KENGE-tet System for Expanding the Repertoire of Optogenetically Targeted Cells

Optogenetics has proven to be a powerful tool capable of manipulating the activity of a specific population of cells in a complex multicellular organism. This approach is enthusiastically pursued in recent neuroscience field and the causal relationship between neural activity and behavior is finally starting to become unveiled. However, most studies utilize virus mediated gene transfer for the induction of light-sensitive proteins, such as channelrhodopsin-2 (ChR2), and such method inevitably introduces surgical injuries and variability of expression between trials. Therefore, transgenic approach has long been sought, however, satisfying the demands of the specificity as well as the abundance of expression were difficult.

In a recent paper published in the Cell Reports, Tanaka and Matsui and their colleagues at the National Institute for Physiological Sciences (Okazaki, Japan) established Knockin-mediated ENhanced Gene Expression by improved tetracycline-controlled gene induction system (KENGE-tet). The authors found that high levels of tTA-mediated transcription can be achieved by knocking in tetO-ChR2 cassette into a locus at a housekeeping gene, beta-actin. The authors crossed this tetO-ChR2 knockin mouse with 7 different tTA lines and achieved ChR2 expression in specific cell-types including sub-populations of neurons, astrocytes, oligodendrocytes, and microglial cells. In all cases, the level of ChR2 expression was high enough to allow manipulation of cell activity.

In this study, a step-function type variant of ChR2 (C128S) was chosen for creating the repertoire of transgenic mice. The C128S variant has two unique characters; slow inactivation of the channel and high sensitivity to light. As neural network is essentially a noisy circuit, exogenous addition of few spikes to neurons would unlikely cause much of an impact to the whole system. Due to the slow inactivation kinetics, a short pulse of light can cause a long-lasting barrage of spikes in neurons and evoke visible behavioral response in in vivo. ChR2 (C128S) variant could also be expressed in “non-excitable” glial cells. As the C128S variant has more than 100 times sensitivity to light compared to the conventional wild-type, light merely penetrating through the skull was sufficient to activate these glial cells, as assessed by the c-fos induction. Such method of totally non-invasive stimulation was of particular importance for studying the glial cells as drilling a small hole in the crania could by itself create a change in the state of glial cell activity.

Superiority of the Knockin-Mediated ENhanced Gene Expression by Improved tet System over Other Transgene Expression Methods. Five cell type-specific tTA lines (columns) were crossed with three tetO lines (rows) coding light-sensitive protein (ChR2 and/or HaloR) and a fluorescent protein (EYFP or EGFP) fusion. Top, middle, and bottom row panels show EYFP immunohistochemistry of tissues from mice crossed with tetO-ChR2(C128S)-EYFP knockin mice (knockin), EYFP from mice crossed with tetO-ChR2(C128S)-EYFP BAC transgenic mice (BAC transgenic), and EGFP from mice crossed with bitetO transgenic line (BTR6; transgenic). tTA lines specific for neurons (αCaMKII and orexin promoter; first and second columns, respectively), astrocytes (Mlc1 promoter; third column), oligodendrocytes (PLP promoter; fourth column), and microglia (Iba1 promoter; fifth column) were used. The first, fourth, and fifth columns are from tissues of the hippocampus, the second column is from the lateral hypothalamus, and the third column is from the cerebellar lobe. Note that crossing of the cell type-specific tTA lines with tetO knockin mice (KENGE-tet system) provided the highest levels of gene expression in all cell types. Scale: 200 μm.

This technological improvement, KENGE-tet, enables us to expand the repertoire of optogenetically targeted cells in which both the cell-type specificity and the abundance of expression are realized. Further generation of new lines of either tetO or tTA line would add even more repertoire for scientists to choose from. Such repertoire would benefit large community of scientists, both in neuroscience as well as other biomedical field, who are actively pursuing the causal relationship between cellular activity and behavior of complex organism.

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