We conducted experiments to understand the role of Ventral Hippocampus (vHPC) projections to the Nucleus Accumbens (NAc) in exploratory locomotion, and to determine if the reduced vHPC parvalbumin neuron activity can result in behaviors associated with schizophrenia. Through the use of optogenetics, we activated vHPC neurons and vHPC terminals in the NAc. Both manipulations significantly increased locomotor activity in the open field. Selective inhibition of vHPC terminals in the NAc during a test for novel environment exploration significantly reduced preference for novel environments over familiar environments. DREADD-mediated inhibition of activation of vHPC parvalbumin neuron activity did not significantly alter amphetamine-induced locomotion. Overall, these experiments provide support for the role of the vHPC-NAc pathway in mediating exploratory behavior in novel environments, but it remains inconclusive whether dysregulated vHPC activity due to the loss of parvalbumin neurons leads to behaviors associated with schizophrenia.

Cellular lipid membranes can – and often do – support a transmembrane electric field, serving as biological capacitors that maintain a voltage difference between their two sides. It isn't hard to see why these voltage gradients matter; the electrical spiking of neurons gives rise to our thoughts and actions, and the voltage dynamics of cardiomyocytes keep our hearts beating. Studies of bioelectricity have historically relied on electrode-based techniques to perturb and measure membrane potential, but these techniques have inherent limitations. I present new optogenetic methods of studying membrane potential that will broaden the scope of electrophysiological investigations by complementing traditional approaches. I introduce the microbial rhodopsin Archaerhodopsin-3 (Arch), a transmembrane protein from Halorubrum sodomense. The fluorescence of Arch is a function of membrane potential, allowing it to serve as an optical voltage reporter. We use time-dependent pump-probe spectroscopy to interrogate the light- and voltage- dependent conformational dynamics of this protein, to elucidate the mechanism of voltage-dependent fluorescence in Arch. I then present two new methods for imaging voltage using engineered variants of Arch. Both techniques take advantage of the unique photophysical properties of Arch(D95X) mutants. The first method, Flash Memory, records a photochemical imprint of the activity state -- firing or not firing -- of a neuron at a user-selected moment in time. The Flash Memory technique decouples the recording of neural activity from its readout, and can potentially allow us to take large-scale snapshots of voltage (e.g. maps of activity in a whole mouse brain). The second method allows for the quantitative optical measurement of membrane potential. This technique overcomes the problems that typically hinder intensity-based measurements by encoding a measurement of voltage in the time domain. Finally, I present a method to visualize cellular responses to changes in membrane potential. I engineer mutants of Channelrhodopsin-2 (ChR2), a light-gated cation channel from Chlamydomonas reinhardtii that is used for optical control of neural activity, and use these optogenetic actuators in conjunction with GFP-based sensors to study the activity-dependent behavior of cultured neurons.

We conducted experiments to understand the role of Ventral Hippocampus (vHPC) projections to the Nucleus Accumbens (NAc) in exploratory locomotion, and to determine if the reduced vHPC parvalbumin neuron activity can result in behaviors associated with schizophrenia. Through the use of optogenetics, we activated vHPC neurons and vHPC terminals in the NAc. Both manipulations significantly increased locomotor activity in the open field. Selective inhibition of vHPC terminals in the NAc during a test for novel environment exploration significantly reduced preference for novel environments over familiar environments. DREADD-mediated inhibition of activation of vHPC parvalbumin neuron activity did not significantly alter amphetamine-induced locomotion. Overall, these experiments provide support for the role of the vHPC-NAc pathway in mediating exploratory behavior in novel environments, but it remains inconclusive whether dysregulated vHPC activity due to the loss of parvalbumin neurons leads to behaviors associated with schizophrenia.

Memory formation and recall depend on a complex circuit that includes the hippocampus and associated cortical regions. The goal of this thesis was to understand how two of the cortical connections, the medial entorhinal cortex (MEC) and medial prefrontal cortex (mPFC), influence spatial and nonspatial activity in the hippocampus. Cells in the MEC exhibit prominent spatially selective activity and have been hypothesized to drive place representation in the hippocampus. In Experiment 1 the MEC was transiently inactivated using the inhibitory opsin ArchaerhodopsinT (ArchT), and simultaneous recordings from CA1 were made as rats ran on an elliptical track. In response to MEC disruption some cells in the hippocampus shifted the preferred location of activity, some changed firing rate and others were unaffected. The new representation that developed following MEC disruption remained stable despite the fact that inhibition was transient. If the MEC is the source of spatial activity in the hippocampus the activity would be either time-locked to periods of inhibition or unstable throughout the period of inconsistent input. These results show that the MEC guides spatial representation in the hippocampus but does not directly drive spatial firing. The mPFC is generally thought to guide behavior in response to contextual elements. Experiment 2 examined the interaction between the mPFC and the hippocampus as rats performed a contextual discrimination task. Recordings were made in CA1, and the mPFC was disrupted using ArchT during the odor sampling phase of the discrimination. As animals perform this task neurons in the hippocampus respond to a conjunction of odor and location which indicates an association of what and where information in the hippocampus. Optogenetic disruption of the mPFC led to a decrease in nonspatial representation. Individual cells showed lower levels of odor selectivity, but there was no change in the level of spatial representation. This indicates that the mPFC is important for determining how the hippocampus represents nonspatial information but does not alter the spatial representation. The results are discussed within a model of memory formation that includes binding spatial and nonspatial information in the hippocampus.

In this dissertation,the membrane protein channelrhodopsin-1 from the green flagellate algae Chlamydomonas agustae (CaChR1) is studied using a variety of spectroscopic techniques developed in the Rothschild Molecular Biophysics Laboratory at Boston University. Over the last decade, channelrhodopsins have proven to be effective optogenetic tools due to their ability to function as light-gated ion channels when expressed in neurons. This ability allows neuroscientists to optically activate an inward directed photocurrent which depolarizes the neuronal membranes and triggers an action potential. Although a variety of channelrhodopsins with different properties have been used, the underlying mechanisms of channelrhodopsin functionality is not yet fully understood. The protein studied here has several advantageous properties compared to the more extensively studied channelrhodopsin-2 from Chlamydomonas reinhardtii including a red shifted visible absorption and slower light inactivation despite having a lower channel current. Elucidating the internal molecular mechanisms underlying the function of CaChR1 provides critical insight into the large class of channelrhodopsin proteins leading toward improved bioengineering for specific optogenetic applications. Here near-IR pre-resonance Raman spectroscopy of CaChR1 provides information on the structure of the unphotolyzed (P0) retinal chromophore, the Schiff base protonation state, and presence of carboxylic acid residues interacting with the Schiff base. Low-temperature FTIR difference spectroscopy combined with site-directed mutagenesis and isotope labeling provide information on changes occurring in the retinal chromophore and protein during the primary phototransition (P0 to P1). This includes information about changes involving protonation state of binding-pocket residues, protein backbone structure, and internal water molecules. Further experiments combining low-temperature and time-resolved FTIR-difference spectroscopy reveal additional information about structural changes during the transition from the unphotolyzed state to the active (open channel) state of the protein (P0 to P2). This work has resulted in an initial model that describes key proton transfer events which occur between the Schiff base and carboxylic acid residues inside the active site of CaChR1. The model raises the possibility that ion channel gating and ion specificity is regulated by the protonation changes of two key residues (Glu 169 and Asp299) located near the Schiff base.

In the 1980s, it was widely accepted that new neurons are continuously generated in the dentate gyrus of the mammalian hippocampus. Since its acceptance, researchers have employed various techniques and behavioral paradigms to study the proliferation, differentiation, and functional role of adult-born neurons. This literature thesis aims to discuss how optogenetics is able to overcome the limitations of past techniques and provide the field with new insights into the functional role of neurogenesis. We will review the current knowledge on both adult hippocampal neurogenesis and optogenetics, present representative studies using optogenetics to investigate neurogenesis and discuss potential limitations and concerns involved in using optogenetics.

Les récepteurs canaux P2X, sélectifs des cations, sont activés par l'ATP extracellulaire. Les récepteurs P2X remplissent de nombreux rôles physiologiques allant de la nociception à la neuromodulation. L'étude du rôle physiopathologique de ces récepteurs souffre d'un manque d'outils pharmacologiques sélectifs. L'optogénétique pharmacologique serait une méthode pour palier ce manque. Mes travaux se divisent en deux parties, l'une concernant l'étude structurale des récepteurs P2X et l'autre présentant le développement d'outils optogénétiques chimiques pour contrôler l'activité des récepteurs P2X. Dans une première série d'expériences nous avons identifié le site de liaison de l'ATP par marquage d'affinité dirigé à l'aide d'un analogue de l'ATP thiol réactif. Ensuite,nous avons démontré le mécanisme d'activation des récepteurs P2X dans une étude utilisant la bioinformatique et l'ingénierie de site zinc. Ainsi nous avons établi une corrélation entre l'ouverture du canal et le rétrécissement du site de liaison suite à la fixation de l'ATP. Enfin nous avons mis au point une nouvelle stratégie optogénétique chimique appelée « optogating » permettant de reprogrammer un canal ionique afin de le contrôler par la lumière. Nous avons montré qu'un récepteur canal modifié au niveau transmembranaire, par un réactif contenant un azobenzène, peut être activé réversiblement par la lumière sans recourir au ligand endogène. Nous avons réussi à photocontrôler l'activité neuronale à l'aide d'un récepteur P2X activé par la lumière,dans lequel, la sensibilité à l'ATP a été génétiquement supprimée. Cet outil est prometteur pour l'étude du rôle physiologique des récepteurs P2X in vivo. / The ATP-gated P2X receptors are trimeric ion channels that are selective to cations.These ion channels are involved in various physiological processes such as nociception and neuromodulation. The study of P2XR physiology suffers from a lack of selective pharmacological molecules. Optogenetic pharmacology could solve this problem. ln thiswork, 1 performed structural studies of P2X receptors and developed an original optochemical tool in order to contrai P2X activity. First, we localized the ATP-binding sites by creating, through a proximity-dependent"tethering" reaction, covalent bonds between a synthesized ATP-derived thiol-reactiveP2X2 agonist (NCS-ATP) and single cysteine mutants engineered in the putativebinding cavities of the P2X2 receptor. Next, we demonstrated that tightening of the ATP-binding sites correlates precisely with channel opening in the P2X2 receptor. Finally, we developed a unique and versatile method, in which the gating machinery of the P2X2 receptor was reprogrammed to respond to light. We found that channels covalently modified by azobenzene-containing reagents at the transmembrane segments could be reversibly turned on and off by light, without the need of the natural ligand (here ATP). We demonstrated photocontrol of neuronal activity by a light-gatedP2X receptor, in which the natural sensitivity to ATP was genetically removed. These light-gated P2X receptors represent valuable tools for investigating the physiological functions of P2X receptors.

Chronic pain induced by nerve damage due to trauma or invasion of cancer to the bone elicits severe ongoing pain as well as hyperalgesia and allodynia likely reflecting adaptive changes within central circuits that amplify nociceptive signals. The present study explored the possible contribution of the mesolimbic dopaminergic circuit in promoting allodynia related to neuropathic and cancer pain. Mice with ligation of the sciatic nerve or treated with intrafemoral osteosarcoma cells showed allodynia to a thermal stimulus applied to the paw on the injured side. Patch clamp electrophysiology revealed that the intrinsic neuronal excitability of ventral tegmental area (VTA) dopamine neurons projecting to the nucleus accumbens (N.Acc.) was significantly reduced in those mice. We used tyrosine hydroxylase (TH)-cre mice that were microinjected with adeno-associated virus (AAV) to express channelrhodopsin-2 (ChR2) to allow optogenetic stimulation of VTA dopaminergic neurons in the VTA or in their N.Acc. terminals. Optogenetic activation of these cells produced a significant but transient anti-allodynic effect in nerve injured or tumor-bearing mice without increasing response thresholds to thermal stimulation in sham-operated animals. Suppressed activity of mesolimbic dopaminergic neurons is likely to contribute to decreased inhibition of N.Acc. output neurons and to neuropathic or cancer pain-induced allodynia suggesting strategies for modulation of pathological pain states.

La cécité ou la très grande malvoyance peut résulter de différentes pathologies comme les dystrophies rétiniennes héréditaires (DRH) caractérisées par la perte des photorécepteurs. Une des approches pour traiter les DRH est la thérapie génique spécifique, c’est à dire le remplacement du gène défectueux par un gène sain. Des études chez des modèles animaux de DRH ont démontré l’efficacité de la thérapie géniques spécifique, et conduit au lancement d’essais cliniques.Malgré des résultats encourageants, la thérapie génique spécifique n’est pas toujours applicable, en particulier quand la dégénérescence est trop avancée ou si le gène muté est inconnu. Pour traiter tous les cas de DRH, un nouvel axe de thérapie génique est envisagé : le transfert d’optogène.Cette stratégie consiste à réactiver la rétine devenue aveugle par l’expression de protéines photosensibles dans la rétine. Notre objectif est d'évaluer l'efficacité du transfert d'optogène chez un modèle canin naturellement déficient pour le gène Rpe65, provoquant une forme sévère de DRH proche de celles retrouvées chez l’homme. La stratégie thérapeutique retenue est : L'injection intravitréenne, après vitrectomie, d'un vecteur recombinant dérivé du virus adéno-associé de sérotype2 (rAAV2/2), portant le transgène optogénétique sous contrôle d'un promoteur fort et spécifique des tissus neuronaux : hSyn. Le but de ce projet est de transduire efficacement les cellules ganglionnaires rétiniennes d'un modèle canin déficient pour le gène Rpe65 et d'évaluer la photosensibilité des cellules transduites. / Inherited retinal diseases (IRD) affect about 2 million people worldwide, leading to severe visual impairment.Specific gene addition therapy is one of the most promising strategies to treat these patients. Howevermany of them are not eligible for specific gene therapy,such as.1) Patients with unknown deficient genes.2) Patients beyond the therapeutic window.3) Patients whose the deficient gene is too large forAAV encapsidation.4) Patients undergoing a dominant form of IRD.Therefore, the aim of this project is to develop analternative strategy, independent of the mutation and the retinal degeneration kinetic: the optogene transfer. In context of IRD, it will consist to convert survivingretinal ganglion cells into sensitive light cells followingthe transfer of ChR2 or Opn4 optogene. Several rodentmodels of IRD have been successfully treated usingthese optogenes. Nevertheless, this approach hasnever been evaluated in large animal models. The objective of our study will be to define the feasibility ofoptogene transfer to restore vision in blind patients by evaluating the safety and the efficacy of AAV-mediated gene transfer of ChR2, eNpHR or Opn4, after vitrectomy, in ganglion cells of a canine model of IRD, the Rpe65-deficient dog.

京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第21610号 / 理博第4517号 / 新制||理||1648(附属図書館) / 京都大学大学院理学研究科生物科学専攻 / (主査)准教授 今元 泰, 教授 高田 彰二, 教授 杤尾 豪人 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM