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1.
Figure 4.

Figure 4. From: Physiological Features of the S- and M-cone Photoreceptors of Wild-type Mice from Single-cell Recordings.

Dim-flash responses of cone photoreceptors of WT and Gtα−/− mice. Cones were classified as S-cones or M-cones according to Fig. 3 B. (A) Dim-flash responses to 361-nm flashes of individual S-cones (gray noisy traces) and their average (purple trace). (B) Dim-flash responses of individual M-cones to 510-nm flashes (gray traces) and their average (green trace). (C) Average dim-flash response of S-cones to 361-nm flashes (purple trace, repeated from A) and to 510-nm flashes (thinner, green trace). (D) Average dim-flash response of M-cones to 510-nm flashes (green trace, repeated from B) and to 361-nm flashes (thinner, purple trace). (E) Comparison of the average response of S-cones to 361-nm flashes (purple trace) and M-cones to 510-nm flashes (green trace). (F) Comparison of the grand average dim-flash responses to 361-nm flashes of WT S-cones (purple trace), Gtα−/− S-cones (blue), Nrl−/− cones (cyan, n = 7, recorded in the “OS-out” configuration), and 26 rods (gray trace) recorded under the same conditions (Nikonov et al., 2005). Each trace is scaled to unity at its peak.

Sergei S. Nikonov, et al. J Gen Physiol. 2006 April;127(4):359-374.
2.
Figure 7.

Figure 7. From: Physiological Features of the S- and M-cone Photoreceptors of Wild-type Mice from Single-cell Recordings.

Bleached S- and M-cone opsins and rhodopsin persistently activate phototransduction to different degrees. (A) Saturating responses of a rod, S-cone, and M-cone before (black trace) and after (colored traces; a, b, c) light exposures that bleached substantial fractions of their respective visual pigment. The fractions of the pigments bleached at the time of the second response were calculated with Eq. 3, and were 60% (rhodopsin in rod), 53% (S-opsin in S-cone), and 99% (M-opsin in M-cone). (B) Normalized circulating current as a function of the fraction p(T) (Eq. 3) of the total opsin in the unbleached state at the time T when a saturating flash was delivered to WT S-cones (purple symbols), M-cones (green symbols), and rods (gray symbols). The absolute amplitudes of the initial responses (black traces) in the top panels were 40 pA (Rod), 5.0 pA (S-cone), and 12.5 pA (M-cones). (The rod response likely arose from two nuclei in the suction pipette, as confirmed single rods were rarely found to yield >25 pA.) The smaller amplitude traces in the top panels are labeled with letters, a, b, c; these letters have been placed next to the points in B to which they correspond.

Sergei S. Nikonov, et al. J Gen Physiol. 2006 April;127(4):359-374.
3.
Figure 1.

Figure 1. From: Physiological Features of the S- and M-cone Photoreceptors of Wild-type Mice from Single-cell Recordings.

Isolation of WT cone responses. (A) Combined infra-red and fluorescence images of a retinal slice of a mouse expressing EGFP under the human LWS/MWS cone promoter (Fei and Hughes, 2001), as seen in the recording chamber. (B) Fluorescence image alone, revealing details of the cone in A. (C) Infra-red image of another slice illustrating method of drawing several “inner segments” into the suction pipette. (D) Recordings from experiment, as illustrated in C: averaged step responses to 4 different intensities of 500-nm light with superimposed 361-nm flash. At 1 s after step onset, the flash was presented; the step was terminated 2 s later; this timing of the background step and flash were used in all experiments. (E) Amplitudes of step responses from D (colored symbols correspond to colored traces), renormalized by the amplitude of the step response to the most intense step (red trace). Gray symbols are data from individual rods. The smooth curve plots Eq. 6 with I1/2 = 350 photons μm2s−1.

Sergei S. Nikonov, et al. J Gen Physiol. 2006 April;127(4):359-374.
4.
Figure 5.

Figure 5. From: Physiological Features of the S- and M-cone Photoreceptors of Wild-type Mice from Single-cell Recordings.

Effect of M-opsin expression level on kinetic features of the dim-flash response. (A) Time to peak of the dim-flash response (c.f., Fig. 4, Table I) plotted as a function of the estimated M-opsin coexpression ratio. (B) Flash sensitivity (% saturating response per photon μm−2). The M-opsin coexpression ratio ρ was estimated as the relative sensitivity to 501- and 361-nm flashes, i.e., for each cone ρ = S501/S361 (compare Fig. 3) was measured. Each point plotted without error bars represents data of a single cone: for S-cones (red filled circles) ρ < 1; for M-cones (green filled circles) ρ > 1; error bars are 95% confidence intervals for pooled data (data for several S-cones with ρ ≤ 0.001 were pooled and plotted at ρ ≈ 0.001; the points in the M-cone sector with bidirectional error bars provide the mean and 95% confidence intervals for the entire M-cone populations). The gray bars plot 95% confidence region for the time to peak (tpeak) and dim-flash sensitivity of Gtα−/− cones; since there were no trends in these cones, their results were pooled (see Table I). The smooth (gray) curve in A plots an empirical relation, tpeak (ρ) = tpeak,dark − Δtpeakn/ ], with tpeak,dark = 96 ms, Δtpeak = 36 ms, n = 2, ρ0 = 0.048; 96 ms is the overall mean tpeak for Gtα−/− cones, while Δtpeak was selected so the curve would run through tpeak for the M-cones. The gray curve in B plots the upper envelope of Eqs. 7a and 7b; the extension of the individual curves for S361 and S501 are shown as dashed lines. Eq. 7 has two parameters SF,dark and Idark*; we set SF,dark = 0.034, close to the average value for Gtα−/− cones (Table I), and Idark* = 1200 s−1.

Sergei S. Nikonov, et al. J Gen Physiol. 2006 April;127(4):359-374.
5.
Figure 6.

Figure 6. From: Physiological Features of the S- and M-cone Photoreceptors of Wild-type Mice from Single-cell Recordings.

Responses of WT and Gtα−/− mouse cone photoreceptors to steps of light of graded intensity. A, C, E, and G present the responses of cones of mice of the genotype indicated on the figure to steps of light, while B, D, F, and H present the response amplitude vs. step intensity relation for the same cone. Thus, each point plotted in the righthand panels corresponds to the average amplitude of the step response in the portion of the plot at left illustrated on a gray background. For the S-cones (A, B; E, F), steps of 361-nm light were used; for the M-cones (C, D; G, H), steps of 501-nm light. The timing of the delivery steps and flashes in experiments with WT cones is illustrated in Fig. 1 D; in these experiments, the steps also suppressed rod activity, but as the initial re sponse to the step need not reflect cone activity alone, it is omitted. In the experiments with Gtα−/− retinas there was no rod activity, and so the initial response to the step reflects the time course of the cone response (note the different time scales in A, C vs. E, F). To accurately determine the fractional response (i.e., the fraction of the cone's circulating current suppressed by the background), a very strong flash was delivered in each cycle of presentation of the steps: the data in A, C, E, and G are aligned with respect to this flash (t = 0). The response vs. intensity data were fitted with a hyperbolic saturation relation (Eq. 6); the fitted curve (black smooth trace) and the estimated intensity I1/2 of the step that produces a response of half-maximal amplitude in each case are given on the panels to the right of the data. Each response illustrated corresponds to between 10 and 20 repetitions of the light step, and the traces are the average, normalized by the response to the saturating flash presented in the presence of the dimmest step (the standard background) (A and C) or presented in darkness (E and G). For the WT cones, the averaged response to the saturating flash in the presence of the standard background after the series of step presentations is shown as the cyan trace. The gray symbols replot the response amplitude data on an intensity axis adjusted for the decrease in collecting area due to the depletion (bleaching) of the cone pigment by light stimuli presented before the steps were delivered, calculated by applying Eq. 3 to the data. The hyperbolic saturation relations fitted to these “bleach-corrected” data are shown as the smooth gray traces, with I1/2 given to the left of the data and smooth curve.

Sergei S. Nikonov, et al. J Gen Physiol. 2006 April;127(4):359-374.
6.
Figure 3.

Figure 3. From: Physiological Features of the S- and M-cone Photoreceptors of Wild-type Mice from Single-cell Recordings.

Spectral properties of WT and Gtα−/− mouse cone photoreceptors. (A) Spectral sensitivities. Data of all cones have been normalized at either 361 or 501 nm, depending on the wavelength of maximal sensitivity. Purple filled circles with error bars plot sensitivity results of a single WT cone: the data are fitted with the sum of two pigment templates (Lamb, 1995), the mouse cone S-opsin (λmax = 360 nm, smooth purple curve) and the mouse cone M-opsin (λmax = 508 nm), with the latter scaled by 0.039 (lower smooth green curve). The upper green curve is the same template, normalized; the dotted portion of the curve is a possible extension of the template (Govardovskii et al., 2000). For other cones, results from only 361 and 501 nm are plotted. Data of WT mice are plotted as circles, and data from Gtα−/− mice as triangles. Data from cones in retinal slices dissected from the most ventral portion of the retina (see MATERIALS AND METHODS) were invariably maximally sensitive at 361 nm and are plotted as purple filled circles, while data from cones in retinal slices from the most dorsal portion of the retina were invariably more sensitive at 501 nm and are plotted as green filled circles. Data obtained from cones in retinal slices of unknown location are colored according to the wavelength of maximal sensitivity 361 (purple) or 501 nm (green), but are shown with embedded white cross-hairs. One cone maximally sensitive at 361 nm had a sensitivity at 501 nm that matched the template (arrow), and thus is inferred to express only S-opsin. (B) Classification of WT, Gtα−/−, and Nrl−/− cones according to their relative sensitivities at 361 and 501 nm. Each point plots the absolute sensitivity of one cone at 501 nm (ordinate) vs.its absolute sensitivity at 361 nm, with sensitivity expressed in percentage of circulating current suppressed per photon μm−2. The same symbol scheme is used as in A; data from Nrl−/− cones recorded in the “OS-out” configuration (Nikonov et al., 2005) are plotted as filled blue circles. The diagonal (unity slope line) plots the locus of cones that would be equally sensitive to 361- and 501-nm light; cones that plot above this line are considered “M-cones” and those lying below it “S-cones.” The dashed line plots a locus defined by S361 + S501 = 0.03, which would describe the data if total cone opsin expression were conserved and both opsins drove phototransduction with equal efficiency. The data of the one “pure S-cone” is again identified by an arrow. (The gray hatched bars have been added to emphasize the nearly 2 log10 unit break in the axes.)

Sergei S. Nikonov, et al. J Gen Physiol. 2006 April;127(4):359-374.
7.
Figure 2.

Figure 2. From: Physiological Features of the S- and M-cone Photoreceptors of Wild-type Mice from Single-cell Recordings.

Kinetics and amplification of WT and Gtα−/− mouse cone photoreceptors. Each row of three panels presents results obtained from a single mouse cone. The first column of panels presents families of light responses to a series of 20 μs (A, D, G) or 7 ms (J) flashes of graduated intensity. The second column of figure (B, E, H, and K) replots three to five of the traces in the first column on an expanded time base, but with the same vertical scaling; in these panels the amplification constant, A, of the responses is extracted by fitting the “LP” model of phototransduction (MATERIALS AND METHODS, Eq. 4) (thickened gray traces) to the rising phase of the responses (thinner black traces). As all cone responses exhibited a “nose” current (which decays rapidly after the peak of the responses to the most intense flashes), for the LP analysis the data were renormalized at the level of the dotted line, corresponding to 80% of the full response amplitude. The analyses in the third column of panels (C, F, I, and L) extract two additional kinetic parameters characterizing the response families: the half-saturating flash intensity Q1/2 (photons μm−2), obtained by fitting a hyperbolic saturation function (Eq. 5) to the response amplitude vs. intensity data (open circles, left ordinate)and the dominant recovery time constant, τD (ms), obtained from a “Pepperberg” analysis applied to the recovery times of the first three saturating responses (spanning ∼1 log10 unit of intensity) of each response family (filled circles, right ordinate). The data in the first two rows of panels (A, B, C; D, E, F) were obtained from retinal slices of WT mice in the presence of a rod-saturating background, while those in the third and fourth rows (G, H, I; J, K, L) were obtained from slices of Gtα−/− in the absence of the background. The data in the first and third rows were obtained from cones maximally sensitive at ∼360 nm (“S-cones”) and were recorded in response to 361-nm flashes, while those in the second and fourth rows were from cones maximally sensitive at ∼510 nm (“M-cones”) and were recorded in response to 501-nm flashes. All responses were filtered during acquisition with an 8-pole low pass analogue filter set at 20 Hz and digitized at 200 Hz. At least 15 responses to the same flash intensity were averaged for each trace, and at least 30 for responses to the dimmest flashes. The saturating response amplitudes were 6 pA (A), 15 pA (D), 11 pA (G), and 4 pA (J).

Sergei S. Nikonov, et al. J Gen Physiol. 2006 April;127(4):359-374.

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