Heim N, Garaschuk O, Friedrich MW, Mank M, Milos RI, Kovalchuk Y, Konnerth A, Griesbeck O.
Improved calcium imaging in transgenic mice expressing a troponin C-based biosensor.
Nat Methods. 2007 Feb;4(2):127-9.
PubMed.
Grace Stutzmann Rosalind Franklin University/The Chicago Medical School
Posted:
This study introduces a transgenic mouse expressing a novel, genetically encoded calcium indicator named CerTN-L15. At first pass, the utility of the mouse met with skepticism as past attempts to endogenously express fluorescent indicators have not been particularly successful. Although past constructs would work well in vitro or in non-neuronal cells, equivalent functional expression in neurons proved problematic, often due to binding proteins interfering with the base calcium sensor, calmodulin, and to marked reduction in fluorescent intensity. Here Oliver Griesbeck smartly circumvents many of the previous hurdles by basing the calcium sensor on troponin-C, which is expressed in skeletal and cardiac muscle, and will therefore have fewer competing elements when expressed in neurons. In addition, improvements in, and modifications to, the choice of donor and acceptor FRET pairs improved brightness and facilitated protein folding dynamics. This approach appears to have rectified many of the historical problems, and results in a convincing presentation of a genetically encoded calcium indicator that lives up to its in vitro reputation.
It is refreshing to see the characterization of this construct in an in vitro setting, in acute transfected neurons, in brain slices, and in vivo, with functional properties appearing consistent across platforms. The Kd is 1.2 μM, which is of relatively lower affinity than many of the commonly used calcium indicators. Although this reduces the likelihood of dye saturation (as demonstrated), it could introduce some limitations for accurately measuring calcium dynamics at lower concentration ranges. However, neurons expressing CerTN-L15 show that calcium signals can be generated by single action potentials over large volume areas. Detectable signals from fine dendritic processes in vivo were also demonstrated, and this greatly expands the range of possible research applications.
For the neurodegeneration research world, this transgenic mouse opens many interesting possibilities for crossing with existing mouse models of AD. Interactions between plaque pathology, tangle deposition, and calcium signaling dysregulation can be studied directly across many brain regions, and long-term changes in calcium dynamics can be measured over the organism’s lifetime. The CerTN-L15 mice were heterozygous, and it would be interesting to compare the calcium sensor dynamics with a homozygous line, both to observe the effect of gene dose, and to facilitate crossing with other transgenics.
An additional query is the level of expression, or brightness, between neurons. In the two-photon images of hippocampal and cortical neurons (supplemental figure 2e and f), it appears there are substantial differences in the relative brightness between neurons. This may reflect differences in actual calcium concentrations, in expression levels of the construct, or other factors affecting fluorescence intensity. Fluorescence intensity appears homogenous within neurons, but between neurons it is not clear if the differences are physiological, promoter-driven, or due to some other factor that may change the properties of the calcium sensor. While these issues will likely be worked out in future experiments, the development of a transgenic mouse expressing a stable and robust calcium sensor will likely prove invaluable to understanding neuronal signaling mechanisms affected in AD and other neurodegenerative diseases.
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Rosalind Franklin University/The Chicago Medical School
This study introduces a transgenic mouse expressing a novel, genetically encoded calcium indicator named CerTN-L15. At first pass, the utility of the mouse met with skepticism as past attempts to endogenously express fluorescent indicators have not been particularly successful. Although past constructs would work well in vitro or in non-neuronal cells, equivalent functional expression in neurons proved problematic, often due to binding proteins interfering with the base calcium sensor, calmodulin, and to marked reduction in fluorescent intensity. Here Oliver Griesbeck smartly circumvents many of the previous hurdles by basing the calcium sensor on troponin-C, which is expressed in skeletal and cardiac muscle, and will therefore have fewer competing elements when expressed in neurons. In addition, improvements in, and modifications to, the choice of donor and acceptor FRET pairs improved brightness and facilitated protein folding dynamics. This approach appears to have rectified many of the historical problems, and results in a convincing presentation of a genetically encoded calcium indicator that lives up to its in vitro reputation.
It is refreshing to see the characterization of this construct in an in vitro setting, in acute transfected neurons, in brain slices, and in vivo, with functional properties appearing consistent across platforms. The Kd is 1.2 μM, which is of relatively lower affinity than many of the commonly used calcium indicators. Although this reduces the likelihood of dye saturation (as demonstrated), it could introduce some limitations for accurately measuring calcium dynamics at lower concentration ranges. However, neurons expressing CerTN-L15 show that calcium signals can be generated by single action potentials over large volume areas. Detectable signals from fine dendritic processes in vivo were also demonstrated, and this greatly expands the range of possible research applications.
For the neurodegeneration research world, this transgenic mouse opens many interesting possibilities for crossing with existing mouse models of AD. Interactions between plaque pathology, tangle deposition, and calcium signaling dysregulation can be studied directly across many brain regions, and long-term changes in calcium dynamics can be measured over the organism’s lifetime. The CerTN-L15 mice were heterozygous, and it would be interesting to compare the calcium sensor dynamics with a homozygous line, both to observe the effect of gene dose, and to facilitate crossing with other transgenics.
An additional query is the level of expression, or brightness, between neurons. In the two-photon images of hippocampal and cortical neurons (supplemental figure 2e and f), it appears there are substantial differences in the relative brightness between neurons. This may reflect differences in actual calcium concentrations, in expression levels of the construct, or other factors affecting fluorescence intensity. Fluorescence intensity appears homogenous within neurons, but between neurons it is not clear if the differences are physiological, promoter-driven, or due to some other factor that may change the properties of the calcium sensor. While these issues will likely be worked out in future experiments, the development of a transgenic mouse expressing a stable and robust calcium sensor will likely prove invaluable to understanding neuronal signaling mechanisms affected in AD and other neurodegenerative diseases.
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