to simultaneously probe and control neuronal activity.

The nerve terminals found in the body wall of Drosophila melanogaster larvae are readily accessible to experimental manipulation. We used the light-activated ion channel, channelrhodopsin-2, which is expressed by genetic manipulation in Type II varicosities to study octopamine release in Drosophila. We report the development of a method to measure neurotransmitter release from exocytosis events at individual varicosities in the Drosophila larval system by amperometry. A microelectrode was placed in a region of the muscle containing a varicosity and held at a potential sufficient to oxidize octopamine and the terminal stimulated by blue light. Optical stimulation of Type II boutons evokes exocytosis of octopamine, which is detected through oxidization at the electrode surface. We observe 22700±4200 molecules of octopamine released per vesicle. This system provides a genetically accessible platform to study the regulation of amine release at an intact synapse.

© The Author(s), 2013. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Frontiers in Neural Circuits 7 (2013): 160, doi:10.3389/fncir.2013.00160., Here we characterize several new lines of transgenic mice useful for optogenetic analysis of brain circuit function. These mice express optogenetic probes, such as enhanced halorhodopsin or several different versions of channelrhodopsins, behind various neuron-specific promoters. These mice permit photoinhibition or photostimulation both in vitro and in vivo. Our results also reveal the important influence of fluorescent tags on optogenetic probe expression and function in transgenic mice., This work was supported by a CRP grant from the National Research Foundation of Singapore and by the World Class Institute (WCI )Program of the National Research Foundation of Korea (NRF )funded by the Ministry of Education, Science and Technology of Korea (MEST) (NRF Grant Number: WCI2009-003).

© The Author(s), 2013. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Frontiers in Neural Circuits 7 (2013): 160, doi:10.3389/fncir.2013.00160., Here we characterize several new lines of transgenic mice useful for optogenetic analysis of brain circuit function. These mice express optogenetic probes, such as enhanced halorhodopsin or several different versions of channelrhodopsins, behind various neuron-specific promoters. These mice permit photoinhibition or photostimulation both in vitro and in vivo. Our results also reveal the important influence of fluorescent tags on optogenetic probe expression and function in transgenic mice., This work was supported by a CRP grant from the National Research Foundation of Singapore and by the World Class Institute (WCI )Program of the National Research Foundation of Korea (NRF )funded by the Ministry of Education, Science and Technology of Korea (MEST) (NRF Grant Number: WCI2009-003).

Kanalrhodpsine (ChRs) sind lichtaktivierbare Kationenkanäle, welche als primäre Fotorezeptoren in Grünalgen dienen. In der Optogenetik werden ChRs verwendet um neuronale Membranen zu depolarisieren und mit Licht Aktionspotentiale auszulösen. Das mit blauem Licht aktivierte Chlamydomonas Kanalrhodopsin 2 (C2) und effiziente Mutanten wie C2 H134R stellen die am häufigsten genutzten depolarisierenden, optogenetischen Werkzeuge dar. Komplementär zu ChRs werden Protonen- und Chloridpumpen aus Archaebakterien zur neuronalen Inhibierung durch lichtinduzierte Hyperpolarisation verwendet. In der vorliegenden Arbeit untersuchten wir die ChR-Chimäre C1V1, ein grünlichtaktiviertes ChR, das sich durch hervorragende Membranlokalisierung und hohe Fotoströme in HEK-Zellen auszeichnet. C1V1 und C1V1-Mutanten mit feinabgestimmten spektralen und kinetischen Eigenschaften ermöglichen die neuronale Aktivierung mit Wellenlängen bis 620 nm sowie die unabhängige Aktivierung zweier Zellpopulationen in Kombination mit C2. Um die strukturelle Basis von Kanalöffnung und Ionentransport in ChRs zu verstehen, wurden gezielt Mutationen in C2 und C1V1 eingeführt. Die Fotoströme der entsprechenden Mutanten wurden auf Kationenselektivität und kinetische Veränderungen untersucht. Während Aminosäuren, die den Kanal an der zytosolischen Seite begrenzen, die Kationenfreisetzung und Einwärtsgleichrichtung der ChRs bestimmen, spielen zentral im Kanal gelegende Aminosäuren ein entscheidende Rolle für Kationenselektivität und -kompetition. Ein enzymkinetisches Modell ermöglichte außerdem die Zerlegung der Fotoströme in Beiträge der verschiedenen, konkurrierenden Kationen. Im letzten Teil der Arbeit wurde pHoenix, ein optogenetisches Werkzeug zur Ansäuerung synaptischer Vesikel, entwickelt. In Neuronen des Hippocampus wurde pHoenix verwendet, um die treibenden Kräfte für die vesikuläre Neurotransmitteraufnahme sowie den Zusammenhang zwischen Vesikelfüllstand und Freisetzungswahrscheinlichkeit zu analysieren., Channelrhodopsins (ChRs) are light-activated cation channels functioning as primary photoreceptors in green algae. In the emerging field of optogenetics, ChRs are used to depolarize neuronal membranes, thus allowing for light-induced action-potential firing. The blue light-activated Chlamydomonas channelrhodopsin 2 (C2) and high-efficiency mutants such as C2 H134R represent the most commonly used depolarizing optogenetic tools. Complementary to ChRs, green to yellow light-activated proton and chloride pumps originating from archea enable neuronal inhibition by membrane hyperpolarization. In the present work, we developed the chimeric ChR C1V1, a green-light activated ChR with excellent membrane targeting and high photocurrents in HEK cells. Action spectrum and kinetic properties of C1V1 were further fine-tuned by site-directed mutagenesis. The ensemble of C1V1 variants allows for neuronal activation with wavelengths up to 620 nm and can be used in two-color optogenetic experiments in combination with C2 derivatives. In order to understand the structural motifs involved in channel gating and ion transport, conserved residues in C2 and C1V1 were mutated and photocurrents of the respective mutants were analyzed for kinetic characteristics and cation selectivity. In these experiments, residues of the inner gate region were shown to alter cytosolic cation release and inward rectification, whereas central gate residues determine cation competition and selectivity, as well as the equilibrium between the two open channel conformations. Moreover, an enzyme-kinetic model was used to quantitatively dissect ChR photocurrents into the contribution of different competing cations. Finally, we designed pHoenix, an optogenetic tool enabling green-light induced acidification of synaptic vesicles. In hippocampal neurons, pHoenix was used to study both the energetics of vesicular neurotransmitter uptake and the impact of the vesicular contents on synaptic vesicle release.

Kanalrhodpsine (ChRs) sind lichtaktivierbare Kationenkanäle, welche als primäre Fotorezeptoren in Grünalgen dienen. In der Optogenetik werden ChRs verwendet um neuronale Membranen zu depolarisieren und mit Licht Aktionspotentiale auszulösen. Das mit blauem Licht aktivierte Chlamydomonas Kanalrhodopsin 2 (C2) und effiziente Mutanten wie C2 H134R stellen die am häufigsten genutzten depolarisierenden, optogenetischen Werkzeuge dar. Komplementär zu ChRs werden Protonen- und Chloridpumpen aus Archaebakterien zur neuronalen Inhibierung durch lichtinduzierte Hyperpolarisation verwendet. In der vorliegenden Arbeit untersuchten wir die ChR-Chimäre C1V1, ein grünlichtaktiviertes ChR, das sich durch hervorragende Membranlokalisierung und hohe Fotoströme in HEK-Zellen auszeichnet. C1V1 und C1V1-Mutanten mit feinabgestimmten spektralen und kinetischen Eigenschaften ermöglichen die neuronale Aktivierung mit Wellenlängen bis 620 nm sowie die unabhängige Aktivierung zweier Zellpopulationen in Kombination mit C2. Um die strukturelle Basis von Kanalöffnung und Ionentransport in ChRs zu verstehen, wurden gezielt Mutationen in C2 und C1V1 eingeführt. Die Fotoströme der entsprechenden Mutanten wurden auf Kationenselektivität und kinetische Veränderungen untersucht. Während Aminosäuren, die den Kanal an der zytosolischen Seite begrenzen, die Kationenfreisetzung und Einwärtsgleichrichtung der ChRs bestimmen, spielen zentral im Kanal gelegende Aminosäuren ein entscheidende Rolle für Kationenselektivität und -kompetition. Ein enzymkinetisches Modell ermöglichte außerdem die Zerlegung der Fotoströme in Beiträge der verschiedenen, konkurrierenden Kationen. Im letzten Teil der Arbeit wurde pHoenix, ein optogenetisches Werkzeug zur Ansäuerung synaptischer Vesikel, entwickelt. In Neuronen des Hippocampus wurde pHoenix verwendet, um die treibenden Kräfte für die vesikuläre Neurotransmitteraufnahme sowie den Zusammenhang zwischen Vesikelfüllstand und Freisetzungswahrscheinlichkeit zu analysieren., Channelrhodopsins (ChRs) are light-activated cation channels functioning as primary photoreceptors in green algae. In the emerging field of optogenetics, ChRs are used to depolarize neuronal membranes, thus allowing for light-induced action-potential firing. The blue light-activated Chlamydomonas channelrhodopsin 2 (C2) and high-efficiency mutants such as C2 H134R represent the most commonly used depolarizing optogenetic tools. Complementary to ChRs, green to yellow light-activated proton and chloride pumps originating from archea enable neuronal inhibition by membrane hyperpolarization. In the present work, we developed the chimeric ChR C1V1, a green-light activated ChR with excellent membrane targeting and high photocurrents in HEK cells. Action spectrum and kinetic properties of C1V1 were further fine-tuned by site-directed mutagenesis. The ensemble of C1V1 variants allows for neuronal activation with wavelengths up to 620 nm and can be used in two-color optogenetic experiments in combination with C2 derivatives. In order to understand the structural motifs involved in channel gating and ion transport, conserved residues in C2 and C1V1 were mutated and photocurrents of the respective mutants were analyzed for kinetic characteristics and cation selectivity. In these experiments, residues of the inner gate region were shown to alter cytosolic cation release and inward rectification, whereas central gate residues determine cation competition and selectivity, as well as the equilibrium between the two open channel conformations. Moreover, an enzyme-kinetic model was used to quantitatively dissect ChR photocurrents into the contribution of different competing cations. Finally, we designed pHoenix, an optogenetic tool enabling green-light induced acidification of synaptic vesicles. In hippocampal neurons, pHoenix was used to study both the energetics of vesicular neurotransmitter uptake and the impact of the vesicular contents on synaptic vesicle release.

Author Posting. © The Author(s), 2012. This is the author's version of the work. It is posted here by permission of Elsevier B.V. for personal use, not for redistribution. The definitive version was published in Neuroscience Research 75 (2013): 76-81, doi:10.1016/j.neures.2012.11.006., Recent advances in our understanding of brain function have come from using light to either control or image neuronal activity. Here we describe an approach that combines both techniques: a micromirror array is used to photostimulate populations of presynaptic neurons expressing channelrhodopsin-2, while a red-shifted voltage-sensitive dye allows optical detection of resulting postsynaptic activity. Such technology allowed us to control the activity of cerebellar interneurons while simultaneously recording inhibitory responses in multiple Purkinje neurons, their postsynaptic targets. This approach should substantially accelerate our understanding of information processing by populations of neurons within brain circuits., This work was supported by a Grass Foundation fellowship, National Institutes of Health (NIH grant: R01 EB001963), Duke‐NUS Signature Research Program (SRP) block grant, CRP grant from the National Research Foundation (Singapore) and by the World Class Institute (WCI) Program of the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology of Korea (MEST) (NRF Grant Number: WCI 2009-003).

Author Posting. © The Author(s), 2012. This is the author's version of the work. It is posted here by permission of Elsevier B.V. for personal use, not for redistribution. The definitive version was published in Neuroscience Research 75 (2013): 76-81, doi:10.1016/j.neures.2012.11.006., Recent advances in our understanding of brain function have come from using light to either control or image neuronal activity. Here we describe an approach that combines both techniques: a micromirror array is used to photostimulate populations of presynaptic neurons expressing channelrhodopsin-2, while a red-shifted voltage-sensitive dye allows optical detection of resulting postsynaptic activity. Such technology allowed us to control the activity of cerebellar interneurons while simultaneously recording inhibitory responses in multiple Purkinje neurons, their postsynaptic targets. This approach should substantially accelerate our understanding of information processing by populations of neurons within brain circuits., This work was supported by a Grass Foundation fellowship, National Institutes of Health (NIH grant: R01 EB001963), Duke‐NUS Signature Research Program (SRP) block grant, CRP grant from the National Research Foundation (Singapore) and by the World Class Institute (WCI) Program of the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology of Korea (MEST) (NRF Grant Number: WCI 2009-003).

Animals are exposed to a variety of odor cues that serve as environmental guides for their exploratory and social behaviors. Two distinct but complementing pathways process chemosensory cues: the Main and the Accessory olfactory System (AOS). Sensory neurons send their axons to the olfactory bulb (OB), specifically to the main and the accessory olfactory bulb (MOB and AOB, respectively) where they synapse onto principal neurons, the mitral (MCs). The OB is the only relay center between sensory neurons and cortical and limbic structures and therefore important aspects of odor processing occur in this region. Specifically, a distinctive mechanism used for olfactory processing is a strict regulation of MCs output by inhibitory neurons called granule cells (GCs). Importantely, inhibition of MCs is a dynamic process; it is regulated by the constant addition of new GCs to the OB circuit throughout life, in a process known as adult neurogenesis. Little is known, however, about the contribution of adult born neurons to the processing of olfactory cues, known as pheromones. Detection of pheromones by the AOS is critical for proper display of social behaviors such as hierarchical dominance and mate recognition. Here, we studied how the integration of new-born neurons could be regulated. We found that the arrival of new neurons into the adult AOB increases after animals are exposed to aggression and mate cues, suggesting that these newly arrived neurons can add important plasticity to the AOB circuitry and modify olfactory processing under different behavioral contexts. In addition, GCs mediated inhibition in the OB is precisely controlled by an extensive centrifugal innervation. For example, cortical feedback projections and neuromodulatory afferents originating in the midbrain and basal forebrain excite GC, inhibiting MCs' and decreasing their output. Regulation of of GCs by inhibition has also been reported, however, the source of this inhibition and its relevance to olfact

Epilepsy is a common disease that affects about 1 % of the population. The neurological disease is characterised by episodes of neural hyperactivity (seizures) and neural synchronization that can have behavioural manifestations such as convulsions. Anti-epileptic drugs (AEDs) can be used for treating most cases of epilepsy, but since especially temporal lobe epilepsy (TLE) has proven to be drug resistant, there is still a need to develop novel treatment methods. The research towards such new treatments requires animal models that mimic the pathology of the human disorder. In this project, optogenetics is used in evoking seizure-like afterdischarges (ADs) in anaesthetised mice by stimulating the CA1 region of the hippocampus. The mice are transgenic, expressing channelrhodopsin 2 – green fluorescent protein (ChR2-GFP) under the Thy1 promoter. An optrode is constructed and used to both stimulate with 463 nm light in 10 Hz pulses and to record an intracranial electroencephalogram (EEG). The tissue response and amount of ADs that follows a light stimulation train is evaluated. Also, spontaneous activity is assessed by calculating the burst suppression rate (BSR). Isoflurane and ketamine anaesthesia is compared and it is concluded that ketamine at the dose of 80mg/kg blocks the development of ADs following a stimulation train. On the other hand, the isoflurane anesthetised animals developed ADs which seemed to escalate in a kindling like manner.

Larval zebrafish are emerging as a model for describing the development and function of simple neural circuits. To analyze somatosensory neuron diversity in larval zebrafish, we identified several enhancers from the zebrafish and pufferfish genomes and used them to create five new reporter transgenes. Sequential deletions of three of these enhancers identified small sequence elements sufficient to drive expression in zebrafish trigeminal and Rohon-Beard (RB) neurons. One of these reporters highlighted a somatosensory neuron subtype that expressed both the p2rx3a and pkca; genes, as well as a previously described trpA1b reporter. To determine whether neurons of this subtype possess characteristic peripheral branching morphologies or central axon projection patterns, we analyzed the morphology of single neurons. Surprisingly, although these analyses revealed diversity in peripheral axon branching and central axon projection, PKCa/p2rx3a/trpA1b-expressing RB cells did not possess characteristic morphological features, suggesting that even within this molecularly defined subtype, individual neurons may possess distinct properties. Due to their external fertilization, rapid development, and optical clarity, zebrafish larvae are particularly well suited for optogenetic approaches to investigate neural circuit function. Here we demonstrate a procedure for expressing a light-sensitive ion channel in larval zebrafish somatosensory neurons, photo-activating single cells, and recording the resulting behaviors. Specifically, we created a transgene using a somatosensory neuron enhancer to drive the expression of the tagged channelrhodopsin variant, ChEF-tdTomato. Illuminating identified cells in these animals with light from a 473 nm DPSS laser, guided through a fiber optic cable, elicited behaviors that can be recorded with a high-speed video camera and analyzed quantitatively. By introducing a few modifications to previously established methods, this approach was used to elici

Larval zebrafish are emerging as a model for describing the development and function of simple neural circuits. To analyze somatosensory neuron diversity in larval zebrafish, we identified several enhancers from the zebrafish and pufferfish genomes and used them to create five new reporter transgenes. Sequential deletions of three of these enhancers identified small sequence elements sufficient to drive expression in zebrafish trigeminal and Rohon-Beard (RB) neurons. One of these reporters highlighted a somatosensory neuron subtype that expressed both the p2rx3a and pkca; genes, as well as a previously described trpA1b reporter. To determine whether neurons of this subtype possess characteristic peripheral branching morphologies or central axon projection patterns, we analyzed the morphology of single neurons. Surprisingly, although these analyses revealed diversity in peripheral axon branching and central axon projection, PKCa/p2rx3a/trpA1b-expressing RB cells did not possess characteristic morphological features, suggesting that even within this molecularly defined subtype, individual neurons may possess distinct properties. Due to their external fertilization, rapid development, and optical clarity, zebrafish larvae are particularly well suited for optogenetic approaches to investigate neural circuit function. Here we demonstrate a procedure for expressing a light-sensitive ion channel in larval zebrafish somatosensory neurons, photo-activating single cells, and recording the resulting behaviors. Specifically, we created a transgene using a somatosensory neuron enhancer to drive the expression of the tagged channelrhodopsin variant, ChEF-tdTomato. Illuminating identified cells in these animals with light from a 473 nm DPSS laser, guided through a fiber optic cable, elicited behaviors that can be recorded with a high-speed video camera and analyzed quantitatively. By introducing a few modifications to previously established methods, this approach was used to elici

Channelrhodopsin-2 ist ein lichtaktivierter Kationenkanal, der zur nichtinvasiven Steuerung neuronaler Aktivität verwendet wird. Einige grundlegende Eigenschaften dieses Proteins sind bereits bekannt, aber die molekularen Mechanismen des Ionentransports und der Aktivierung liegen noch weitgehend im Dunkeln. Ziel dieser Studie war es, anhand von Mutationsstudien die Funktion einzelner Aminosäuren zu bestimmen. Dazu habe ich gezielt potentiell wichtige Reste substituiert und die Channelrhodopsin-2-Varianten elektrophysiologisch untersucht. Um die aufgetretenen Änderungen beim Ionentransport und den Kanalkinetiken zu erklären, habe ich verschiedene mathematische Modelle an die experimentellen Daten angepasst. Dabei stellte sich heraus, dass die Reste H134 und E90 Schlüsselpositionen für den Protonentransport sind. Außerdem haben auch die Reste E235 und D253 einen großen Einfluss auf den Ladungstransport. Dagegen wird die Kanalöffnung von C128 und D156 kontrolliert. Des Weiteren kontrolliert E123 die Übergänge zwischen leitenden und nichtleitenden Zuständen von Channelrhodopsin-2. Aus der zielgerichteten Mutation von Aminosäuren resultierten Varianten, die langsamere oder schnellere Kinetiken hatten oder eine bessere Expression zeigten als der Wildtyp. Das Anwendungspotential der modifizierten Kanäle wurde in Kooperationen mit neurophysiologischen Arbeitsgruppen untersucht. Dadurch konnten drei neue Typen von Channelrhodopsinen in die Neurophysiologie eingeführt werden. Die step-functions opsins führen zu einer anhaltenden Membrandepolarisation, die die Erregbarkeit von Neuronen gegenüber synaptischen Inputs erhöht. ChETA erlaubt das zeitlich präzise Auslösen von Aktionspotentialen auch bei sehr hohen Anregungsfrequenzen. T159C und E123T/T159C ermöglichen durch ihre großen Photoströme und optimierten Kinetiken eine hohe Zuverlässigkeit bei der optischen Steuerung neuronaler Aktivität. Dadurch wird das Anwendungsspektrum von Channelrhodopsin-2 erheblich erweitert., Channelrhodopsin-2 is a light-activated cation channel which has become a very useful tool in neurophysiology, since it allows the noninvasive control of neural activity. Some of the basic features of this channel are known from previous studies, but the molecular mechanisms of ion translocation and activation are largely unknown. The aim of my thesis is to elucidate the function of single amino acids by mutational studies. I replaced potentially important residues and probed the constructs by electrophysiological measurements under various conditions. Additionally, I fitted the experimental data to several mathematical models in order to explain changes in ion permeabilities and channel kinetics and I assigned particular functions to the mutated residues. Apparently, H134 and E90 are key positions for the proton transportation. Mutations at E235 and D253 also strongly influence ion translocation, whereas C128 and D156 obviously control the channel opening. Moreover, I found that E123 is a key element for the channel activation which controls the transitions between conducting and non-conducting states of Channelrhodopsin-2. The genetically modified Channelrhodopsin-2-variants provide several favorable features, such as, a slower or faster channel opening and closing or an optimized expression. Therefore, we tested the potential of promising constructs for applications in collaboration with neurophysiology laboratories. Finally, we introduced three new tools. First, step-function opsins induce a sustained membrane depolarization which sensitizes neurons to native synaptic inputs. Second, the ChETA variant allows the temporally precise generation of action potentials even at high stimulation frequencies. Third, T159C and E123T/T159C provide large photocurrents and optimized kinetics resulting in an improved performance in the noninvasive control of neural activity. In summary, this significantly broadens the range of application for channelrhodopsin-2.

Channelrhodopsin-2 ist ein lichtaktivierter Kationenkanal, der zur nichtinvasiven Steuerung neuronaler Aktivität verwendet wird. Einige grundlegende Eigenschaften dieses Proteins sind bereits bekannt, aber die molekularen Mechanismen des Ionentransports und der Aktivierung liegen noch weitgehend im Dunkeln. Ziel dieser Studie war es, anhand von Mutationsstudien die Funktion einzelner Aminosäuren zu bestimmen. Dazu habe ich gezielt potentiell wichtige Reste substituiert und die Channelrhodopsin-2-Varianten elektrophysiologisch untersucht. Um die aufgetretenen Änderungen beim Ionentransport und den Kanalkinetiken zu erklären, habe ich verschiedene mathematische Modelle an die experimentellen Daten angepasst. Dabei stellte sich heraus, dass die Reste H134 und E90 Schlüsselpositionen für den Protonentransport sind. Außerdem haben auch die Reste E235 und D253 einen großen Einfluss auf den Ladungstransport. Dagegen wird die Kanalöffnung von C128 und D156 kontrolliert. Des Weiteren kontrolliert E123 die Übergänge zwischen leitenden und nichtleitenden Zuständen von Channelrhodopsin-2. Aus der zielgerichteten Mutation von Aminosäuren resultierten Varianten, die langsamere oder schnellere Kinetiken hatten oder eine bessere Expression zeigten als der Wildtyp. Das Anwendungspotential der modifizierten Kanäle wurde in Kooperationen mit neurophysiologischen Arbeitsgruppen untersucht. Dadurch konnten drei neue Typen von Channelrhodopsinen in die Neurophysiologie eingeführt werden. Die step-functions opsins führen zu einer anhaltenden Membrandepolarisation, die die Erregbarkeit von Neuronen gegenüber synaptischen Inputs erhöht. ChETA erlaubt das zeitlich präzise Auslösen von Aktionspotentialen auch bei sehr hohen Anregungsfrequenzen. T159C und E123T/T159C ermöglichen durch ihre großen Photoströme und optimierten Kinetiken eine hohe Zuverlässigkeit bei der optischen Steuerung neuronaler Aktivität. Dadurch wird das Anwendungsspektrum von Channelrhodopsin-2 erheblich erweitert., Channelrhodopsin-2 is a light-activated cation channel which has become a very useful tool in neurophysiology, since it allows the noninvasive control of neural activity. Some of the basic features of this channel are known from previous studies, but the molecular mechanisms of ion translocation and activation are largely unknown. The aim of my thesis is to elucidate the function of single amino acids by mutational studies. I replaced potentially important residues and probed the constructs by electrophysiological measurements under various conditions. Additionally, I fitted the experimental data to several mathematical models in order to explain changes in ion permeabilities and channel kinetics and I assigned particular functions to the mutated residues. Apparently, H134 and E90 are key positions for the proton transportation. Mutations at E235 and D253 also strongly influence ion translocation, whereas C128 and D156 obviously control the channel opening. Moreover, I found that E123 is a key element for the channel activation which controls the transitions between conducting and non-conducting states of Channelrhodopsin-2. The genetically modified Channelrhodopsin-2-variants provide several favorable features, such as, a slower or faster channel opening and closing or an optimized expression. Therefore, we tested the potential of promising constructs for applications in collaboration with neurophysiology laboratories. Finally, we introduced three new tools. First, step-function opsins induce a sustained membrane depolarization which sensitizes neurons to native synaptic inputs. Second, the ChETA variant allows the temporally precise generation of action potentials even at high stimulation frequencies. Third, T159C and E123T/T159C provide large photocurrents and optimized kinetics resulting in an improved performance in the noninvasive control of neural activity. In summary, this significantly broadens the range of application for channelrhodopsin-2.

Expression of halorhodopsin (NpHR), a light-driven microbial chloride pump, allows for optical control of membrane potential and reversible silencing of transfected neurons and muscle cells ("optogenetics"). Zebrafish are ideal model organisms for experiments involving localized light delivery, since they are optically clear, genetically tractable and display many behaviors within the first two weeks of life. Here, the optogenetic approach was adapted to zebrafish by creating transgenic zebrafish lines coding for light-activatable pumps (NpHR) and channels (channelrhodopsin-2, ChR2) and by implementing optical methods for the precise application of light. We studied the neural circuits underlying the generation of fast eye movements (saccades) during the optokinetic response (OKR). The OKR is a visual behavior, which minimizes retinal slip during rotating motion. Applying light through thin optic fibers positioned above the animal's head enabled us to target small groups of cells and to simultaneously test the effect of their silencing on the generation of saccades during the OKR. We identified a cell population in rhombomere 5 that is required and sufficient for the generation of saccades in zebrafish larvae. The discovered similarities between zebrafish and mammalian oculomotor circuitry are in line with a possible homology of the saccade generator in fish and mammals. Furthermore, we studied the cardiac conduction system throughout development using these tools. By automated sequential illumination of small areas of the developing heart with a digital micromirror device (DMD), while simultaneously monitoring heart contractions, we identified the regions responsible for initiating and relaying cardiac conduction. By directly targeting the pacemaker areas with well-defined light pulses, we were able to induce disease-like states (tachycardia, bradycardia, AV blocks). Together, our studies have introduced a versatile optogenetic toolkit for precise loss-of-function

Expression of halorhodopsin (NpHR), a light-driven microbial chloride pump, allows for optical control of membrane potential and reversible silencing of transfected neurons and muscle cells ("optogenetics"). Zebrafish are ideal model organisms for experiments involving localized light delivery, since they are optically clear, genetically tractable and display many behaviors within the first two weeks of life. Here, the optogenetic approach was adapted to zebrafish by creating transgenic zebrafish lines coding for light-activatable pumps (NpHR) and channels (channelrhodopsin-2, ChR2) and by implementing optical methods for the precise application of light. We studied the neural circuits underlying the generation of fast eye movements (saccades) during the optokinetic response (OKR). The OKR is a visual behavior, which minimizes retinal slip during rotating motion. Applying light through thin optic fibers positioned above the animal's head enabled us to target small groups of cells and to simultaneously test the effect of their silencing on the generation of saccades during the OKR. We identified a cell population in rhombomere 5 that is required and sufficient for the generation of saccades in zebrafish larvae. The discovered similarities between zebrafish and mammalian oculomotor circuitry are in line with a possible homology of the saccade generator in fish and mammals. Furthermore, we studied the cardiac conduction system throughout development using these tools. By automated sequential illumination of small areas of the developing heart with a digital micromirror device (DMD), while simultaneously monitoring heart contractions, we identified the regions responsible for initiating and relaying cardiac conduction. By directly targeting the pacemaker areas with well-defined light pulses, we were able to induce disease-like states (tachycardia, bradycardia, AV blocks). Together, our studies have introduced a versatile optogenetic toolkit for precise loss-of-function

During the first year of our funding, we have discovered, using our optical neural activation techniques, that optical activation of neurons of the infralimbic (IL) cortex, in a mouse model of post-traumatic stress disorder (PTSD), during exposure therapy, results in rapid and enduring remission of the symptoms of PTSD. To accomplish this, in the first year we invented and deployed several novel technologies: we developed viruses that sensitize cortical neurons to being driven and inhibited by light, we developed arrays of optical fibers for delivering light into 3-D patterns in the brain, we secured approval for all behavioral experiments in animals, we developed precision fear-conditioning equipment optimized for optical control, we invented injector arrays that facilitate the injection of viruses throughout the brain, and we developed new neural silencers to improve the screening process. We are continuing to refine these techniques, and to screen for other targets in the brain that can modulate fear symptoms, including cortical areas and the amygdala., The original document contains color images.