Monthly Archives: August 2012

Optogenetic voltage imaging in slices and living mice using VSFP-Butterfly

In a recent paper published in the Journal of Neurophysiology, the Knöpfel lab (Riken, Japan) introduced a new design variant of FRET-based voltage sensitive fluorescent proteins, termed VSFP-Butterflies, along with an extensive series of application examples in brain slices and living mice. VSFP-Butterfly 1.2 uses red shifted FP, allowing excitation at 488 to 510 nm with acceptor emission > 600 nm. While the sensitivity of VSFP-Butterfly 1.2 is similar to that of VSFP2.3 (around 22% ΔF/F at half maximal activation, V1/2), its fluorescence to voltage relationship is left shifted, resulting in a more sensitive detection of subthreshold potentials as well as of action potentials (fig. 1). VSFP-Butterfly imaging of voltage signals over the cortex of living mice revealed traveling waves generated by the activity of layer 2/3 pyramidal cells (fig 2). The application examples in this report demonstrate that cell class-specific voltage imaging is practical with VSFP-Butterflies. The authors discuss how VSFP-based voltage imaging will opening new avenues towards a better understanding of the neuronal computations reflected in the dynamics of cortical circuits.

Posted in Journal Club | Tagged , | Leave a comment

Transcriptional amplification with two rAAVs using tetracycline-controlled gene induction

Cell-type specific expression of genetically encoded indicators or optogenetic probes is often hampered by the use of promoters that are specific but drive expression only weakly. In a recent paper of the Wang Lab at Princeton University, the authors describe a means of introducing a calcium indicator using the TET promoter system in the cerebellar cortex to boost expression by about ten-fold, as determined by quantitative determination of intracellar concentration. Kuhn et al. show specific labeling of Purkinje cells and all interneuron types. Together with a previous paper of the Wang Lab where glial cells were targeted, now nearly all cell types of the cerebellar cortex can be selectively labeled. Additionally, Kuhn et al. overcome cell toxicity associated with rAAV injection and/or local GECI overexpression by systemic pre-injection of hyperosmotic D-mannitol, doubling the time window for functional imaging.

Original article by Kuhn et al.

Posted in Journal Club | Tagged , | Leave a comment

Novel AAVs for Cre-dependent control of transgene expression

Recombinant adeno-associated viruses (rAAVs) designed to activate transgene expression in only those cells expressing Cre-recombinase (Cre-On) are widely used to introduce optogenetics constructs into specific cell types and brain regions. In many experiments, what functionally distinguishes Cre-expressing cells from their non-Cre expressing neighbors is not fully understood. A recent paper by Saunders et al. (Sabatini lab, Harvard Medical School) published in Frontiers in Neural Circuits describes two rAAV strategies that allow for simultaneous Cre-On and Cre-Off transgene expression. One strategy (Cre-Switch) achieves differential transgene expression with a single rAAV. The second strategy introduces a Cre-Off vector (FAS), built with lox sites that do not efficiently recombine with loxP or lox2272 sites, which allow FAS rAAVs to be used simultaneously with popular Cre-On DIO (double-floxed inverted ORF) a.k.a FLEX (flip-excision) rAAVs. All Cre-On, Cre-Off, and Cre-Switch rAAV vectors in Saunders et al. are freely available from Addgene.

Original article by Saunders et al.

Posted in Journal Club | Tagged , | Leave a comment

Expanding the color palette of channelrhodopsins

In a recent paper published in the Journal of Biological Chemistry, the Hegemann and Deisseroth labs introduced several color-shifted Channelrhodopsins (ChRs) with different absorption and kinetic properties. Prigge et al. mutated several key amino acids in ChR2 and C1V1 to further separate action spectra of those two existing ChRs. The resulting colour-variants are separated by 30 nm from each other and show peak absorption at 460 ,490, 520 and 550 nm respectively (Fig. 1). All color-variants exhibit two times larger photocurrents in HEK-cells then the wild type ChR2. Further engineering yielded off-kinetics spanning the range from ms to s for each colour variant. The two most spectrally separated variants (ChR2 T159C and C1V1-triple) were used to show the feasibility of a separate, wavelength-dependent activation of a HEK cell population expressing on those variants (Fig. 2). In addition the blue absorbing mutant ChR2 T159C-L132C exhibits 3 times larger photocurrents then ChR2 H134R, has a small inactivation and a reduced proton permeation making this variant the most efficient ChR for blue activation so far.

Posted in Journal Club | Tagged , | Leave a comment