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Characterization of Zebrafish Green Cone Photoresponse Recorded with Pressure-Polished Patch Pipettes, Yielding Efficient Intracellular Dialysis.

Aquila M, Benedusi M, Fasoli A, Rispoli G - PLoS ONE (2015)

Bottom Line: Sub-saturating flashes elicited responses in different cells with similar rising phase kinetics but with very different recovery kinetics, suggesting the existence of physiologically distinct cones having different Ca2+ dynamics.Theoretical considerations demonstrate that the different recovery kinetics can be modelled by simulating changes in the Ca2+-buffering capacity of the outer segment.Importantly, the Ca2+-buffer action preserves the fast response rising phase, when the Ca2+-dependent negative feedback is activated by the light-induced decline in intracellular Ca2+.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Science and Biotechnology, University of Ferrara, Ferrara, Italy.

ABSTRACT
The phototransduction enzymatic cascade in cones is less understood than in rods, and the zebrafish is an ideal model with which to investigate vertebrate and human vision. Therefore, here, for the first time, the zebrafish green cone photoresponse is characterized also to obtain a firm basis for evaluating how it is modulated by exogenous molecules. To this aim, a powerful method was developed to obtain long-lasting recordings with low access resistance, employing pressure-polished patch pipettes. This method also enabled fast, efficient delivery of molecules via a perfusion system coupled with pulled quartz or plastic perfusion tubes, inserted very close to the enlarged pipette tip. Sub-saturating flashes elicited responses in different cells with similar rising phase kinetics but with very different recovery kinetics, suggesting the existence of physiologically distinct cones having different Ca2+ dynamics. Theoretical considerations demonstrate that the different recovery kinetics can be modelled by simulating changes in the Ca2+-buffering capacity of the outer segment. Importantly, the Ca2+-buffer action preserves the fast response rising phase, when the Ca2+-dependent negative feedback is activated by the light-induced decline in intracellular Ca2+.

No MeSH data available.


Related in: MedlinePlus

Methods.A, The cytosolic perfusion was realized with a commercial instrument (a) that applied a positive pressure to the perfusion tube and a simultaneous depression in the standard side-port (c); perfusion tube was inserted in the pipette lumen (g and h; white scale bar is 20 μm) via a custom side-port drilled in the holder, (b), and filled with the intracellular solution containing the exogenous molecules. Alternatively, the positive pressure was applied with a precision syringe (d) connected to the perfusion tube by means of a three-way valve (red disk), whose piston was moved with a micromanipulator (not shown); in this case no depression was applied. The standard side port could also be connected, with two three-way valves, to a 50 ml syringe (e) to apply strong positive pressure to clean the pipette, or to a mouth piece (f), to attain the seal on the cone outer segment (g and B) or on its inner segment (h and C). B, fluorescence intensity vs time of lucifer yellow (40 μM, injected at time 0 in the outer segment, a; white scale bars are 10 μm; Ra~4.1 MΩ), integrated in the black, red, and green regions of a, right panel, normalized to the maximal intensity recorded in the site of dye injection (red region). Data (black, dark red and dark green curves in b) are fitted with a biexponential equation (red dashed line, τf = 0.6 s, Af = 0.29, τs = 19 s, As = 0.8) and with a monoexponential one (grey dashed line, τs = 17.5 s, As = 0.37; red dotted line, τs = 10 s, As = 1; green dashed line, τs = 13 s, As = 0.55). Fluorescence image after 50 s of lucifer yellow perfusion is shown in a, left panel. C, same experiment of A on a morphologically different green cone, with the patch pipette sealed on the inner segment (a; Ra~6.6 MΩ); data (same colour coding of A) are fitted with a biexponential equation (red dashed line, τf = 1.9 s, Af = 0.27, τs = 22 s, As = 0.31; green dashed line, τf = 1.3 s, Af = 0.5, τs = 20 s, As = 0.56) and with a monoexponential one (grey dashed line, τs = 23 s, As = 0.33).
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pone.0141727.g001: Methods.A, The cytosolic perfusion was realized with a commercial instrument (a) that applied a positive pressure to the perfusion tube and a simultaneous depression in the standard side-port (c); perfusion tube was inserted in the pipette lumen (g and h; white scale bar is 20 μm) via a custom side-port drilled in the holder, (b), and filled with the intracellular solution containing the exogenous molecules. Alternatively, the positive pressure was applied with a precision syringe (d) connected to the perfusion tube by means of a three-way valve (red disk), whose piston was moved with a micromanipulator (not shown); in this case no depression was applied. The standard side port could also be connected, with two three-way valves, to a 50 ml syringe (e) to apply strong positive pressure to clean the pipette, or to a mouth piece (f), to attain the seal on the cone outer segment (g and B) or on its inner segment (h and C). B, fluorescence intensity vs time of lucifer yellow (40 μM, injected at time 0 in the outer segment, a; white scale bars are 10 μm; Ra~4.1 MΩ), integrated in the black, red, and green regions of a, right panel, normalized to the maximal intensity recorded in the site of dye injection (red region). Data (black, dark red and dark green curves in b) are fitted with a biexponential equation (red dashed line, τf = 0.6 s, Af = 0.29, τs = 19 s, As = 0.8) and with a monoexponential one (grey dashed line, τs = 17.5 s, As = 0.37; red dotted line, τs = 10 s, As = 1; green dashed line, τs = 13 s, As = 0.55). Fluorescence image after 50 s of lucifer yellow perfusion is shown in a, left panel. C, same experiment of A on a morphologically different green cone, with the patch pipette sealed on the inner segment (a; Ra~6.6 MΩ); data (same colour coding of A) are fitted with a biexponential equation (red dashed line, τf = 1.9 s, Af = 0.27, τs = 22 s, As = 0.31; green dashed line, τf = 1.3 s, Af = 0.5, τs = 20 s, As = 0.56) and with a monoexponential one (grey dashed line, τs = 23 s, As = 0.33).

Mentions: Patch pipettes were filled with an intracellular solution containing (in mM): 40 KCl, 70 K-Asp, 5 MgCl2, 1 GTP, 5 ATP, 5 HEPES (buffered to pH = 7.6 with KOH; osmolality: 260 mM mOsm/Kg). All chemicals were purchased from Sigma (St. Louis, MO, USA). The intracellular dialysis of zebrafish cones was performed by using pressure-polished pipette and an intrapipette perfusion system (Fig 1A). Besides improving the electrical recordings, the enlarged shank of pressure-polished pipettes [28, 31] can accommodate pulled quartz or plastic perfusion tubes close to the pipette tip (Fig 1A), allowing the fast and controlled cytosolic incorporation of exogenous molecules. The perfusion tube was filled with an intracellular solution containing the molecules under study, which was injected in the cytosol during whole-cell recording by the controlled application of pressure to the capillary lumen. The pressure was delivered by a commercially available perfusion pressure/vacuum generator (2PK+, ALA scientific instruments, New York, New York; applied pressure: ~40 PSI (~280kPa)), or with a 1 ml precision syringe coupled to a micromanipulator (Fig 1A).


Characterization of Zebrafish Green Cone Photoresponse Recorded with Pressure-Polished Patch Pipettes, Yielding Efficient Intracellular Dialysis.

Aquila M, Benedusi M, Fasoli A, Rispoli G - PLoS ONE (2015)

Methods.A, The cytosolic perfusion was realized with a commercial instrument (a) that applied a positive pressure to the perfusion tube and a simultaneous depression in the standard side-port (c); perfusion tube was inserted in the pipette lumen (g and h; white scale bar is 20 μm) via a custom side-port drilled in the holder, (b), and filled with the intracellular solution containing the exogenous molecules. Alternatively, the positive pressure was applied with a precision syringe (d) connected to the perfusion tube by means of a three-way valve (red disk), whose piston was moved with a micromanipulator (not shown); in this case no depression was applied. The standard side port could also be connected, with two three-way valves, to a 50 ml syringe (e) to apply strong positive pressure to clean the pipette, or to a mouth piece (f), to attain the seal on the cone outer segment (g and B) or on its inner segment (h and C). B, fluorescence intensity vs time of lucifer yellow (40 μM, injected at time 0 in the outer segment, a; white scale bars are 10 μm; Ra~4.1 MΩ), integrated in the black, red, and green regions of a, right panel, normalized to the maximal intensity recorded in the site of dye injection (red region). Data (black, dark red and dark green curves in b) are fitted with a biexponential equation (red dashed line, τf = 0.6 s, Af = 0.29, τs = 19 s, As = 0.8) and with a monoexponential one (grey dashed line, τs = 17.5 s, As = 0.37; red dotted line, τs = 10 s, As = 1; green dashed line, τs = 13 s, As = 0.55). Fluorescence image after 50 s of lucifer yellow perfusion is shown in a, left panel. C, same experiment of A on a morphologically different green cone, with the patch pipette sealed on the inner segment (a; Ra~6.6 MΩ); data (same colour coding of A) are fitted with a biexponential equation (red dashed line, τf = 1.9 s, Af = 0.27, τs = 22 s, As = 0.31; green dashed line, τf = 1.3 s, Af = 0.5, τs = 20 s, As = 0.56) and with a monoexponential one (grey dashed line, τs = 23 s, As = 0.33).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4626105&req=5

pone.0141727.g001: Methods.A, The cytosolic perfusion was realized with a commercial instrument (a) that applied a positive pressure to the perfusion tube and a simultaneous depression in the standard side-port (c); perfusion tube was inserted in the pipette lumen (g and h; white scale bar is 20 μm) via a custom side-port drilled in the holder, (b), and filled with the intracellular solution containing the exogenous molecules. Alternatively, the positive pressure was applied with a precision syringe (d) connected to the perfusion tube by means of a three-way valve (red disk), whose piston was moved with a micromanipulator (not shown); in this case no depression was applied. The standard side port could also be connected, with two three-way valves, to a 50 ml syringe (e) to apply strong positive pressure to clean the pipette, or to a mouth piece (f), to attain the seal on the cone outer segment (g and B) or on its inner segment (h and C). B, fluorescence intensity vs time of lucifer yellow (40 μM, injected at time 0 in the outer segment, a; white scale bars are 10 μm; Ra~4.1 MΩ), integrated in the black, red, and green regions of a, right panel, normalized to the maximal intensity recorded in the site of dye injection (red region). Data (black, dark red and dark green curves in b) are fitted with a biexponential equation (red dashed line, τf = 0.6 s, Af = 0.29, τs = 19 s, As = 0.8) and with a monoexponential one (grey dashed line, τs = 17.5 s, As = 0.37; red dotted line, τs = 10 s, As = 1; green dashed line, τs = 13 s, As = 0.55). Fluorescence image after 50 s of lucifer yellow perfusion is shown in a, left panel. C, same experiment of A on a morphologically different green cone, with the patch pipette sealed on the inner segment (a; Ra~6.6 MΩ); data (same colour coding of A) are fitted with a biexponential equation (red dashed line, τf = 1.9 s, Af = 0.27, τs = 22 s, As = 0.31; green dashed line, τf = 1.3 s, Af = 0.5, τs = 20 s, As = 0.56) and with a monoexponential one (grey dashed line, τs = 23 s, As = 0.33).
Mentions: Patch pipettes were filled with an intracellular solution containing (in mM): 40 KCl, 70 K-Asp, 5 MgCl2, 1 GTP, 5 ATP, 5 HEPES (buffered to pH = 7.6 with KOH; osmolality: 260 mM mOsm/Kg). All chemicals were purchased from Sigma (St. Louis, MO, USA). The intracellular dialysis of zebrafish cones was performed by using pressure-polished pipette and an intrapipette perfusion system (Fig 1A). Besides improving the electrical recordings, the enlarged shank of pressure-polished pipettes [28, 31] can accommodate pulled quartz or plastic perfusion tubes close to the pipette tip (Fig 1A), allowing the fast and controlled cytosolic incorporation of exogenous molecules. The perfusion tube was filled with an intracellular solution containing the molecules under study, which was injected in the cytosol during whole-cell recording by the controlled application of pressure to the capillary lumen. The pressure was delivered by a commercially available perfusion pressure/vacuum generator (2PK+, ALA scientific instruments, New York, New York; applied pressure: ~40 PSI (~280kPa)), or with a 1 ml precision syringe coupled to a micromanipulator (Fig 1A).

Bottom Line: Sub-saturating flashes elicited responses in different cells with similar rising phase kinetics but with very different recovery kinetics, suggesting the existence of physiologically distinct cones having different Ca2+ dynamics.Theoretical considerations demonstrate that the different recovery kinetics can be modelled by simulating changes in the Ca2+-buffering capacity of the outer segment.Importantly, the Ca2+-buffer action preserves the fast response rising phase, when the Ca2+-dependent negative feedback is activated by the light-induced decline in intracellular Ca2+.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Science and Biotechnology, University of Ferrara, Ferrara, Italy.

ABSTRACT
The phototransduction enzymatic cascade in cones is less understood than in rods, and the zebrafish is an ideal model with which to investigate vertebrate and human vision. Therefore, here, for the first time, the zebrafish green cone photoresponse is characterized also to obtain a firm basis for evaluating how it is modulated by exogenous molecules. To this aim, a powerful method was developed to obtain long-lasting recordings with low access resistance, employing pressure-polished patch pipettes. This method also enabled fast, efficient delivery of molecules via a perfusion system coupled with pulled quartz or plastic perfusion tubes, inserted very close to the enlarged pipette tip. Sub-saturating flashes elicited responses in different cells with similar rising phase kinetics but with very different recovery kinetics, suggesting the existence of physiologically distinct cones having different Ca2+ dynamics. Theoretical considerations demonstrate that the different recovery kinetics can be modelled by simulating changes in the Ca2+-buffering capacity of the outer segment. Importantly, the Ca2+-buffer action preserves the fast response rising phase, when the Ca2+-dependent negative feedback is activated by the light-induced decline in intracellular Ca2+.

No MeSH data available.


Related in: MedlinePlus