Challenges & Opportunities
Although in the young field of optogenetics
spectacular results have been obtained, many improvements are necessary with
respect to 1.) Transfection methods, 2.) Optogenetic tools, and 3.) Development
of appropriate light sources for studies in the brain or the retina of
mammals.
1.) Transfection methods
Transfection methods are focused on the construction of vectors and promoters for cell specific expression of the microbial rhodopsins. The molecular biology aspect is a challenge, because of the enormous variety of constructs, which have to be tested by time consuming screening. As carriers for gene transfer different viruses have been used, preferably Lentiviruses and Adeno Associated Viruses (AAV´s). The virus approach is quick and efficient and has a biomedical implication, whereas the construction of transgenic animals (rodents, fruitflies, zebrafish and C. elegans) are time consuming but have been proven to be ideal for a variety of different experiments in basic research.
2.) Improvement of the optogenetic tools
Although the wild type ChR2 and NphR work quite well, an improved light sensitivity is important for experiments in the mammalian brain because of its the low transmittance. All microbial rhodopsins utilize retinal as their chromophore, whose sensitivity for light can be influenced only slightly by changing the surrounding pocket within the protein. Certain ChR2 mutants, however, show an improved light sensitivity by an increased lifetime up to minutes in the open state of the channel. These channels can be switched on and off at variable time intervals by different wavelengths, but the increased life time and light-induced off response makes these mutants too slow for many neurobiological applications. In addition a search for other channelrhodopsins could be helpful. For the Cl- pump NphR the chances to increase the activity are low, because in this case the photo (reaction) cycle has to be accelerated substantially for a more efficient Cl- pumping in the cell. To find such mutants is not probable, because in the field of the light-activated ion pumps those have not been identified in the last 25 years of research on bacterial rhodopsins.
3.) Improvement of appropriate light sources
For high spatial resolution in the micrometer range light emission diodes (LED´s) arrays, which are separately addressable are necessary for the optimal mapping of the brain and the retina. An alternate method consists of the design of arrays of micro light pipes. Such devices are sufficient for light stimulation on the surface of the brain or in the environment of the light source within the brain in the submillimeter range. For applications on the dense tissue in the brain, however, the two photon excitation would be desirable to increase the transmittance. (Source: http://www.mpg.de/36227/bm06_Optogenetics-basetext.pdf)
1.) Transfection methods
Transfection methods are focused on the construction of vectors and promoters for cell specific expression of the microbial rhodopsins. The molecular biology aspect is a challenge, because of the enormous variety of constructs, which have to be tested by time consuming screening. As carriers for gene transfer different viruses have been used, preferably Lentiviruses and Adeno Associated Viruses (AAV´s). The virus approach is quick and efficient and has a biomedical implication, whereas the construction of transgenic animals (rodents, fruitflies, zebrafish and C. elegans) are time consuming but have been proven to be ideal for a variety of different experiments in basic research.
2.) Improvement of the optogenetic tools
Although the wild type ChR2 and NphR work quite well, an improved light sensitivity is important for experiments in the mammalian brain because of its the low transmittance. All microbial rhodopsins utilize retinal as their chromophore, whose sensitivity for light can be influenced only slightly by changing the surrounding pocket within the protein. Certain ChR2 mutants, however, show an improved light sensitivity by an increased lifetime up to minutes in the open state of the channel. These channels can be switched on and off at variable time intervals by different wavelengths, but the increased life time and light-induced off response makes these mutants too slow for many neurobiological applications. In addition a search for other channelrhodopsins could be helpful. For the Cl- pump NphR the chances to increase the activity are low, because in this case the photo (reaction) cycle has to be accelerated substantially for a more efficient Cl- pumping in the cell. To find such mutants is not probable, because in the field of the light-activated ion pumps those have not been identified in the last 25 years of research on bacterial rhodopsins.
3.) Improvement of appropriate light sources
For high spatial resolution in the micrometer range light emission diodes (LED´s) arrays, which are separately addressable are necessary for the optimal mapping of the brain and the retina. An alternate method consists of the design of arrays of micro light pipes. Such devices are sufficient for light stimulation on the surface of the brain or in the environment of the light source within the brain in the submillimeter range. For applications on the dense tissue in the brain, however, the two photon excitation would be desirable to increase the transmittance. (Source: http://www.mpg.de/36227/bm06_Optogenetics-basetext.pdf)