Improved expression of halorhodopsin for light-induced silencing of neuronal activity
The ability to control and manipulate neuronal activity within an intact mammalian brain is of key importance for mapping functional connectivity and for dissecting the neural circuitry underlying behaviors. We have previously generated transgenic mice that express channelrhodopsin-2 for light-induced activation of neurons and mapping of neural circuits. Here we describe transgenic mice that express halorhodopsin (NpHR), a light-driven chloride pump that can be used to silence neuronal activity via light. Using the Thy-1 promoter to target NpHR expression to neurons, we found that neurons in these mice expressed high levels of NpHR-YFP and that illumination of cortical pyramidal neurons expressing NpHR-YFP led to rapid, reversible photoinhibition of action potential firing in these cells. However, NpHR-YFP expression led to the formation of numerous intracellular blebs, which may disrupt neuronal function. Labeling of various subcellular markers indicated that the blebs arise from retention of NpHR-YFP in the endoplasmic reticulum. By improving the signal peptide sequence and adding an ER export signal to NpHR-YFP, we eliminated the formation of blebs and dramatically increased the membrane expression of NpHR-YFP. Thus, the improved version of NpHR should serve as an excellent tool for neuronal silencing in vitro and in vivo.
- Gradinaru, V., Thompson, K. R., and Deisseroth, K. (2008). eNpHR: a Natronomonas halorhodopsin enhanced for optogenetic applications. Brain Cell Biol. 2008 Aug 2 [Epub ahead of print]Google Scholar
- Li, X., Gutierrez, D. V., Hanson, M. G., Han, J., Mark, M. D., Chiel, H., Hegemann, P., Landmesser, L. T., and Herlitze, S. (2005) Fast noninvasive activation and inhibition of neural and network activity by vertebrate rhodopsin and green algae channelrhodopsin. Proc. Natl. Acad. Sci. USA 102, 17816–17821PubMedCrossRefGoogle Scholar
- Schroll, C., Riemensperger, T., Bucher, D., Ehmer, J., Völler, T., Erbguth, K., Gerber, B., Hendel, T., Nagel, G., Buchner, E., and Fiala, A. (2006) Light-induced activation of distinct modulatory neurons triggers appetitive or aversive learning in Drosophila larvae. Curr. Biol. 16, 1741–1747PubMedCrossRefGoogle Scholar
- Wang, H., Peca, J., Matsuzaki, M., Matsuzaki, K., Noguchi, J., Qiu, L., Wang, D., Zhang, F., Boyden, E., Deisseroth, K., Kasai, H., Hall, W. C., Feng, G., and Augustine, G. J. (2007) High-speed mapping of synaptic connectivity using photostimulation in Channelrhodopsin-2 transgenic mice. Proc. Natl. Acad. Sci. USA 104, 8143–8148PubMedCrossRefGoogle Scholar
- Wulff, P., Goetz, T., Leppä, E., Linden, A. M., Renzi, M., Swinny, J. D., Vekovischeva, O. Y., Sieghart, W., Somogyi, P., Korpi, E. R., Farrant, M., and Wisden, W. (2007) From synapse to behavior: rapid modulation of defined neuronal types with engineered GABAA receptors. Nat. Neurosci. 10, 923–929PubMedCrossRefGoogle Scholar