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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

Flash response waveforms.The responses reported in panels A, B, and C of Fig 3 were averaged together and the corresponding six traces were aligned and normalized in panel A. To these, four other responses were added to flashes delivering 1.14·102 (9 responses averaged), 1.16·103, 9.55·104, and 1.85·105 photons/μm2. B, Response waveform had either a prominent dual component recovery (black traces, that are the responses to flashes of 2.32·103, 4.49·103, 8.68·103, and 1.77·104 photons/μm2 of panel A), or had a single kinetic component recovery (red traces, responses from a different cell to the same flash intensities), or a recovery in between these two types (thick blue trace). C, response amplitude vs light intensity (each data point, black dots and error bars, is the average of at least 75 responses to the same flash; n = 25). Hill equation fit (m = 1, I0 = 2·103 photons/μm2, blue trace; m = 1.4, I0 = 2.3·103 photons/μm2, green trace) and exponential saturation equation fit (I0 = 3·103 photons/μm2, red trace) to the data points. D, black, green, red, and blue traces are the responses (of panel A) to flashes delivering 1.77·104, 9.55·104, 1.85·105, and 3.76·105 photons/μm2, respectively, but unsmoothed; yellow trace is the linear fit to the rising phase of the two fastest responses (evoked by flashes delivering 1.85·105 and 3.76·105 photons/μm2) having a slope of 64 s-1 (correlation coefficient: 0.97); flash timing is the dark purple trace.
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pone.0141727.g004: Flash response waveforms.The responses reported in panels A, B, and C of Fig 3 were averaged together and the corresponding six traces were aligned and normalized in panel A. To these, four other responses were added to flashes delivering 1.14·102 (9 responses averaged), 1.16·103, 9.55·104, and 1.85·105 photons/μm2. B, Response waveform had either a prominent dual component recovery (black traces, that are the responses to flashes of 2.32·103, 4.49·103, 8.68·103, and 1.77·104 photons/μm2 of panel A), or had a single kinetic component recovery (red traces, responses from a different cell to the same flash intensities), or a recovery in between these two types (thick blue trace). C, response amplitude vs light intensity (each data point, black dots and error bars, is the average of at least 75 responses to the same flash; n = 25). Hill equation fit (m = 1, I0 = 2·103 photons/μm2, blue trace; m = 1.4, I0 = 2.3·103 photons/μm2, green trace) and exponential saturation equation fit (I0 = 3·103 photons/μm2, red trace) to the data points. D, black, green, red, and blue traces are the responses (of panel A) to flashes delivering 1.77·104, 9.55·104, 1.85·105, and 3.76·105 photons/μm2, respectively, but unsmoothed; yellow trace is the linear fit to the rising phase of the two fastest responses (evoked by flashes delivering 1.85·105 and 3.76·105 photons/μm2) having a slope of 64 s-1 (correlation coefficient: 0.97); flash timing is the dark purple trace.

Mentions: Since the image resolution of the bright field microscope used could not unambiguously ascertain the morphology of small cells, it seemed likely that some recording might instead be derived from the red cone counterpart of a double red and green cone [33]. To ensure that the recordings presented here were from green cones only, red and green stimuli were routinely delivered (Fig 2A and 2B). As a rationale, a cone is considered green sensitive if its response to a green (525 nm) flash 1.14·103 photons/μm2 in intensity suppressed ~40% of the dark current (average suppression: 0.38±0.03, 334 flashes averaged in 25 cells) but it did not respond to a red flash (627.5 nm, Fig 2A and 2B) of same intensity. A typical recording using pressure-polished pipettes, lasting more than 20 min, is illustrated in Fig 2A on a very slow time scale: light flashes of increasing intensity (range: 1.14·102 to 3.76·106 photons/μm2), were delivered in triplets in repeated sequences. The small drift in the baseline was due to small changes in the current flowing through the inner segment channels rather than in changes in the light sensitive (or dark) current flowing through the cGMP channels, because the amount of current suppressed by saturating flashes (with intensities ≥1.8·104 photons/μm2) was the same (average: 18.7±1.8 pA; n = 25) from the beginning to the end of these recordings. Responses to the same flash intensities were averaged and smoothed (see below and Methods): they were reproducible over a recording time of up to more than 20 min. This reproducibility is exemplified by the recording of Fig 3A–3C: consecutive flashes of the same intensities, delivered within ~2, ~5 ~10 and ~15 min from the beginning of whole-cell recording, did not reveal any significant differences with respect to dark current amplitude and response kinetics. Therefore these responses were all averaged, aligned, and normalized as shown in Fig 4A and 4B. Steps of light (1.8·105 photons/(μm2·sec), lasting 8 s) were occasionally delivered during this recording (Fig 2A, 2C and 2D) at the following times: 227 s (black trace), 285 s (green), 583 s (red), 977 s (blue), 1100 s (pink), and 1136 s (cyan). These steps gave a constant fractional suppression of 83% (averaging the response between 2.5 and 6 s, where current was most stable) for at least 5 min of recording, and declined to 69% (Fig 2C) after 20 min, but after having delivered repetitive supersaturating flashes (Figs 2A, 4A and 4D). Light sensitive current declined from 20.6 to 19.8 pA during this 20 min recording, i.e. 3.9%, while sensitivity in the same period declined of 17.3%. The decline in sensitivity and dark current was in general not larger than 5% and 10%, respectively, in the first 12.5±1.4 min (n = 20; range: 4–23 min) of recording. Responses in Fig 4A recovered from the maximum amplitude with a characteristic waveform, consisting of dual kinetic components, that was particularly evident for flash intensities in the range 2·103 to 104 photons/μm2. This feature was observed clearly in ~40% of the recordings (n = 25), while others had a faster recovery with a single kinetic component, (Fig 4B, red traces, compared with the corresponding four responses with a dual component recovery, in black, on an expanded time scale). In fact, a continuum of behavior in between these two recovery types (as the thick blue trace, that is the blue trace of Fig 5C) was observed.


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)

Flash response waveforms.The responses reported in panels A, B, and C of Fig 3 were averaged together and the corresponding six traces were aligned and normalized in panel A. To these, four other responses were added to flashes delivering 1.14·102 (9 responses averaged), 1.16·103, 9.55·104, and 1.85·105 photons/μm2. B, Response waveform had either a prominent dual component recovery (black traces, that are the responses to flashes of 2.32·103, 4.49·103, 8.68·103, and 1.77·104 photons/μm2 of panel A), or had a single kinetic component recovery (red traces, responses from a different cell to the same flash intensities), or a recovery in between these two types (thick blue trace). C, response amplitude vs light intensity (each data point, black dots and error bars, is the average of at least 75 responses to the same flash; n = 25). Hill equation fit (m = 1, I0 = 2·103 photons/μm2, blue trace; m = 1.4, I0 = 2.3·103 photons/μm2, green trace) and exponential saturation equation fit (I0 = 3·103 photons/μm2, red trace) to the data points. D, black, green, red, and blue traces are the responses (of panel A) to flashes delivering 1.77·104, 9.55·104, 1.85·105, and 3.76·105 photons/μm2, respectively, but unsmoothed; yellow trace is the linear fit to the rising phase of the two fastest responses (evoked by flashes delivering 1.85·105 and 3.76·105 photons/μm2) having a slope of 64 s-1 (correlation coefficient: 0.97); flash timing is the dark purple trace.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4626105&req=5

pone.0141727.g004: Flash response waveforms.The responses reported in panels A, B, and C of Fig 3 were averaged together and the corresponding six traces were aligned and normalized in panel A. To these, four other responses were added to flashes delivering 1.14·102 (9 responses averaged), 1.16·103, 9.55·104, and 1.85·105 photons/μm2. B, Response waveform had either a prominent dual component recovery (black traces, that are the responses to flashes of 2.32·103, 4.49·103, 8.68·103, and 1.77·104 photons/μm2 of panel A), or had a single kinetic component recovery (red traces, responses from a different cell to the same flash intensities), or a recovery in between these two types (thick blue trace). C, response amplitude vs light intensity (each data point, black dots and error bars, is the average of at least 75 responses to the same flash; n = 25). Hill equation fit (m = 1, I0 = 2·103 photons/μm2, blue trace; m = 1.4, I0 = 2.3·103 photons/μm2, green trace) and exponential saturation equation fit (I0 = 3·103 photons/μm2, red trace) to the data points. D, black, green, red, and blue traces are the responses (of panel A) to flashes delivering 1.77·104, 9.55·104, 1.85·105, and 3.76·105 photons/μm2, respectively, but unsmoothed; yellow trace is the linear fit to the rising phase of the two fastest responses (evoked by flashes delivering 1.85·105 and 3.76·105 photons/μm2) having a slope of 64 s-1 (correlation coefficient: 0.97); flash timing is the dark purple trace.
Mentions: Since the image resolution of the bright field microscope used could not unambiguously ascertain the morphology of small cells, it seemed likely that some recording might instead be derived from the red cone counterpart of a double red and green cone [33]. To ensure that the recordings presented here were from green cones only, red and green stimuli were routinely delivered (Fig 2A and 2B). As a rationale, a cone is considered green sensitive if its response to a green (525 nm) flash 1.14·103 photons/μm2 in intensity suppressed ~40% of the dark current (average suppression: 0.38±0.03, 334 flashes averaged in 25 cells) but it did not respond to a red flash (627.5 nm, Fig 2A and 2B) of same intensity. A typical recording using pressure-polished pipettes, lasting more than 20 min, is illustrated in Fig 2A on a very slow time scale: light flashes of increasing intensity (range: 1.14·102 to 3.76·106 photons/μm2), were delivered in triplets in repeated sequences. The small drift in the baseline was due to small changes in the current flowing through the inner segment channels rather than in changes in the light sensitive (or dark) current flowing through the cGMP channels, because the amount of current suppressed by saturating flashes (with intensities ≥1.8·104 photons/μm2) was the same (average: 18.7±1.8 pA; n = 25) from the beginning to the end of these recordings. Responses to the same flash intensities were averaged and smoothed (see below and Methods): they were reproducible over a recording time of up to more than 20 min. This reproducibility is exemplified by the recording of Fig 3A–3C: consecutive flashes of the same intensities, delivered within ~2, ~5 ~10 and ~15 min from the beginning of whole-cell recording, did not reveal any significant differences with respect to dark current amplitude and response kinetics. Therefore these responses were all averaged, aligned, and normalized as shown in Fig 4A and 4B. Steps of light (1.8·105 photons/(μm2·sec), lasting 8 s) were occasionally delivered during this recording (Fig 2A, 2C and 2D) at the following times: 227 s (black trace), 285 s (green), 583 s (red), 977 s (blue), 1100 s (pink), and 1136 s (cyan). These steps gave a constant fractional suppression of 83% (averaging the response between 2.5 and 6 s, where current was most stable) for at least 5 min of recording, and declined to 69% (Fig 2C) after 20 min, but after having delivered repetitive supersaturating flashes (Figs 2A, 4A and 4D). Light sensitive current declined from 20.6 to 19.8 pA during this 20 min recording, i.e. 3.9%, while sensitivity in the same period declined of 17.3%. The decline in sensitivity and dark current was in general not larger than 5% and 10%, respectively, in the first 12.5±1.4 min (n = 20; range: 4–23 min) of recording. Responses in Fig 4A recovered from the maximum amplitude with a characteristic waveform, consisting of dual kinetic components, that was particularly evident for flash intensities in the range 2·103 to 104 photons/μm2. This feature was observed clearly in ~40% of the recordings (n = 25), while others had a faster recovery with a single kinetic component, (Fig 4B, red traces, compared with the corresponding four responses with a dual component recovery, in black, on an expanded time scale). In fact, a continuum of behavior in between these two recovery types (as the thick blue trace, that is the blue trace of Fig 5C) was observed.

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