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The discovery of Channelrhodopsins (ChRs) allows their application in the field of neuroscience, so called optogenetics, for exciting or inhibiting neuronal activity. Nowadays optogenetics technology is very promising for decoding neuronal circuits associated with specific behaviors, as well as neurological disorders. However, many questions still need to be addressed, such as the lack of cell type-specific gene transfer methods for ChRs delivery or powerful and more efficient optogenetic tools. In the first part of my thesis, recombinant adeno-associated virus (rAAV) serotypes are screened for their ability to transfer genes to neurons. rAAV6 was identified as the most efficient serotype for transduction of cortical-glial mixed cultures. First, we used the constitutively active cytomegalovirus (CMV) promoter to drive gene expression of the Channelrhodopsin 2 variant ChR2opt. The protein was detected in neurons as well as in glia cells. After exchanging the CMV promoter for the human synapsin (hSyn) promoter, ChR2opt expression was restricted to neurons. Notably, using blue light illumination, I could control electrical activity in primary cortical neurons expressing ChR2opt and succeeded in triggering action potentials by ChR2opt stimulation. Using this strategy, I was able to register responses at the single cell level with high temporal accuracy. Based on the system of rAAV6-hSyn-ChR2opt investigated in the first part, the second part successfully achieved optical control of neuronal networks with both random and patterned connectivity in vitro. In both networks, optogenetic mapping of cortical microcircuits was performed. Most important, using two kinds of branched patterns with defined structure, optical control of neural activity at the single cell level was successfully achieved. Only stimulating the small area of axons or dendrites was sufficient to effectively elicit action potentials in the branched patterns. These results provide the next steps in increasing the resolution of optical control of neuronal activity. In the third part of my thesis, one novel powerful tool with super light-sensitivity, long open-state and large photocurrent, termed ChR2-XXL, was employed in primary rat cortical neurons in vitro. Its two important features have been confirmed in neurons with whole cell patch clamp together with blue laser illumination. On the one hand, stable depolarization block could be achieved in ChR2-XXL expressing neurons; on the other hand, neurotransmission can be successfully induced when stimulating the presynaptic neurons that expressed ChR2-XXL. The last part addressed a naturally occurring anion channelrhodopsin (GtACR1) in primary rat cortical neurons. Light-gated chloride conduction of GtACR1 was verified in primary cortical neurons. Subsequently, the efficient photosuppression of neuronal action potentials, including electrically stimulated and spontaneous activity, was performed using blue laser illumination. In addition, my work implies that the chloride concentration in neurons decreases during neural development. This work provides strong evidence that GtACR1 can not only inhibit neuronal signals, including spontaneous activity, but may also be used to manipulate Cl- based cell maturation systems.In summary, my work offers strong evidence to support two main aims. First, rAAV6 with hSyn promoter driven constructs provides a useful tool for specifically manipulating neuronal activity and mapping microcircuits. Employing this system to express ChR2opt, allowed both random and patterned networks to be manipulated with specificity of stimulation down to single neurites. Second, new tools have been validated for use in neurons. ChR2-XXL is able to generate stable depolarization block and shape powerful neurotransmission. Also, ACRs provide a method to achieve the rapid and efficient suppression of neuronal activity by hyperpolarization. These new tools, and the optimized method of gene transfer are promising candidates for in vivo applications of optogenetic control. |
