Chapter 6 Optogenetic actuation, inhibition, modulation and readout for neuronal networks generating behavior in the nematode Caenorhabditis elegans

Optogenetics combines genetic engineering with optics to observe and control the function of cells using light, with clinical implications for restoration of vision and treatment of neurological diseases. As a new discipline much of the basic science and methods are currently under investigation and active development, thus there is a strong need for introductory literature in this field. This graduate level textbook provides an overview of the field of optogenetics in 5 concise chapters: Optogenetic tools, Applications in cellular systems, Mapping neuronal networks, Clinical applications and Restoration of vision and hearing. The concept and content was developed with top international researchers and students at a prestigious Dahlem Conference workshop. * A concise overview of the methodology, basic research and new clinical applications for optogenetics * Content developed with top international scientists and students in a "Dahlem Conference" workshop * Color illustrations for better visualization of complex structures List of contributing authors Introduction 1 The biophysics and engineering of signaling photoreceptors 1.1 Photoreceptors 1.1.1 Novel photoreceptors 1.1.2 Biophysics of photoreceptors and signal transduction 1.2 Engineering of photoreceptors 1.2.1 Approaches to designing light-regulated biological processes 1.3 Case study – transcriptional control in cells by light 1.4 Conclusion Acknowledgements References 2 Current challenges in optogenetics 2.1 Introduction 2.2 Background: current functionality of tools 2.3 Unsolved problems and open questions: technology from cell biology, optics, and behavior 2.4 Unsolved problems and open questions: genomics and biophysics 2.5 Conclusion References 3 Challenges and opportunities for optochemical genetics 3.1 Introduction 3.2 Photosensitizing receptors 3.3 PCL and PTL development and applications 3.4 Advantages and disadvantages of PCLs and PTLs 3.5 Conclusion References 4 Optogenetic imaging of neural circuit dynamics using voltage-sensitive fluorescent proteins: potential, challenges and perspectives 4.1 Introduction 4.2 The biological problem 4.3 The large scale challenge of circuit neurosciences 4.4 The current approach to the large-scale integration problem 4.5 Large-scale recordings of neuronal activities using optogenetic approaches 4.6 Genetically encoded voltage indicators: state of development and application 4.7 Unsolved methodological / technical challenges References 5 Why optogenetic “control” is not (yet) control Acknowledgments References 6 Optogenetic actuation, inhibition, modulation and readout for neuronal networks generating behavior in the nematode Caenorhabditis elegans 6.1 Introduction – the nematode as a genetic model in systems neurosciencesystems neuroscience 6.2 Imaging of neural activity in the nematode 6.2.1 Genetically encoded Ca2+ indicators (GECIs) 6.2.2 Imaging populations of neurons in immobilized animals 6.2.3 Imaging neural activity in freely moving animals 6.2.4 Other genetically encoded indicators of neuronal function 6.3 Optogenetic tools established in the nematode 6.3.1 Channelrhodopsin (ChR2) and ChR variants with different functional properties for photodepolarization 6.3.2 Halorhodopsin and light-triggered proton pumps for photohyperpolarization 6.3.3 Photoactivated Adenylyl Cyclase (PAC) for phototriggered cAMPdependent effects that facilitate neuronal transmission 6.3.4 Other optogenetic approaches 6.3.5 Stimulation of single neurons by optogenetics in freely behaving C. elegans 6.4 Examples for optogenetic applications in C. elegans 6.4.1 Optical control of synaptic transmission at the neuromuscular junction and between neurons 6.4.2 Optical control of neural network activity in the generation of behavior 6.5 Future challenges 6.5.1 Closed-loop optogenetic control and optical feedback from behavior and individual neurons 6.5.2 Requirements for integrated optogenetics in the nematode References 7 Putting genetics into optogenetics: knocking out proteins with light 7.1 Introduction 7.2 Protein degradation 7.3 Light stimulation References 8 Optogenetic approaches in behavioral neuroscience 8.1 Introduction 8.2 Approaches to dissect neuronal circuits: determining physiological correlations, requirement and sufficiency of neurons 8.3 Optogenetic analysis of simple stimulus-response-connections 8.4 Optogenetic and thermogenetic analysis of modulatory neurons: artificial mimicry of relevance 8.5 Conclusion References 9 Combining genetic targeting and optical stimulation for circuit dissection in the zebrafish nervous system 9.1 Introduction 9.2 Zebrafish neuroscience: Genetics + Optics + Behavior 9.3 Genetic targeting of optogenetic proteins to specific neurons 9.4 Optical stimulation in behaving zebrafish 9.5 Annotating behavioral functions of genetically-identified neurons by optogenetics 9.5.1 Spinal cord neurons (Rohon–Beard and Kolmer–Agduhr cells) 9.5.2 Hindbrain motor command neurons 9.5.3 Tangential neurons in the vestibular system 9.5.4 Size filtering neurons in the tectum 9.5.5 Whole-brain calcium imaging of motor adaptation at single-cell resolution 9.6 Future directions References 10 Optogenetic analysis of mammalian neural circuits 10.1 Introduction 10.2 Optogenetic approaches to probe integrative properties at the cellular level 10.2.1 Excitatory signal integration at dendrites 10.2.2 Control of excitatory signal integration by inhibition or neuromodulation 10.2.3 Long-term analysis of synaptic function 10.3 Circuits and systems level 10.4 Optogenetics and behavior: testing causal relationships in freely moving animals References 11 Optogenetics to benefit human health: opportunities and challenges 11.1 Introduction 11.2 Opportunities for translational applications 11.3 Safety challenges 11.4 Need for feedback 11.5 Conclusion References 12 Optogenetic tools for controlling neural activity: molecules and hardware 12.1 Overview 12.2 Molecular tools for sensitizing neural functions to light 12.3 Hardware for delivery of light into intact brain circuits References 13 In vivo application of optogenetics in rodents 13.1 Introduction 13.2 Sleep / wake regulation 13.3 Addiction 13.4 Fear, anxiety and depression 13.5 Autism and schizophrenia 13.6 Aggression 13.7 Breathing 13.8 Seizures 13.9 Conclusion Acknowledgments References 14 Potential of optogenetics in deep brain stimulation 14.1 DBS history and indications 14.2 Electrical DBS: advantages and drawbacks 14.3 Potential of optogenetic stimulation 14.4 Conclusion References 15 Optogenetic approaches for vision restoration 15.1 Introduction 15.2 Proof-of-concept studies 15.3 Light sensors 15.4 rAAV-mediated retinal gene delivery 15.5 Retinal cell-type specific targeting 15.6 Summary References Further reading 16 Restoration of vision – the various approaches 16.1 Introduction 16.2 The various conditions to be treated 16.3 State of the various restorative approaches 16.3.1 Neuroprotection 16.3.1.1 Encapsulated cell technology (ECT) 16.3.1.2 Electrostimulation 16.3.1.3 Visual Cycle modulators 16.3.1.4 Gene replacement therapy 16.3.1.5 Stem cell approaches 16.3.1.6 Optogenetic approaches 16.3.1.7 Electronic retinal prosthesis 16.3.2 Cortical prosthesis 16.3.3 Tongue stimulators 16.4 The current situation 16.5 Open Questions 16.6 Conclusion References Selected registered clinical trials as by February 2013 17 Optogenetic approaches to cochlear prosthetics for hearing restoration 17.1 Background and state of the art 17.2 Current research on cochlear optogenetics 17.2.1 Current and future work on cochlear optogenetics aims at 17.3 Potential and risks of cochlear optogenetics for auditory prosthetics References 18 History in the making: the ethics of optogenetics References 19 Optogenetics as a new therapeutic tool in medicine? A view from the principles of biomedical ethics 19.1 Principles of optogenetics 19.2 Principles of biomedical ethics 19.2.1 Respect for the patient’s autonomy 19.2.2 Nonmaleficence 19.2.3 Beneficence 19.2.4 Justice 19.3 Conclusion References Appendix : Dahlem-Conference (Berlin, September 2–5, 2012): “Optogenetics. Challenges and Perspectives.” Index Elucidating the mechanisms by which nervous systems process information and generate behavior is among the fundamental problems of biology. The complexity of our brain and plasticity of our behaviors make it challenging to understand even simple human actions in terms of molecular mechanisms and neural activity. However the molecular machines and operational features of our neural circuits are often found in invertebrates, so that studying flies and worms provides an effective way to gain insights into our nervous system. Caenorhabditis elegans offers special opportunities to study behavior. Each of the 302 neurons in its nervous system can be identified and imaged in live animals [1, 2], and manipulated transgenically using specific promoters or promoter combinations [3, 4, 5, 6]. The chemical synapses and gap junctions made by every neuron are known from electron micrograph reconstruction [1]. Importantly, forward genetics can be used to identify molecules that modulate C. elegans' behavior. Forward genetic dis-section of behavior is powerful because it requires no prior knowledge. It allows molecules to be identified regardless of in vivo concentration, and focuses attention on genes that are functionally important. The identity and expression patterns of these molecules then provide entry points to study the molecular mechanisms and neural circuits controlling the behavior. Genetics does not provide the temporal resolution required to study neural circuit function directly. However, neural activity can be monitored using genetically encoded sensors for Ca2+ (e.g., GCaMP and cameleon) [7, 8, 9, 10] and voltage (e.g., mermaid, arclight or VSFP- Butterfly) [11, 12, 13]. In C. elegans, imaging studies have focused largely on single neurons in immobilized animals [14]. However, it is now becoming possible to image the activity of single neurons in freely moving animals, and of multiple neurons in three dimensions. Additionally, increasingly sophisticated hardware allows precise spatial control of neural activity in freely moving C. elegans, using light activated channels and pumps (see Section 6.2). From a reductionist perspective, the worm model is very exciting because it has the potential to reveal how neural circuits work in enormous detail. This potential has fostered collaborations between physicists, engineers, and neuroscientists. Here we try to convey some of the excitement in this fast moving field The transmembrane proteins that underlie neural processing are now known at a level of detail that has greatly increased our understanding of these sophisticated molecular machines. Starting with MacKinnon's seminal structure of a potassium channel, several voltage-gated ion channels and ionotropic receptors have been revealed with atomic resolution (Figure 3.1) [2, 3, 4, 5, 6]. This has been complemented by structures of G-protein coupled receptors, adding opsins and metabotropic receptors to the ever-increasing repertoire of transmembrane proteins elucidated with structural biology [7, 8, 9, 10]. As a consequence of this structural revolution and recent advances in pharmacology, Nature's molecular machines can now be manipulated with relative ease. This can be done, for instance, via synthetic on-off switches or tuning elements that are attached to the signaling protein of interest to allow for its orthogonal control with non-natural input signals. Amongst these signals, light is particularly useful, since it is unmatched in terms of temporal and spatial precision and techniques for the delivery and control of light are highly developed.

Optogenetics combines genetic engineering with optics to observe and control the function of cells using light, with clinical implications for restoration of vision and treatment of neurological diseases. As a new discipline much of the basic science and methods are currently under investigation and active development, thus there is a strong need for introductory literature in this field. This graduate level textbook provides an overview of the field of optogenetics in 5 concise chapters: Optogenetic tools, Applications in cellular systems, Mapping neuronal networks, Clinical applications and Restoration of vision and hearing. The concept and content was developed with top international researchers and students at a prestigious Dahlem Conference workshop.