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 Optogenetics......Page 4 List of contributing authors......Page 6 Content......Page 10 Introduction......Page 16 1.1 Photoreceptors......Page 22 1.1.2 Biophysics of photoreceptors and signal transduction......Page 25 1.2 Engineering of photoreceptors......Page 27 1.2.1 Approaches to designing light-regulated biological processes......Page 28 1.3 Case study – transcriptional control in cells by light......Page 32 1.4 Conclusion......Page 33 References......Page 35 2.2 Background: current functionality of tools......Page 38 2.3 Unsolved problems and open questions: technology from cell biology, optics, and behavior......Page 40 2.4 Unsolved problems and open questions: genomics and biophysics......Page 43 2.5 Conclusion......Page 46 References......Page 48 3.1 Introduction......Page 50 3.2 Photosensitizing receptors......Page 51 3.3 PCL and PTL development and applications......Page 54 3.4 Advantages and disadvantages of PCLs and PTLs......Page 56 References......Page 57 4.3 The large scale challenge of circuit neurosciences......Page 62 4.4 The current approach to the large-scale integration problem......Page 63 4.6 Genetically encoded voltage indicators: state of development and application......Page 64 References......Page 67 5 Why optogenetic “control” is not (yet) control......Page 70 References......Page 74 6.1 Introduction – the nematode as a genetic model in systems neurosciencesystems neuroscience......Page 76 6.2.2 Imaging populations of neurons in immobilized animals......Page 77 6.2.3 Imaging neural activity in freely moving animals......Page 78 6.3.1 Channelrhodopsin (ChR2) and ChR variants with different functional properties for photodepolarization......Page 79 6.3.3 Photoactivated Adenylyl Cyclase (PAC) for phototriggered cAMP-dependent effects that facilitate neuronal transmission......Page 80 6.3.5 Stimulation of single neurons by optogenetics in freely behaving C. elegans......Page 81 6.4.1 Optical control of synaptic transmission at the neuromuscular junction and between neurons......Page 83 6.4.2 Optical control of neural network activity in the generation of behavior......Page 84 6.5.1 Closed-loop optogenetic control and optical feedback from behavior and individual neurons......Page 85 6.5.2 Requirements for integrated optogenetics in the nematode......Page 87 References......Page 89 7.2 Protein degradation......Page 94 7.3 Light stimulation......Page 100 References......Page 103 8.1 Introduction......Page 106 8.2 Approaches to dissect neuronal circuits: determining physiological correlations, requirement and sufficiency of neurons......Page 107 8.3 Optogenetic analysis of simple stimulus-response-connections......Page 108 8.4 Optogenetic and thermogenetic analysis of modulatory neurons: artificial mimicry of relevance......Page 110 References......Page 112 9.2 Zebrafish neuroscience: Genetics + Optics + Behavior......Page 116 9.3 Genetic targeting of optogenetic proteins to specific neurons......Page 117 9.5.1 Spinal cord neurons (Rohon–Beard and Kolmer–Agduhr cells)......Page 118 9.5.3 Tangential neurons in the vestibular system......Page 119 9.5.5 Whole-brain calcium imaging of motor adaptation at single-cell resolution......Page 120 References......Page 121 10.1 Introduction......Page 124 10.2.1 Excitatory signal integration at dendrites......Page 125 10.2.2 Control of excitatory signal integration by inhibition or neuromodulation......Page 126 10.2.3 Long-term analysis of synaptic function......Page 128 10.3 Circuits and systems level......Page 129 10.4 Optogenetics and behavior: testing causal relationships in freely moving animals......Page 135 References......Page 136 11.2 Opportunities for translational applications......Page 142 11.3 Safety challenges......Page 144 References......Page 145 12.2 Molecular tools for sensitizing neural functions to light......Page 148 12.3 Hardware for delivery of light into intact brain circuits......Page 151 References......Page 152 13.2 Sleep / wake regulation......Page 158 13.3 Addiction......Page 160 13.4 Fear, anxiety and depression......Page 163 13.6 Aggression......Page 165 13.8 Seizures......Page 166 References......Page 167 14.2 Electrical DBS: advantages and drawbacks......Page 172 14.3 Potential of optogenetic stimulation......Page 173 References......Page 174 15.1 Introduction......Page 176 15.2 Proof-of-concept studies......Page 177 15.3 Light sensors......Page 179 15.4 rAAV-mediated retinal gene delivery......Page 181 15.5 Retinal cell-type specific targeting......Page 182 References......Page 184 Further reading......Page 186 16.2 The various conditions to be treated......Page 188 16.3.1.1 Encapsulated cell technology (ECT)......Page 189 16.3.1.3 Visual Cycle modulators......Page 190 16.3.1.5 Stem cell approaches......Page 191 16.3.1.7 Electronic retinal prosthesis......Page 192 16.3.3 Tongue stimulators......Page 196 16.5 Open Questions......Page 197 References......Page 198 Selected registered clinical trials as by February 2013......Page 200 17.1 Background and state of the art......Page 202 17.2 Current research on cochlear optogenetics......Page 204 17.3 Potential and risks of cochlear optogenetics for auditory prosthetics......Page 205 References......Page 206 18 History in the making: the ethics of optogenetics......Page 208 References......Page 214 19.1 Principles of optogenetics......Page 216 19.2 Principles of biomedical ethics......Page 217 19.2.1 Respect for the patient’s autonomy......Page 220 19.2.2 Nonmaleficence......Page 221 19.2.3 Beneficence......Page 222 19.2.4 Justice......Page 223 19.3 Conclusion......Page 224 References......Page 225 Appendix : Dahlem-Conference (Berlin, September 2–5, 2012): “Optogenetics. Challenges and Perspectives.”......Page 228 Index......Page 238 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.