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Tools, methods, and applications for optophysiology in neuroscience.

Smedemark-Margulies N, Trapani JG - Front Mol Neurosci (2013)

Bottom Line: A large number of genetically encoded protein sensors have also been developed to optically track cellular activity in real time, including membrane-voltage-sensitive fluorophores and fluorescent calcium and pH indicators.The development of techniques for controlled expression of these proteins has also increased their utility by allowing the study of specific populations of cells.Additionally, recent advances in optics technology have enabled both activation and observation of target proteins with high spatiotemporal fidelity.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology and Neuroscience Program, Amherst College Amherst, MA, USA.

ABSTRACT
The advent of optogenetics and genetically encoded photosensors has provided neuroscience researchers with a wealth of new tools and methods for examining and manipulating neuronal function in vivo. There exists now a wide range of experimentally validated protein tools capable of modifying cellular function, including light-gated ion channels, recombinant light-gated G protein-coupled receptors, and even neurotransmitter receptors modified with tethered photo-switchable ligands. A large number of genetically encoded protein sensors have also been developed to optically track cellular activity in real time, including membrane-voltage-sensitive fluorophores and fluorescent calcium and pH indicators. The development of techniques for controlled expression of these proteins has also increased their utility by allowing the study of specific populations of cells. Additionally, recent advances in optics technology have enabled both activation and observation of target proteins with high spatiotemporal fidelity. In combination, these methods have great potential in the study of neural circuits and networks, behavior, animal models of disease, as well as in high-throughput ex vivo studies. This review collects some of these new tools and methods and surveys several current and future applications of the evolving field of optophysiology.

No MeSH data available.


Related in: MedlinePlus

Diagram of DIO transgenic system in mice. The double-floxed inverse open reading frame (DIO) construct comprises inverted terminal repeats (ITR), the EF1α promoter, an eYFP-ChR2 fusion gene surrounded by a pair of LoxP sites and a pair of Lox2722 sites oriented inward, a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) and a human growth hormone polyadenylation signal (hGH polyA). The eYFP-ChR2 gene starts in an inverted, inactive orientation. Expression of Cre recombinase will cause serial recombination resulting in the active, fixed orientation of the transgene (bottom; figure adapted from Sohal et al., 2009).
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Figure 1: Diagram of DIO transgenic system in mice. The double-floxed inverse open reading frame (DIO) construct comprises inverted terminal repeats (ITR), the EF1α promoter, an eYFP-ChR2 fusion gene surrounded by a pair of LoxP sites and a pair of Lox2722 sites oriented inward, a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) and a human growth hormone polyadenylation signal (hGH polyA). The eYFP-ChR2 gene starts in an inverted, inactive orientation. Expression of Cre recombinase will cause serial recombination resulting in the active, fixed orientation of the transgene (bottom; figure adapted from Sohal et al., 2009).

Mentions: For optophysiology with genetically encoded proteins, specific over-expression is generally very important. This is both for ensuring adequate activation when using photoactuators, and for increasing the signal-to-noise ratio of experiments with photosensors. Several modular transgene systems have been developed to facilitate cell-type specificity and strong expression. The double-floxed inverse open reading frame (DIO) strategy devised by Karl Deisseroth’s group provides one such method for minimizing so called “transcriptional leakage” while simultaneously achieving high expression levels (Figure 1; Sohal et al., 2009). This strategy improves on a common two-part scheme for transgene expression, wherein a promoter with high cell-specificity drives the expression of Cre recombinase, while a promoter with stronger expression drives a desired transgene with a floxed stop codon. In this traditional formulation of the floxed-stop scheme, the researcher can combine the spatiotemporal specificity of the first promoter with the transcriptional strength of the second. Excision of the stop codon and subsequent expression of the transgene will ideally occur only following Cre expression at the appropriate developmental time point or within cells of interest. Practically speaking, though, there can be transcriptional leakage when the stop codon fails to completely prevent transcription of the transgene. This can be particularly detrimental in the case of a high-expression promoter that is generally ubiquitously expressed and constitutively active. The innovation of Sohal et al.’s (2009) modification is that the transgene is initially inverted and truly inactive in its “off” state. Expression is accomplished using two pairs of incompatible lox sites that flank the inverted transgene. Serial recombination will first reorient the transgene and then excise one of the two lox sites from each pair, and the final state of the gene contains only an incompatible pair of lox sites that are no longer responsive to Cre. This reorientation mechanism can be used as a Cre-on system where the initial orientation of the gene is inactive, or Cre-off where the initial orientation of the gene is active. To add even more degrees of control, this mechanism can be used as a Cre-switch system if the double-floxed region contains two genes in opposite orientations with a stop codon between, though transcriptional leakage of the stop codon again becomes a factor in this setup (Saunders et al., 2012).


Tools, methods, and applications for optophysiology in neuroscience.

Smedemark-Margulies N, Trapani JG - Front Mol Neurosci (2013)

Diagram of DIO transgenic system in mice. The double-floxed inverse open reading frame (DIO) construct comprises inverted terminal repeats (ITR), the EF1α promoter, an eYFP-ChR2 fusion gene surrounded by a pair of LoxP sites and a pair of Lox2722 sites oriented inward, a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) and a human growth hormone polyadenylation signal (hGH polyA). The eYFP-ChR2 gene starts in an inverted, inactive orientation. Expression of Cre recombinase will cause serial recombination resulting in the active, fixed orientation of the transgene (bottom; figure adapted from Sohal et al., 2009).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3713398&req=5

Figure 1: Diagram of DIO transgenic system in mice. The double-floxed inverse open reading frame (DIO) construct comprises inverted terminal repeats (ITR), the EF1α promoter, an eYFP-ChR2 fusion gene surrounded by a pair of LoxP sites and a pair of Lox2722 sites oriented inward, a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) and a human growth hormone polyadenylation signal (hGH polyA). The eYFP-ChR2 gene starts in an inverted, inactive orientation. Expression of Cre recombinase will cause serial recombination resulting in the active, fixed orientation of the transgene (bottom; figure adapted from Sohal et al., 2009).
Mentions: For optophysiology with genetically encoded proteins, specific over-expression is generally very important. This is both for ensuring adequate activation when using photoactuators, and for increasing the signal-to-noise ratio of experiments with photosensors. Several modular transgene systems have been developed to facilitate cell-type specificity and strong expression. The double-floxed inverse open reading frame (DIO) strategy devised by Karl Deisseroth’s group provides one such method for minimizing so called “transcriptional leakage” while simultaneously achieving high expression levels (Figure 1; Sohal et al., 2009). This strategy improves on a common two-part scheme for transgene expression, wherein a promoter with high cell-specificity drives the expression of Cre recombinase, while a promoter with stronger expression drives a desired transgene with a floxed stop codon. In this traditional formulation of the floxed-stop scheme, the researcher can combine the spatiotemporal specificity of the first promoter with the transcriptional strength of the second. Excision of the stop codon and subsequent expression of the transgene will ideally occur only following Cre expression at the appropriate developmental time point or within cells of interest. Practically speaking, though, there can be transcriptional leakage when the stop codon fails to completely prevent transcription of the transgene. This can be particularly detrimental in the case of a high-expression promoter that is generally ubiquitously expressed and constitutively active. The innovation of Sohal et al.’s (2009) modification is that the transgene is initially inverted and truly inactive in its “off” state. Expression is accomplished using two pairs of incompatible lox sites that flank the inverted transgene. Serial recombination will first reorient the transgene and then excise one of the two lox sites from each pair, and the final state of the gene contains only an incompatible pair of lox sites that are no longer responsive to Cre. This reorientation mechanism can be used as a Cre-on system where the initial orientation of the gene is inactive, or Cre-off where the initial orientation of the gene is active. To add even more degrees of control, this mechanism can be used as a Cre-switch system if the double-floxed region contains two genes in opposite orientations with a stop codon between, though transcriptional leakage of the stop codon again becomes a factor in this setup (Saunders et al., 2012).

Bottom Line: A large number of genetically encoded protein sensors have also been developed to optically track cellular activity in real time, including membrane-voltage-sensitive fluorophores and fluorescent calcium and pH indicators.The development of techniques for controlled expression of these proteins has also increased their utility by allowing the study of specific populations of cells.Additionally, recent advances in optics technology have enabled both activation and observation of target proteins with high spatiotemporal fidelity.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology and Neuroscience Program, Amherst College Amherst, MA, USA.

ABSTRACT
The advent of optogenetics and genetically encoded photosensors has provided neuroscience researchers with a wealth of new tools and methods for examining and manipulating neuronal function in vivo. There exists now a wide range of experimentally validated protein tools capable of modifying cellular function, including light-gated ion channels, recombinant light-gated G protein-coupled receptors, and even neurotransmitter receptors modified with tethered photo-switchable ligands. A large number of genetically encoded protein sensors have also been developed to optically track cellular activity in real time, including membrane-voltage-sensitive fluorophores and fluorescent calcium and pH indicators. The development of techniques for controlled expression of these proteins has also increased their utility by allowing the study of specific populations of cells. Additionally, recent advances in optics technology have enabled both activation and observation of target proteins with high spatiotemporal fidelity. In combination, these methods have great potential in the study of neural circuits and networks, behavior, animal models of disease, as well as in high-throughput ex vivo studies. This review collects some of these new tools and methods and surveys several current and future applications of the evolving field of optophysiology.

No MeSH data available.


Related in: MedlinePlus