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Kinetic evaluation of photosensitivity in bi-stable variants of chimeric channelrhodopsins.

Hososhima S, Sakai S, Ishizuka T, Yawo H - PLoS ONE (2015)

Bottom Line: The turning-on rate constant of each photocurrent followed a linear relationship to 0-0.12 mW mm(-2) of blue LED light or to 0-0.33 mW mm(-2) of cyan LED light.Each photocurrent of bi-stable ChR was shut off to the non-conducting state by yellow or orange LED light in a manner dependent on the irradiance.On the other hand, in another group of neurons, the threshold irradiance was not dependent on the irradiation time.

View Article: PubMed Central - PubMed

Affiliation: Department of Developmental Biology and Neuroscience, Tohoku University Graduate School of Life Sciences, Sendai, Japan; Core Research of Evolutional Science & Technology (CREST), Japan Science and Technology Agency (JST), Tokyo, Japan.

ABSTRACT
Channelrhodopsin-1 and 2 (ChR1 and ChR2) form cation channels that are gated by light through an unknown mechanism. We tested the DC-gate hypothesis that C167 and D195 are involved in the stabilization of the cation-permeable state of ChRWR/C1C2 which consists of TM1-5 of ChR1 and TM6-7 of ChR2 and ChRFR which consists of TM1-2 of ChR1 and TM3-7 of ChR2. The cation permeable state of each ChRWR and ChRFR was markedly prolonged in the order of several tens of seconds when either C167 or D195 position was mutated to alanine (A). Therefore, the DC-gate function was conserved among these chimeric ChRs. We next investigated the kinetic properties of the ON/OFF response of these bi-stable ChR mutants as they are important in designing the photostimulation protocols for the optogenetic manipulation of neuronal activities. The turning-on rate constant of each photocurrent followed a linear relationship to 0-0.12 mW mm(-2) of blue LED light or to 0-0.33 mW mm(-2) of cyan LED light. Each photocurrent of bi-stable ChR was shut off to the non-conducting state by yellow or orange LED light in a manner dependent on the irradiance. As the magnitude of the photocurrent was mostly determined by the turning-on rate constant and the irradiation time, the minimal irradiance that effectively evoked an action potential (threshold irradiance) was decreased with time only if the neuron, which expresses bi-stable ChRs, has a certain large membrane time constant (eg. τm > 20 ms). On the other hand, in another group of neurons, the threshold irradiance was not dependent on the irradiation time. Based on these quantitative data, we would propose that these bi-stable ChRs would be most suitable for enhancing the intrinsic activity of excitatory pyramidal neurons at a minimal magnitude of irradiance.

No MeSH data available.


Related in: MedlinePlus

Differential sensitivity to irradiance and duration among neurons.A, The threshold irradiance to evoke an action potential was related to the membrane time constant (τm) for the blue LED pulse of either 0.1 s (closed diamonds) or 1 s (open circles). B, Reduction of threshold irradiance of the type-1 neurons with prolongation of the pulse duration. C, The latency to evoke an action potential was related to τm. D, The delayed firing of type-1 neurons with prolongation of the pulse duration. Statistical significance was evaluated with Mann-Whitney U test; *, P < 0.05 and **, P < 0.005.
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pone.0119558.g006: Differential sensitivity to irradiance and duration among neurons.A, The threshold irradiance to evoke an action potential was related to the membrane time constant (τm) for the blue LED pulse of either 0.1 s (closed diamonds) or 1 s (open circles). B, Reduction of threshold irradiance of the type-1 neurons with prolongation of the pulse duration. C, The latency to evoke an action potential was related to τm. D, The delayed firing of type-1 neurons with prolongation of the pulse duration. Statistical significance was evaluated with Mann-Whitney U test; *, P < 0.05 and **, P < 0.005.

Mentions: As shown in Fig. 6A, which shows the relationship between the threshold irradiance and τm, the longer irradiation more effectively reduced the threshold irradiance in neurons with larger τm, but was less effective in neurons with smaller τm. For convenience, the cortical neurons were classified into two groups: type 1 (τm > 20 ms, 38 ± 4.7 ms, n = 11) and type 2 (τm < 20 ms, 12 ± 3.1 ms, n = 5) with the significant difference of τm (P < 0.001). Indeed, the threshold irradiance was reduced by the prolongation of the irradiation time in the case of type-1 neurons, whereas it was less dependent in the case of type-2 neurons (Fig. 6B). However, the reduction of the threshold irradiance was often accompanied by prolongation of the latency to evoke the first action potential in the case of type-1 neurons (Fig. 6C and 6D).


Kinetic evaluation of photosensitivity in bi-stable variants of chimeric channelrhodopsins.

Hososhima S, Sakai S, Ishizuka T, Yawo H - PLoS ONE (2015)

Differential sensitivity to irradiance and duration among neurons.A, The threshold irradiance to evoke an action potential was related to the membrane time constant (τm) for the blue LED pulse of either 0.1 s (closed diamonds) or 1 s (open circles). B, Reduction of threshold irradiance of the type-1 neurons with prolongation of the pulse duration. C, The latency to evoke an action potential was related to τm. D, The delayed firing of type-1 neurons with prolongation of the pulse duration. Statistical significance was evaluated with Mann-Whitney U test; *, P < 0.05 and **, P < 0.005.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4366085&req=5

pone.0119558.g006: Differential sensitivity to irradiance and duration among neurons.A, The threshold irradiance to evoke an action potential was related to the membrane time constant (τm) for the blue LED pulse of either 0.1 s (closed diamonds) or 1 s (open circles). B, Reduction of threshold irradiance of the type-1 neurons with prolongation of the pulse duration. C, The latency to evoke an action potential was related to τm. D, The delayed firing of type-1 neurons with prolongation of the pulse duration. Statistical significance was evaluated with Mann-Whitney U test; *, P < 0.05 and **, P < 0.005.
Mentions: As shown in Fig. 6A, which shows the relationship between the threshold irradiance and τm, the longer irradiation more effectively reduced the threshold irradiance in neurons with larger τm, but was less effective in neurons with smaller τm. For convenience, the cortical neurons were classified into two groups: type 1 (τm > 20 ms, 38 ± 4.7 ms, n = 11) and type 2 (τm < 20 ms, 12 ± 3.1 ms, n = 5) with the significant difference of τm (P < 0.001). Indeed, the threshold irradiance was reduced by the prolongation of the irradiation time in the case of type-1 neurons, whereas it was less dependent in the case of type-2 neurons (Fig. 6B). However, the reduction of the threshold irradiance was often accompanied by prolongation of the latency to evoke the first action potential in the case of type-1 neurons (Fig. 6C and 6D).

Bottom Line: The turning-on rate constant of each photocurrent followed a linear relationship to 0-0.12 mW mm(-2) of blue LED light or to 0-0.33 mW mm(-2) of cyan LED light.Each photocurrent of bi-stable ChR was shut off to the non-conducting state by yellow or orange LED light in a manner dependent on the irradiance.On the other hand, in another group of neurons, the threshold irradiance was not dependent on the irradiation time.

View Article: PubMed Central - PubMed

Affiliation: Department of Developmental Biology and Neuroscience, Tohoku University Graduate School of Life Sciences, Sendai, Japan; Core Research of Evolutional Science & Technology (CREST), Japan Science and Technology Agency (JST), Tokyo, Japan.

ABSTRACT
Channelrhodopsin-1 and 2 (ChR1 and ChR2) form cation channels that are gated by light through an unknown mechanism. We tested the DC-gate hypothesis that C167 and D195 are involved in the stabilization of the cation-permeable state of ChRWR/C1C2 which consists of TM1-5 of ChR1 and TM6-7 of ChR2 and ChRFR which consists of TM1-2 of ChR1 and TM3-7 of ChR2. The cation permeable state of each ChRWR and ChRFR was markedly prolonged in the order of several tens of seconds when either C167 or D195 position was mutated to alanine (A). Therefore, the DC-gate function was conserved among these chimeric ChRs. We next investigated the kinetic properties of the ON/OFF response of these bi-stable ChR mutants as they are important in designing the photostimulation protocols for the optogenetic manipulation of neuronal activities. The turning-on rate constant of each photocurrent followed a linear relationship to 0-0.12 mW mm(-2) of blue LED light or to 0-0.33 mW mm(-2) of cyan LED light. Each photocurrent of bi-stable ChR was shut off to the non-conducting state by yellow or orange LED light in a manner dependent on the irradiance. As the magnitude of the photocurrent was mostly determined by the turning-on rate constant and the irradiation time, the minimal irradiance that effectively evoked an action potential (threshold irradiance) was decreased with time only if the neuron, which expresses bi-stable ChRs, has a certain large membrane time constant (eg. τm > 20 ms). On the other hand, in another group of neurons, the threshold irradiance was not dependent on the irradiation time. Based on these quantitative data, we would propose that these bi-stable ChRs would be most suitable for enhancing the intrinsic activity of excitatory pyramidal neurons at a minimal magnitude of irradiance.

No MeSH data available.


Related in: MedlinePlus