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The Journal of Neuroscience, January 1, 1999, 19(1):442-455
UV- and Midwave-Sensitive Cone-Driven Retinal Responses of the
Mouse: A Possible Phenotype for Coexpression of Cone
Photopigments
A. L.
Lyubarsky1, 2,
B.
Falsini3,
M. E.
Pennesi1, 2,
P.
Valentini3, and
E. N.
Pugh Jr1, 2
1 Department of Psychology and 2 Institute
of Neurological Sciences, University of Pennsylvania, Philadelphia,
Pennsylvania 19104-6196, and 3 Institute of Ophthalmology,
Catholic University, Rome, Italy 00168
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ABSTRACT |
Molecular biological, histological and flicker electroretinographic
results have established that mice have two cone photopigments, one
peaking near 350 nm (UV-cone pigment) and a second near 510 nm
[midwave (M)-cone pigment]. The goal of this investigation was
to measure the action spectra and absolute sensitivities of the
UV-cone- and M-cone-driven b-wave responses of C57BL/6 mice. To achieve
this goal, we suppressed rod-driven signals with steady or flashed
backgrounds and obtained intensity-response relations for cone-driven
b-waves elicited by narrowband flashes between 340 and 600 nm. The
derived cone action spectra can be described as retinal1
pigments with peaks at 355 and 508 nm. The UV peak had an absolute
sensitivity of ~8 nV/(photon µm2) at the cornea,
approximately fourfold higher than the M peak. In an attempt to isolate
UV-cone-driven responses, it was discovered that an orange conditioning
flash ( > 530 nm) completely suppressed ERG signals driven by both
M pigment- and UV pigment-containing cones. Analysis showed that the
orange flash could not have produced a detectable response in the
UV-cone pathway were their no linkage between M pigment- and UV
pigment-generated signals. Because cones containing predominantly the
UV and M pigments have been shown to be located largely in separate
parts of the mouse retina (Szel et al., 1992 ), the most probable
linkage is coexpression of M pigment in cones primarily expressing UV
pigment. New histological evidence supports this interpretation
(Gloesman and Ahnelt, 1998). Our data are consistent with an upper
bound of ~3% coexpression of M pigment in the cones that express
mostly the UV pigment.
Key words:
cone photoreceptors; cones; cone pigments; murine retina; retinal sensitivity; electroretinogram; retinal circuitry; gene
coexpression
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INTRODUCTION |
The mouse has become an increasingly
important preparation for the investigation of retinal function and
retinal disease. This importance comes from general use of the mouse as
a model system for mammalian gene mapping and manipulation, and from
the specific development of lines of mice having transgenes for
retinally expressed proteins (Lem and Makino, 1996). Because
rodents such as the mouse have traditionally been characterized as
having "rod-dominated" retinas, and because signals originating in
cone photoreceptors are so important in human vision, concern has been
expressed that the mouse may be an inadequate model system for
investigation of the retinal processing of cone signals. That mice have
cones is certainly incontrovertible.
Carter-Dawson and LaVail (1979a,b) performed a definitive histological
assessment of the photoreceptor types of the mouse retina and estimated
that cones constitute 3-3.5% of the total number of photoreceptors.
Subsequent studies using flicker ERG photometry under cone-isolation
conditions found that the mouse retina has two distinct spectral peaks
of sensitivity, one at 360-365 nm and a second at 511 nm (Jacobs et
al., 1991; Deegan and Jacobs, 1993). Experiments with regional flicker
stimulation later confirmed the UV sensitivity to be higher in ventral
retina and responsiveness to green light to be localized mostly in its dorsal part (Calderone and Jacobs, 1995).
Immunocytochemical studies also provided evidence of two
distinguishable cone types in the murine retina (Szel et al., 1992). One type was labeled by a monoclonal antibody specific to the midwave-
to long-wave-sensitive visual pigment of the mammals, whereas the other
type was stained by a short-wave (S) cone-specific monoclonal antibody.
The same study provided the first evidence that the
midwave-sensitive cones are located exclusively in the dorsal
half of the mouse retina, whereas the overwhelming majority of the
short-wave-sensitive cones occupy the ventral half (Szel et al.,
1992).
Molecular biological screening has also confirmed the existence and
expression of two distinct cone pigments in mouse, and sequence and
spectral analyses have shown one of these to be a member of the
"LW" cone photopigment family [of which the human L- and
midwave (M) cone pigments are members] and the second to be a member
of the "S" cone photopigment family (of which the human
S-cone pigment is a member) (Chiu et al., 1994; Sun et al., 1997). The S-cone family contains members whose peak sensitivity ranges
from 350 to 450 nm or higher (Chiu et al., 1994).
In summary, three bodies of evidence electroretinographic,
histochemical, and molecular biologicalhave converged in showing the
mouse to have cones of two distinct classes and thus in providing support for use of the mouse as a model for the investigation of
signals originating in cones. We undertook this investigation to
isolate ERG responses driven by murine UV- and midwave-sensitive cones,
and to characterize more fully than before the absolute flash
sensitivities, the spectral sensitivities, and the magnitudes and
kinetics of these responses.
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MATERIALS AND METHODS |
Animal maintenance and light regimen. All
experimental procedures were done in compliance with National
Institutes of Health guidelines as approved by the University of
Pennsylvania's Institutional Animal Care and Use Committee. Animals
used in the study were born and maintained under strictly controlled
ambient illumination throughout their lives. Breeding pairs were set in
light-tight, ventilated enclosures lit from inside by four 25 W
tungsten lamps situated behind a diffusing screen; the lamps were
powered by a regulated power supply and controlled by a timer. The
interior of the containers was painted with a flat-white epoxy paint,
with the result that interior illumination was practically isotropic. Animals were maintained on a 12 hr light/dark cycle, with the illumination level at 2.5 photopic lux ( 7.5 scotopic lux). In a
ganzfeld such illumination has been estimated to produce approximately 650 photoisomerizations/sec in the rods of an adult mouse with a fully
dilated pupil, but no more than 100 photoisomerizations/sec in the rods
of an adult mouse with the pupil fully constricted (Pennesi et al.,
1998). Servicing of the mice was performed three times a week,
exposing them to 7-10 lux illumination for 20-25 min. These
precautions about illumination rearing conditions were taken to obviate
any potential retinal damage from stronger light exposures and also in
an effort to minimize variability between animals.
ERG recording. On the day preceding the experiment animals
were removed from the containers and placed into a completely darkened light-tight ventilated enclosure. By the time of the experiment the
animals had spent 12-20 hr in dark. All of the preparations for
recordings were performed under dim red light. The animals were
anesthetized with an intraperitoneal injection of a solution containing
(in µg/g body weight): 25 ketamine, 10 xylazine, and 1000 urethane.
The average length of time under anesthesia was 1.5 hr, with a range of
1-2 hr. The pupil was dilated with 1% tropicamide saline solution
(Mydriacil; Alconox, New York, NY), and the animal was immobilized in a
holder. A drop of methylcellulose solution (Goniosol; Iolab
Pharmaceutical, Indianapolis, IN) was placed on the eye for protection
and electrical contact, and a recording platinum wire electrode was put
into electrical contact with the cornea while a needle tungsten
reference electrode was inserted subcutaneously on the forehead. After
this the animal was placed into a light-proof Faraday cage and kept in
absolute darkness for 10 min before recordings commenced. The
temperature inside the cage was maintained at 27 ± 1°C. Signals
were amplified by a BMA-200 differential amplifier (GWE Inc., Ardmore,
PA), bandpass-filtered at 0.1-1000 Hz (two-pole Butterworth filter),
sampled at 5 kHz, and stored on the hard drive of a personal computer
using Digidata 1200 acquisition hardware and Axotape2 software (Axon
Instruments, Foster City, CA).
Light stimulation. The Faraday cage in which the recordings
were made was an 8 inch × 8 inch × 14 inch aluminum chamber
whose interior was completely covered with aluminum foil, serving to create a ganzfeld (described further below). The use of aluminum foil
as an interior cover was dictated by the need to minimize the UV
absorbance of the chamber walls, which we found to occur to an
unacceptable degree with commercially available paints. A custom animal
holder and support allowed the mouse to be placed at almost any
location inside the chamber. Thus, the ERGs of either eye could be
recorded, though in the majority of experiments only the right eye was
used. In all of the experiments reported here the holder was adjusted
to keep the optic axis of the eye from which the ERG was being recorded
vertical, at a height about two-thirds above the chamber floor and in
the center of its horizontal cross section.
Light stimuli were delivered through several ports in the walls and
ceiling of the box. Special care was taken to avoid any possibility of
direct illumination of the tested eye from any of these openings. To
achieve this the ceiling ports were protected by foil-wrapped baffles
of appropriate size, and the wall ports were located at the bottom of
the chamber, much lower than the animal holder, which served as a light baffle.
The directional homogeneity of the light field at the location of the
mouse's pupil was checked as follows. A PIN-10 photodiode (UDT,
Waltham, MA) with a custom-made accessory that limited light collection
to an angular subtense of ~2° [for an explanatory drawing, see
Lyubarsky and Pugh (1996), their Fig. 1] was placed at the position occupied by the mouse eye during experiments, and the intensity of light from different directions was measured. For an
angular subtense of 120° about the mouse's optic axis, variations of
light intensity did not exceed ±15% of the average intensity, regardless of the port used to deliver stimulation.
The optical stimulator allowed considerable freedom in the choice of
light sources and protocols of stimulation. In this study we used three
independently powered and triggered flash illuminators built around
U-8538 xenon flash tubes (Mouser Electronics, Randolph, NJ) and one
steady light illuminator. The flash illuminators were powered by
switchable capacitor banks (up to 1000 µF) charged to 450 V, and they
provided up to 100 joules/flash to the lamp. These flashlamps
discharged >85% of their light output in 1 msec. The steady
illuminator used a 12V/50W halogen lamp (HLX 64610, Osram, Berlin,
Germany) powered by a stabilized DC supply; steady illumination was
under the control of a shutter (Uniblitz; Vincent Associates,
Rochester, NY). The illuminators delivered their light directly to the
appropriate ports. The intensity and spectral composition of
illumination were controlled by quartz neutral density, color glass,
and bandwidth interference filters (Ealing Electrooptics, Holliston,
MA) introduced into appropriate places in the beams. The transmission
spectra of all filters over the spectral range 300-700 nm were
measured with a Lambda 20 spectrophotometer (Perkin-Elmer, Norwalk,
CT). Flashes were triggered by a computer with a digital input/output
board (Computer Boards, Mansfield, MA).
Overview of light calibrations, filter spectral transmission
measurements, and estimation of photoisomerization numbers and rates in
rods and cones. Although light calibration is normally straightforward, some requirements of these experiments called for
unexpectedly difficult calibration measures. One such requirement was
that of working with UV stimuli in a ganzfeld. Another was the need to
maximize stimulation intensity in the recording chamber while keeping
stimuli as narrowband as possible. The latter requirement led us to use
noncollimated beams, which broadened and shifted the interference
filter transmission characteristics from the manufacturer's
specifications and forced us to measure the transmission characteristics in a flash beam geometry matching that used in the
experiments. Finally, because of the goal of characterizing the
sensitivity of cone-driven responses in absolute and physiologically meaningful units, we have laid out in detail the steps used to estimate
the numbers and rates of photoisomerizations in rods and cones produced
by the light stimuli. The reader who is not interested in such detail
may prefer to skip to the final section of Materials and Methods.
Measurement and quantification of light stimuli: basic
method. Light from each of the optical stimulators was calibrated
as follows. A factory-calibrated silicon photodiode (PIN-10 or
PIN-5DP/DB, UDT) was placed with working surface up at the position
normally occupied by the mouse's eye during an experiment. Narrowband
interference filters (full width, half-maximum transmission <9 nm)
with transmission maxima ranging from 350 to 600 were positioned in a
beam near the source, and the photodiode current in response to a flash or step of light was measured with a current-to-voltage transducer, with neutral density filters interposed in the beam as needed to keep
the photodiode current below 10% of its saturating magnitude. The
transducer's output signal was sampled at 200 kHz, and the photon flux
density at the pupil plane for light flashes was computed as follows
(Wyszecki and Stiles, 1983):
|
(1)
|
Here nom (nm) is the manufacturer-specified
(nominal) wavelength of the interference filter, D is the
optical density of any neutral density filters interposed in the beam,
Q(nom) is the computed maximal (i.e.,
for D = 0) photon density; i (amps) the
photodiode current, Apd
(µm2) the photodiode surface area,
s() (amps watt 1) the photodiode
sensitivity at wavelength , h = 6.63 × 1034 (joules sec1) Planck's
constant, c = 3 × 1017 nm
sec1 the speed of light. For light steps, the
steady current i was substituted for the integral over time
in Equation 1. The photodiode current i in Equation 1 is
induced by photons whose wavelengths vary over the band of the filter,
and thus an integration over the passband occurs as the photodiode
generates current; the integration over t is performed
explicitly (numerically) offline. Use of Equation 1 embodies the
assumption that the finite bandwidth of the interference filters has
negligible effect on the estimated total quantal flux Q(nom); this assumption is warranted
because s() varies <10% over the band of any specific
filter. However, explicitly mentioning this assumption also raises an
issue that turned out to be critical in the rod and cone spectral
sensitivity measurements, namely, the wavelength nom to
which the measured quantal flux is assigned during the analysis of
spectral sensitivity data. This problem was complicated by the
placement of the interference filters in the flash illuminator beams,
as we now describe.
Measurement of narrowband interference filter transmission
spectra. To achieve adequate intensities for cone-driven responses we found it necessary to use flash tubes whose size made it impossible to collimate their output into parallel light beams. The lack of beam
collimation caused the effective spectral characteristics of the
interference filters to differ significantly from specifications. Therefore, the transmission spectra of all interference filters had to
be measured in an optical geometry identical to that used in the ERG
experiments. Specifically, the filter whose transmission spectrum was
to be measured was placed in the same holder attached to the same flash
unit used in the experiments to deliver light into the recording
chamber. For the spectral transmission determination, however, the
light transmitted through the filter was delivered not to the recording
chamber but rather to one port of an 8-inch-diameter integrating sphere
(Ealing Electrooptics); the light exiting the second port of the
integrating sphere was then passed through a concave grating
monochromator having 1 nm bandwidth (H-20V, J-Y Optical Systems,
Metuchen, NJ). Light intensity was then measured (as described above)
at 1 nm steps with a photodiode attached to the exit slit of the
monochromator. The transmission spectra of the filters were also
obtained with the same monochromator in a configuration generating a
collimated light beam through the filter, for comparison with the
manufacturer's specifications. Figure
1A ( ) presents
transmission spectra obtained as just described of a filter with
manufacturer-specified maximum transmission at nom = 600 nm. It is clear from this figure that the transmission spectrum
obtained with the geometry used in the experiments (In situ)
is both shifted to shorter wavelengths and broadened by the lack of
beam collimation. As shown in Figure 1B, the shift was quantified by determining the wavelength med
corresponding to median of the spectral transmission function: in this
instance, med was shifted from the value 599 nm obtained
with the spectrophotometer measurement to 589 nm by the lack of beam
collimation. Shifts of the same direction and ranging in magnitude from
3 to 10 nm were found for all of the interference filters.
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Figure 1.
Effect of uncollimated sources on an
interference filter transmission spectrum. A, The
thin unbroken line presents the transmission spectrum of
a nominal 600 nm interference filter recorded with a Perkin-Elmer
Lambda-20 spectrophotometer. The open circles are the
transmissivity values measured in situ (i.e., with the
flash geometry used in the ERG investigation), obtained as described in
the text; the filled circles give the transmissivities
of the same filter measured with the same photodiode used to obtain the
open circles, but with a collimated light beam.
B, The thin unbroken line presents the
integral of the transmission spectrum determined with the Lambda-20
spectrophotometer; it reaches 0.5 of its area at 599 nm. The
thickened gray line is the integral of the transmission
spectrum measured in situ, in the uncollimated beam
geometry of the experiments; the 0.5 point () is at 589 nm. The
thickened black line presents the integral used to
determined eff, which is seen to be 583 nm for
the theoretically calculated in situ absorption spectrum
of rhodopsin (see Fig. 6A; Eq. 8).
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Because of the finite bandwidth of the filters, and the effect of
placement in the flash beam on the spectral transmission function, we
must distinguish between the nominal wavelength (manufacturer's specification) of a filter and the wavelength assigned to the flash
energy delivered in situ. In the immediately following
sections in Materials and Methods, we will honor this distinction by
subscripting the when the distinction is material. In particular,
throughout this manuscript, we adopt the convention of referring to the
nominal (or manufacturer-specified) wavelength of a bandbass filter by nom. When not subscripted, the will refer to the
wavelength of an ideal, monochromatic light.
Measurement of spectral densities of white flashes and
flashes filtered by broadband color glass filters. In some
experiments, to generate flashes of sufficiently high intensity we had
to use "white" flashes, or flashes filtered with broad bandwidth
color glass filters. To determine the spectral content of such flashes, we first estimated the emission spectrum of the unfiltered flash source
at 10 nm spacing across the spectrum by making measurements with the
narrowband interference filters and applying the formula:
|
(2)
|
Here Q(med) is the measured
value obtained with Equation 1 (with med substituted for
the nominal wavelength nom),
T med() is the transmission
spectrum of the narrowband filter determined in situ as
described above, and
Esource(med) is the
emission (in photons µm2 at the cornea) at the
wavelength med. The emission E() at all points in the spectrum was then computed by linear interpolation between the discrete measurements.
The efficacy of white flashes or flashes filtered by a broadband filter
having spectrophotometrically measured transmission spectrum
TBB() was calculated in terms of
"equivalent photons at the pigment max" with the
formula:
|
(3)
|
Here  theory() is an expression
for a normalized pigment spectrum, described below (Eq. 8).
Conversion of flash stimuli to numbers of photoisomerizations in
rods and cones. Several factors must be taken into consideration to estimate from the quantal flux at the cornea the retinal flux density, and from the retinal flux density the number ( ) or rate ( ) of photoisomerizations per photoreceptor produced by a light flash or step. For a truly monochromatic flash delivering quantal flux
density Q() (photons µm2 at the
cornea) in a ganzfeld
|
(4)
|
where  () is the transmissivity of the preretinal eye media
at (described below), Apupil,
Aretina the areas of the mouse pupil and retina,
and aC() the end-on collecting area at
the retina of the specific photoreceptor type. The latter can be
expressed (Baylor et al., 1984) as:
![a<SUB>c</SUB>(&lgr;)=f<FR><NU>&pgr;d<SUP>2</SUP></NU><DE>4</DE></FR> [1−10<SUP><UP>−</UP>&Dgr;D(&lgr;)L</SUP>]&ggr;,](/content/vol19/issue1/fulltext/442/img006.gif)
|
(5)
|
where d is the outer segment diameter,
 D() is the specific axial density of the visual
pigment [optical density (o.d.) units µm1],
L (µm) the outer segment length, f  1 a dimensionless factor that accounts for any light funneling by
the inner segment, and  the quantum efficiency of
photoisomerization. Estimates of
aC(max) for mammalian rods
computed with Equation 5 range from 0.9 to 2.3 µm2
(Breton et al., 1994; Schneeweis and Schnapf, 1995; Lyubarsky and Pugh,
1996); we previously adopted the value 1.3 µm2 for
murine rods and will assume the latter value here.
For simplicity one can collapse all of the eye-dependent terms of
Equation 4 into a single parameter; thus:
|
(6)
|
where aC,cornea is the apparent
collecting area of the specific photoreceptor type at the cornea
in a ganzfeld. Previously, we estimated
aC,cornea for mouse rods to be 0.2 µm2 at = 500 nm (Lyubarsky and Pugh, 1996), and we
will adopt that value here. When the spectral density Q()
is not monochromatic, photons of different wavelengths are summed in
the photoreceptor, and so Equation 6 becomes:
|
(7)
|
The collecting areas of mouse cones have not been estimated
previously. We obtained the estimate
aC(max) =2.4
µm2 based largely on information from the
anatomical investigation of Carter-Dawson and LaVail (1979a), as
follows. Mouse cone outer segments were assumed to have length
L = 13 µm and diameter d =1.5 µm at
their base, tapering to 1.0 µm at their tips; in contrast rod outer
segments were assumed to have length L= 25 µm and uniform diameter d = 1.8 µm. Mouse cone inner segments were
assumed to have a maximal diameter of 4 µm, 1.8-fold greater than
mouse rod inner segments, 2.0 µm. The twofold greater maximal inner
segment width of the cones produces a geometrical cross section
fourfold larger for guiding light to the outer segment, on the
assumption that all light entering the waveguide can be effectively
funneled to the outer segment: thus, in Equation 5, f = (4/1.5)2 = 7 for cones and f = (2/1.8)2 = 1.24 for rods. Rod and cones were assumed to
have the same specific (optical) density at their respective
max values: D(max) = 0.015 o.d./µm. The quantum efficiency of photoisomerization was
assumed to be the same for rods and cones, = 2/3. Combining these
factors in Equations 4 and 5, one obtains aC
(max) = 1.3 µm2 for rods and
aC (max) = 2.4 µm2 for cones. [For further discussion of the rod
collecting area, see Lyubarsky and Pugh (1996). We defer analysis of
the cone collecting area "at the cornea" to Discussion.]
Normalized spectral sensitivity. It is useful to
define the normalized spectral sensitivity at the cornea of a
photoreceptor class with peak sensitivity at max in
terms of collecting areas; thus, from Equations 4 and 5:
|
(8)
|
Expressed in these terms, it is clear that provided the spectral
maximum max, the normalized absorbance function
D()/D(max), and
the ocular media transmissivity () are specified, only
Dmax = D(max)L, the total axial
density at the max, remains to be specified to
determine the normalized spectral sensitivity.
ERG a-wave as a measure of the rod circulating current and rod
action spectrum measurements. The primary goal of this
investigation has been the characterization of murine cone-driven
responses. However, because rod-driven ERG responses have been much
more thoroughly quantified than those of cones, we have found it
invaluable to use the rod a-wave as a benchmark for both spectral
sensitivity analysis and quantification of the relative magnitudes of
cone-driven signals. Analysis of the rod a-wave rests on the
biophysical fact that the massed circulating currents of the rods give
rise to a dipole layer field potential across the retina (Hagins et
al., 1970), a potential that "depolarizes" the cornea in direct
proportion to the average rod circulating current. The "a-wave" is
the transient electrical potential resulting from suppression of some
or all of the circulating current. Thus, if at any instant a brief
flash sufficiently intense to saturate the a-wave amplitude is
delivered, then the saturated value amax
satisfies:
|
(9)
|
where Jcirc is the rod circulating
current at the moment immediately preceding the flash. Equation 9 can
be used to recover the dependence of the rod circulating current on
steady illumination and the time course of its recovery after a flash
(Lyubarsky and Pugh, 1996; Pepperberg et al., 1996). It can also be
combined with a well established model of the activation phase of the
rod phototransduction cascade to predict the kinetics and sensitivity of the transient a-wave, a(t), according to:
|
(10)
|
(Lamb and Pugh, 1992; Breton et al., 1994; Lyubarsky and Pugh,
1996). In Equation 10, F(t) = Jcirc(t)/Jcirc,dark,
the circulating current normalized by its dark magnitude, is the
number of photoisomerizations produced by the flash, A
(s2) is the "amplification constant," and
t'eff is a brief delay (2-3 msec).
In this investigation we combined Equations 6 and 10 to determine for
the first time the action spectrum between 340 and 600 nm of the
dark-adapted murine a-wave. Thus, for a monochromatic flash of
wavelength producing a photon density Q() at the
cornea, the predicted time course of the rod a-wave is:
![1−<FENCE><FR><NU>a(t)</NU><DE>a<SUB><UP>max</UP></SUB></DE></FR></FENCE>=<UP>exp</UP>[<UP>−</UP>1/2 Q(&lgr;)a<SUB><UP>C,cornea</UP></SUB>(&lgr;)A(t−t′<SUB><UP>eff</UP></SUB>)<SUP>2</SUP>].](/content/vol19/issue1/fulltext/442/img014.gif)
|
(11)
|
The protocol for determining the rod action spectrum used
Equation 11, as follows. First, a family of ERGs was obtained in response to a series of nom = 500 nm flashes, with
intensities chosen to elicit a-waves whose maximum amplitude (i.e.,
amplitude at time of b-wave intrusion) was 15-50% of the saturated
a-wave amplitude; a flash producing = 280,000 was used to estimate amax. The normalized a-waves were later fitted
as an ensemble with Equation 10 to estimate A at 500 nm
(Breton et al., 1994; Lyubarsky and Pugh, 1996). To determine rod
sensitivity at any other wavelength , a series of flashes of graded
intensity Q() was then delivered that produced a-waves of
15-50% saturated amplitude. With A fixed (as required by
the univariance of phototransduction) at its value at 500 nm,
the ensemble fitting of Equation 11 to the the a-wave data obtained
with light of another wavelength yielded an estimate of
the product aC,cornea() = aC,cornea(max)theory(). Thus, with aC,cornea(max) fixed,
we obtained an estimate of theory(), the rod spectral sensitivity at this wavelength at the cornea (Eq. 8).
We emphasize that the rod action spectrum provided a benchmark against
which we gauged certain features of the cone action spectra, as well as
a general control experiment for all spectral sensitivity determinations.
The reader may wonder why we measured the rod action spectrum with
a-wave responses rather than with the more easily measured scotopic
b-wave. One reason that we decided to estimate the rod spectrum from
a-wave data is that the flash intensities involved were similar to
those used to measure the cone-driven responses; this allowed use of
the same filters and flash units. A second reason is that we feared
that the scotopic b-wave might possibly be contaminated in the UV
region of the spectrum by the relatively sensitive UV cone-driven
b-wave.
Cone-driven signal isolation. Previous work has shown that
cone-driven "b-waves" can be recorded reliably from mice in
response to flashes (Peachey et al., 1993), and here we build on these previous findings. Nonetheless, proof of cone-signal isolation requires
a valid monitor of the rod contribution to any response. This
requirement is all the more acute in mice, because one of the
cone-visual pigments is spectrally very similar to rhodopsin (Chiu et
al., 1994). Because of the large ratio of rods to cones [~30:1 (Carter-Dawson and LaVail, 1979a,b)], the saturated
amplitude of the a-wave of the flash-ERG can be expected to monitor
almost exclusively the rod circulating current at the moment of a probe flash (Eq. 9). However, because the efficacy of using the a-wave amplitude as monitor of the rods is limited by any cone contributions to the a-wave, we will present new evidence concerning the utility and
limitations of this monitor in mice. In particular, we will estimate
the magnitude and activation time course of a putative cone-driven
a-wave and compare its features with those of the total (rod + cone)
a-wave.
We used two protocols to isolate cone-driven ERG responses. In the
first, the eye was exposed to 2.5 sec steps of 520 nm background light
of intensities estimated to produce = 3000-6000
photoisomerizations/sec per rod; a probe flash of controlled
intensity and spectral composition was delivered 2 sec after the onset
of the background. The second approach used an intense conditioning
flash that suppressed the rod circulating current for several seconds;
a probe flash then tested for cone-driven responses at various times
after the conditioning flash before any rod signal recovery.
Measurement of cone action spectra from cone-driven b-waves.
Cone-driven b-waves, isolated as just described, were recorded in
response to flashes of varying wavelengths and intensities. As we shall
show in Results, for flashes of intensities that elicited responses
 20% of the saturating magnitude, the responses were linear in flash
intensity, i.e.:
|
(12)
|
where b(Q(), t) is the
appropriately defined cone b-wave response elicited by a flash of
wavelength and intensity Q(), Sobs() [nV (photon
µm2)1] is the observed
absolute sensitivity of the cone-driven ERG at wavelength
, and  (t) is a scaled
"dim-flash" response having unity amplitude at its peak. The
obedience of cone b-wave responses to Equation 12 is a sufficient
condition for the flash method to yield unique spectral sensitivity
functions for the cones.
Two qualifications to the interpretation of the function
(t) in Equation 12 need be added. The
first qualification concerns the presence of oscillatory potentials. In
our analysis we will interpret (t) as the
function extracted from the cone-driven responses after removal of
oscillatory potentials by appropriate filtering. The second
qualification concerns shape differences in
(t) that may arise depending on the cone
type in which the driving signals originate; thus,
(t) does not need to be identical in shape
for signals driven by different classes of cones. An issue related to
the second qualification concerns the use of a "white" flash to
elicit saturating responses. This use was dictated by the limited
energy available for waveband-limited flashes and the need to drive
into saturation the responses of all b-wave generators receiving input
from the UV- and/or M-cones. The problem of assessing the cone type of
origin of cone-driven signals will be a principal focus of Results.
Because anesthesia limited the duration of the experiments, and because
of the consequent need to use a number of different animals to get
adequate numbers of rod- and cone-driven responses over the whole
spectrum, we found it convenient to adopt nom = 500 nm
as a standard for spectral sensitivity measurements of both rod- and
cone-driven responses. Thus, we always made measurements at 500 nm on
each animal. Furthermore, throughout each experiment at intervals of
5-6 min a saturating white flash was delivered to monitor any secular
changes in maximal signal amplitude; the maximal amplitude was found to
vary no more than 10% between such flashes and no more than 30% over
the time course of an entire experiment. Dim-flash responses were
scaled relative to the amplitude of the nearest saturating response for
the final analysis of sensitivity.
Template analysis of rod and cone action spectra.
Characterization of the action spectra of photoreceptors has been
greatly advanced by the application of photopigment template curves
(Dartnall, 1953; Mansfield, 1985; Lamb, 1995). Such templates predict
the shape of the normalized absorbance (low pigment density) spectrum. We fitted our action spectra with Equation 8, with the normalized absorbance spectrum given by Lamb's (1995) template 2; i.e., we assumed:
![<FR><NU>&Dgr;D(&lgr;)</NU><DE>&Dgr;D(&lgr;<SUB><UP>max</UP></SUB>)</DE></FR>=<FR><NU>1</NU><DE><UP>exp</UP>[a(A−x)]+<UP>exp</UP>[b(B−x)]+<UP>exp</UP>[c(C−x)]+D</DE></FR>,](/content/vol19/issue1/fulltext/442/img017.gif)
|
(13)
|
where D is specific density (compare Eq. 8), is wavelength, x = max/, and
the parameter values are a = 70, b = 28.5, c =  14.1, A = 0.88, B = 0.924, C = 1.104, and
D = 0.655. The template analysis of action spectra data
obtained "at the cornea" requires assumptions about the prereceptor
media transmissivity (). We used values of () obtained by
Alpern et al. (1987) for rats, slightly modified as follows. The
absorption for the neural retina in the mouse was assumed to be the
same as that in rat, whereas absorption by the lens, which occupies
most of the preretinal optical path in the mouse (Remtulla and Hallett, 1985), was taken as half of its value in rat. The prereceptor spectral
transmissivity function () obtained in this way was found to be
well approximated by the expression:
|
(14)
|
where is given in nanometers. Below 410 nm we did not apply
a correction for prereceptor media, because the Alpern et al. (1987)
transmission data extend only to 410 nm.
Graphical presentation of action spectra data. For the
analysis and presentation of action spectra data obtained with
interference filters, each filter was assigned an effective wavelength,
eff, which could differ from the measured filter
spectral density median, med (Fig. 1). The basis for
this assignment is the principle of Univariance, which dictates that
only the number of photoisomerizations generated by the flash in a
given type of photoreceptors, and not their wavelength distribution,
determines the response. Resting on this principle, we defined
eff as the median wavelength of the integral specifying
the total number of photoisomerizations produced by a source/filter
combination. Thus, for an interference filter whose spectral
transmission median in the apparatus is med and
whose transmission spectrum is
Tmed(), we defined eff by the equation:
|
(15)
|
Here 0 is the total number of photoisomerizations
produced by the unattenuated flash in the receptors of the given type, Esource() is the quantal flux density of the
unattenuated, unfiltered source at the cornea,
Tmed() is the transmission
spectrum of the particular filter having transmission median
med, and aC,cornea(max) is the
collecting area of a particular class of photoreceptors at the cornea,
and theory() is the theoretical spectral sensitivity given by Equation 8. Figure 1B
shows the application of Equation 15: it can be seen that whereas the
lack of beam collimation alone shifted the median wavelength from 600 to 589 nm, eff was also predicted to be shifted another
6 nm, to 583 nm, for rods with rhodopsin max at 498 nm.
This 6 nm shift was the largest we found for the combination of filters
and estimated pigment absorption spectrum, and in general shifts of
such magnitude were only predicted to occur in the long-wave regions of
asymptotic slope of the visual pigment absorption spectra. For all
other regions of the photopigment absorption spectra, the
pigment-specific shifts were calculated be <2 nm.
For the analysis and presentation of action spectra data one must
assign the measured light Q(med) of
Equation 1 to a single wavelength [med has been
substituted for nom, because of the analysis of
the filter transmission characteristics in situ (compare Fig. 1)]. Our assignment of the measured light to eff
in Equation 15 is equivalent to making the approximation:
|
(16)
|
where Q(med) is the measured
quantity described in Equation 1, eff is given by
Equation 15, and theory() is the
normalized spectral sensitivity given by Equation 8. In summary,
Equation 16 says that the measured quantal flux
Q(med) at the position of the cornea
is treated as if it were all concentrated at wavelength eff, where eff has the property
that the theoretically computed contributions to the photoreceptor's
total quantum catch from light below and above eff in
the spectral distribution at the retina are equal (Eq. 15).
Rod a-wave as a gauge for estimating magnitudes of generator
currents of other retinal cell types. On the basis of the now certain identification of the rod circulating current as the generator of the field potential whose suppression is the dark-adapted a-wave, the saturated amplitude of the dark-adapted a-wave can provide a gauge
for estimating from field potentials the underlying generator currents
of other retinal cell types. To use this gauge, the proportionality relation of Equation 9 must be expressed more completely. The corneally
recorded potential and the generator current of a specific radially
oriented retinal cell type can be shown to obey approximately the
following relationship (Hagins et al., 1970; Pugh et al., 1998):
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(17)
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In Equation 17 Vcell,cornea
(V) is the electrical potential between corneal and
reference electrodes caused by complete activation (or inactivation) of
the generator current of a particular retinal cell type,
celectrodes is a dimensionless factor
attributable to electrode type and placement,  cell
(cells cm2) is the spatial density of the cell
type in the retina,
 layer( cm2) is the
effective or "average" resistance of the retinal layer in which the
cell is found, and Jcell (A)
is the generator current; the product cell
Jcell is the maximal radial current density generated by the simultaneous activity of all the cells of the specific
type. In general, we can eliminate common terms in Equation 17 that
arise from retinal and recording geometry by forming a ratio for two
cell types. Specifically, because of the identification of the
rod circulating current as the generator of the dark-adapted a-wave we
have the following:
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(18)
|
where we have dropped the subscript "cornea" for simplicity.
In our application of Equation 18, the potentials of the left-hand side
will be taken from the ERG data, and we will assume that the layer
resistances, which are the products of the interstitial resistivities
and layer thickness, are roughly equal for the outer nuclear and inner
nuclear layers. To apply Equation 18, two quantities characterizing
rods are needed: (1) rods, the spatial density of
rods in the retina, and (2) Jcirc,rod, the
average rod circulating current.
We take the average rod circulating (dark) current to be 25 pAa
reasonable compromise between the early estimate of 70 pA by Hagins et
al. (1970) and more recent suction pipette measurements of mammalian
rods (Baylor et al., 1984; Nakatani et al., 1991; Kraft et al., 1993).
Estimates of rod density in rodent retinas converge on the value of
3 × 107 rods cm2 (cf.
Reiser et al., 1996). Thus, the maximal radial current density attributable to rod circulating current is rods
Jcirc,rods = 25 × 1012 × 3 × 107 = 750 × 106 A
cm2; this maximal current density occurs at the
retinal layer corresponding to the junction of the rod outer and inner
segments, because the circulating current sources are all in the rod
inner segment membrane and the sinks are in the outer segment membrane
(Hagins et al., 1970; Pugh et al., 1998). Assuming that the layer
resistances through which flow the signal currents of other radially
oriented retinal cell types are comparable, the "rod gauge" (Eq. 18) indicates that a radial current density in the mouse retina of 750 µA cm2 gives rise to a potential between the
cornea and reference electrode of ~400 µV, the value of
amax, rod [see Table 1; see also Lyubarsky and
Pugh (1996)].
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RESULTS |
Isolation of cone-driven components of the murine ERG:
cone a-wave
Figure 2A shows a
family of ERGs elicited with a white flash estimated to produce 2.8 × 105 photoisomerizations/rod,
sufficiently intense to saturate the a-wave amplitude (Lyubarsky and
Pugh, 1996). The flash was delivered in the fully dark-adapted
condition and in the presence of 520 nm background steps whose
intensity was varied to produce from = 15-3020
photoisomerizations/sec per rod. Each increase in the intensity
of the step up to approximately = 1500 further decreased the
amplitude of the a-wave response to the flash; however, as shown by the
coincidence of traces d,e, the increase from = 1500 to
3020 had no effect on the amplitude. Rather, the flash now evoked a
residual a-wave whose amplitude (25 µV) remained at 6-7% of the
amplitude (395 µV) of the dark-adapted response. In 12 mice exposed
to background steps of = 3000-6000, the residual a-wave amplitude
evoked by intense white flashes such as in Figure 2A
was 21 ± 10 µV (mean ± SD).
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Figure 2.
Separation of cone from rod
signals by steady backgrounds. A, ERGs elicited by a
white flash calculated to produce = 280,000 photoisomerizations/rod, delivered in the dark, and in the presence of
a series of 500 nm steps of light (backgrounds) generating = 15, 30, 76, 150, 300, 760, 1500, and 3020 photoisomerizations/sec per
rod, respectively; the amplitude of the responses diminishes as
the background intensity increases. The traces are averages of two to
three responses, and the flashes were presented 2 sec after the onset
of the light steps. The a-wave is the initial, corneal-negative
response component: the a-wave trace obtained for the dark-adapted
condition is shown thickened. The b-wave is the
corneal-positive-going ERG component that follows and
effectively truncates the a-wave. B, ERGs from
A after subtraction of the average of the traces
(d,e). The filled circle attached to each
trace is its minimum (except for the topmost traces, where the circle
is plotted at the average time of the other circles). The time and
amplitude scales of B are the same as for
A. C, Traces a,b,c from
B and d,e from A
normalized. The terminal portions of the traces have been fitted with
decaying first-order exponential functions (thickened gray
curves): the exponential through traces a,b,c
has a time constant of 0.83 msec, whereas that through traces
d,e has a time constant of 2.5 msec (trace
c was shifted leftward by 0.6 msec to emphasize the
common shape of the terminal portions of
a-c). D, Inferred steady
"photocurrent" response amplitudes of rods to the background light
steps, derived from the data of B ( ) and from data of
two other mice ( ,  ). Each set of symbols gives the maximum
amplitudes of the "cone-corrected" a-wave responses, as shown in
B, by application of Equation 20. Note the exact
amplitude correspondence between the circles plotted in
B and D: the labels a, b,
and c from B have been carried over to
C to show the correspondence. The smooth curves are
hyperbolic or "Naka-Rushton" saturation functions (Eq. 21), with
semisaturation constants of 60 and 250 .
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The residual a-wave component measured in the presence of such
backgrounds was also significantly slower in its "rise" than the
a-wave elicited from the dark-adapted retina: the rising phase of the
saturated dark-adapted a-wave has a time to peak of ~8 msec, whereas
the residual a-wave reaches its peak amplitude in ~14 msec. Further
evidence for this relative slowness is presented in Figure
2C, in which the normalized traces a-c of
B and of the normalized residual traces d,e of
A have been replotted and fitted with exponentials having
time constants of 0.83 and 2.5 msec, respectively. As an alternative
analysis to the fitting of decaying exponential functions, we applied
to the a-wave traces obtained in response to the saturating flash the
model of the a-wave presented by Breton et al. (1994), as modified by
Smith and Lamb (1997) to incorporate analytically the effect of the
photoreceptor membrane time constant. For nine sets of responses from
different mice analyzed in this manner, the estimated time constant was
1.0 ± 0.1 msec (mean ± SD) for the terminal phase of the
dark-adapted a-wave response, and 2.9 ± 0.8 msec for the residual
a-wave recorded in the presence of a background estimated to produce
either 3020 or 6000 photoisomerizations/sec per rod. These
different time constants are consistent with rod and cone origins,
respectively. Specifically, the "rise time" of circulating current
responses to intense flashes are rate-limited by the cell membrane time constant cell (Penn and Hagins, 1972; Cobbs and Pugh,
1987), which have been estimated for mammalian photoreceptors to be
rod 1 msec (Penn and Hagins, 1972; Schneeweis and
Schnapf, 1995), and cone 2-4 msec (Schneeweis and
Schnapf, 1995). Analyses of saturating human rod and cone a-waves have
yielded similar estimates: rod 1 msec and
cone = 2-4 msec (Hood and Birch, 1993, 1995; Cideciyan
and Jacobson, 1996; Smith and Lamb, 1997).
In sum, based on its electrical sign, its relative insensitivity to
background light, and the relative sluggishness in its rise, we
attribute the residual a-wave component (Fig. 2A,C,
traces d, e) mainly to the suppression of the retinal field
potential generated by the circulating currents of cone photoreceptors. In Figure 2B we show the traces of Figure
2A with the presumed cone a-wave component subtracted
out, and in Figure 2D we plot steady-state
response-intensity relations for the rod photocurrent responses to the
background steps, derived from the traces in Figure
2B as follows.
The rod photocurrent response R is the complement of the
circulating current; i.e., the rod photocurrent evoked by a light step
of intensity necessarily satisfies:
|
(19)
|
Combining the defining relation (Eq. 19) with Equation 9, we
arrive at the following expression for the normalized response to the
background steps:
|
(20)
|
Thus, in Figure 2D we plot
(amax,dark amax())/amax,dark as a
function of the step intensity for the data of Figure
2B, along with results obtained in the same way from
two additional mice. The conclusion that the data points represent the
normalized amplitudes of rod photocurrent responses to the light steps
is supported by their dependence on step intensity, which can be characterized with a hyperbolic saturation function:
|
(21)
|
In Equation 21 1/2 is the half-saturating
intensity, a measure of rod light sensitivity to the step intensity.
For the animal whose data are shown in Figure 2B
(), 1/2 = 60, whereas the responses of the other two
animals are reasonably well characterized by Equation 21 with
1/2 =250. This latter value is consistent with
steady-state response versus intensity functions of isolated rods of a
number of mammalian species (Nakatani et al., 1991), which have been
characterized by hyperbolic saturation functions with
1/2 = 150-300 /sec. Taking the value
1/2 250 as a reasonably conservative figure
[because the data tend to saturate somewhat more steeply than the
hyperbolic relation (Eq. 21)], we conclude that >92% of the rod
circulating current should be suppressed for = 3020. We thus chose
to use backgrounds estimated to produce = 3000-6000 as standard
for cone signal isolation, with the goal of keeping the cone signaling
pathways (especially those of the M-cones) maximally sensitive while
simultaneously maximally suppressing rod-driven retinal activity. Below
(see Figs. 4-6) we will present evidence that the b-waves recorded in
the presence of such backgrounds are cone-driven. First, we show a
second method of isolating cone-driven ERG responses, which provides
converging evidence for such isolation.
Isolation of cone-driven components of the murine ERG:
cone b-wave
In Figure 3 the top trace in the
left-hand panel shows the ERG of a fully dark-adapted mouse obtained in
response to an intense white flash estimated to produce = 1.5 × 106 photoisomerizations/rod. The subsequent nine
traces show repeated responses to the same flash, and at various
intervals the response to a second, white "probe" flash one-tenth
as intense, delivered at the times indicated on the graph. The probe
flash generates a corneal-positive (b-wave) response component that
recovers rapidly, during a period when there is no reliably measurable
change in the initial, corneal-negative a-wave.
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Figure 3.
Separation of cone from rod signals by flashed
adapting light. Isolation of cone-driven responses by a
"double-flash" protocol. Ten traces are shown. Initially in each
case a white conditioning flash producing = 1.5 × 106 photoisomerizations/rod was delivered to the
dark-adapted eye, producing an ERG having a 260-300 µV
corneal-negative a-wave, followed immediately by a 530-660 µV
positive-going b-wave, and then a slower, negative-going
potential. At the times indicated by the upward-pointing
arrows, a second flash producing = 1.5 × 105 was delivered to probe recovery from the initial
flash. Inset at bottom right, Symbols plot the peak
amplitude of the cone-driven b-wave responses shown by the
traces.
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The recovery time course of the peak amplitude of the b-wave response
to the probe flashes is shown in the inset at the bottom right of
Figure 3. Here it is seen that there is a rapid phase of b-wave
recovery in the initial second after the conditioning flash, followed
by a more slowly increasing corneal positivity. On the basis of its
rapid recovery time course in the absence of an a-wave component
exceeding a few percent of the initial (dark-adapted) 300 µV
a-wave, we attribute the corneal-positive response recorded in the 3 sec period after the conditioning flash exclusively to cone-driven
cells. In contrast, for the same intensity conditioning flash, the rod
a-wave or circulating current remains in complete saturation for >3
sec and has a recovery half-time of ~10 sec [Penn and Hagins (1972),
their Fig. 8]. In summary, this "double-flash" procedure
provides a complementary method to the use of steady backgrounds for
cone-signal isolation illustrated in Figure 2.
Table 1 presents summary statistics for
some key quantities measured in experiments like those illustrated in
Figures 2-3. One particularly interesting quantity is the ratio of the
saturating b-wave amplitude obtained under cone-signal isolation
conditions to that obtained in the dark-adapted state. In Figure 3, the
peak amplitude of the cone-driven b-wave
(bmax,cone) is seen to be 220-250 µV, ~35%
of the amplitude of the dark-adapted b-wave
(bmax,rod+cone), 660 µV. In similar
experiments on 39 mice, we found the ratio bmax,cone/bmax,rod+cone
to be 0.28 ± 0.09 (mean ± SD; Table 1), a result suggesting
that cone-driven on-bipolar cells generate a maximal current density
nearly half that of rod bipolars (Discussion).
The use of "white" flashes to generate cone-driven responses in
experiments such as that of Figure 3 raises questions in light of the
evidence that the mouse retina has both UV- and M-sensitive cones (see
introductory remarks). For example, the question arises as to whether
the UV- and M-cones are stimulated and adapted to the same degree by
such white flashes, and in particular, whether the b-wave generators
receiving input from each cone type are driven into saturation. We thus
undertook experiments to measure the action spectrum and quantify the
properties of cone-driven b-wave responses.
B-wave response families under cone-isolation conditions
Figure 4 illustrates two families of
ERG responses of a single mouse to a series of flashes of nominal
wavelengths nom = 340 and 520 nm of varying intensity
under conditions of cone-signal isolation. These responses present a
number of well known features of cone-driven ERG responses, including
the progressive appearance of large oscillatory potentials (Heynen et
al., 1985; Peachey et al., 1987).
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Figure 4.
Response families of cone-driven b-waves for 340 and 500 nm flashes. Top panel, Black
traces show the responses of a single mouse (no. 315) to a
series of UV (nom = 340 nm) and midwave
(nom = 520 nm) flashes; each trace is the average of
from 5 to 40 individual responses. The smooth, thickened gray
traces were obtained by digitally filtering the averaged
responses with a Gaussian filter with  = 8 msec (bandwidth 16.6 Hz
at 3 dB cutoff). The UV intensity series delivered (from lowest to
highest intensity) Q(nom) = 1110, 2480, 4390, and 8800 photons µm2 at the cornea,
and the midwave series delivered 4550, 9090, 18700, 37500, and 75000 photons µm2 at the cornea. The 4 msec segment of
the traces immediately after the flash contained a flash artifact, and
so this segment has been omitted for clarity; this segment was also set
to zero before the Gaussian filter was applied. Bottom
panel, Cone-driven oscillatory potentials extracted from the
responses in the top panel by subtracting the
Gaussian-filtered responses from the averaged responses; these
difference traces are shown on the same time and amplitude scales as
the responses in the top panel. The difference traces
were digitally filtered with a Gaussian filter having a bandwidth of
133 Hz.
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We derived the action spectrum of cone-driven signals from data such as
those illustrated in Figure 4. As discussed in Materials and Methods, a
sufficient condition for the b-wave response to have a unique action
spectrum is that it be linear in intensity over some range. Inspection
of the traces in the top panel of Figure 4 leaves some doubt about
linearity, inasmuch as the oscillatory potentials exhibit a strong
nonlinearity, such that the time-to-peak of the individual oscillations
depends on flash intensity even at relatively low intensities. We dealt
with this difficulty by filtering the raw traces digitally with a
Gaussian filter, thereby obtaining the smooth, thickened gray traces.
Although the Gaussian has the defect of being an anticipating filter,
it nonetheless provides a robust, nonsubjective method of extracting a
"running mean" curve that does not exhibit the oscillations. The
Gaussian-filtered traces are nearly invariant in shape at the lowest
intensities, providing support for the derivation of a unique action
spectrum (Eq. 12). A second benefit of applying this filter is that it
gives by subtraction a useful representation of the oscillatory
potentials, as seen in the bottom panels of Figure 4.
Figure 5A shows response peak
amplitude versus intensity functions derived from the filtered traces
of Figure 4. In this double-log plot, response amplitude linearity is
represented by lines of unity slope, which are shown drawn through the
symbols representing the lowest amplitude responses. In Figure
5B the data from A have been replotted with
respect to a normalized amplitude coordinate, along with data of five
other animals obtained with the same and two other wavelengths. Since
the work of Fulton and Rushton (1978), it has been traditional in
presenting such data to fit them over the entire intensity domain with
a single saturation function, such as the hyperbolic function shown in
Figure 2D. We have not fitted these data with a
specific saturation function for three reasons. First, in the UV region
of the spectrum the energy of our flash lamps was insufficient to
generate saturating responses. Second, the filtering is less defensible
in the upper range of intensities where the oscillations are largest
and where the detailed form of the saturation function's compressive
nonlinearity affects the fitting. Third, and most importantly, as we
shall show below, although driven by photons caught by an M- and a
UV-cone pigment, the b-wave responses cannot be explained on the
hypothesis that there are completely separate neural pathways for
signals originating in each cone pigment. Thus, we would not expect any
simple saturation function to describe the amplitude versus intensity
function independent of wavelength.
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Figure 5.
Response amplitude versus intensity functions for
cone-driven b-waves. A, The peak amplitudes of the
Gaussian-filtered traces of Figure 4 (mouse no. 315) are plotted as a
function of the flash intensity in double-logarithmic coordinates.
Lines of slope unity, representing linearity of the peak amplitudes,
have been fitted to the three lowest points in each set. The
dotted line corresponds to a peak response amplitude of
20% of the saturating amplitude. B, Amplitude versus
intensity data for six mice obtained with flashes of five different
wavelengths. Peak amplitudes such as those in A were
first normalized by dividing them by the saturating amplitude; then,
the intensities were scaled by the intensity
I500, estimated (by interpolation) to
produce 20% of the normalized amplitude for nom = 500 nm flashes for the individual mouse. Wavelength
(nom) keys for the data are as follows (from
left to right): 340 nm (black symbols),
400 nm (light gray symbols), 500 nm (symbols with
dots in center), 520 nm (open symbols), 560 nm
(dark gray symbols). Different-shaped symbols are used
for the data of different mice. Note that the open and
filled circles representing the data of mouse no. 315 from A are replotted in B and have the
same relative locations on both x- and
y-axes (this serves to illustrate the method of
x- and y-scaling). (Data obtained at
other wavelengths have been omitted for the sake of clarity. In the
derivation of the cone b-wave action spectrum the response vs intensity
data of each animal were regressed individually, as in
A.)
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It bears emphasizing that the absence of a simple functional
description of cone b-wave saturation in the mouse in no way compromises the derivation of the action spectrum of the responses. Rather, as explained in Materials and Methods, a sufficient condition for determination of a unique spectral sensitivity is an intensity domain of response linearity. In Figure 5, such linearity is manifested in the intensity domains for each wavelength over which unity slope
lines describe the data. In these domains of linearity the derived
sensitivity measure is the ratio of the ordinate amplitude (in
microvolts) divided by the abscissa (in photons
µm2). Before presenting the cone action spectrum
derived from amplitude versus intensity data such as in Figure 5, we
will first describe the rod action spectrum, which provides benchmark
information about several features of the mouse eye media and retina.
Rod a-wave action spectrum
Figure 6A presents
the spectral sensitivity of the a-wave of fully dark-adapted mice over
the spectral range 350-600 nm, derived with the analysis described in
Materials and Methods. The mean amplification constant was
Arod = 3.7 ± 0.9 sec2
(Table 1), 50% lower than that (7.2 ± 1.5 sec2) estimated previously for C57BL/6 mice with a
different method of delivering light (Lyubarsky and Pugh, 1996). We
offer three possible explanations for the discrepancy in
Arod between the studies: (1) the previous
method of light delivery (through a fiber optic coupled to a contact
lens) may have been more effective; in the present, more lengthy
experiments, some corneal drying and opacification oc-curs; (2) an
unaccounted-for systematic error in estimating light delivered to the
retina from different angles in the old and new stimulation geometries;
and (3) differences in the animals.
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Figure 6.
Spectral sensitivities of murine rods and cones
derived from ERGs. A, Spectral sensitivity of the
dark-adapted murine a-wave. Each point is the mean ± SD of two to
five determinations of the sensitivity of the a-wave at the cornea,
estimated by application of Equation 11 to families of a-wave
responses, as described in Materials and Methods. The data of each
animal were individually normalized to a sensitivity of 1.0 at 500 nm.
The dark gray smooth curve (plotted from 460 to 600 nm)
was generated with Equations 8 and 13, with max = 498 nm, and a maximum axial absorbance Dmax = 0.3. The light gray curve drawn through the data below
460 nm is a fifth-order polynomial obtained by least-squares
regression. B, Spectral sensitivity of the cone-driven
b-wave. The data points are sensitivity estimates (mean ± SD)
from two to five animals, each estimate having been derived from a
response versus intensity function such as shown in Figure 7. The
smoothed gray traces were generated with Equations 8 and
13, with max = 355 and 508 nm, respectively, and
Dmax = 0.1 (negligible self-screening). The
light gray curve was obtained from the fifth-order
polynomial describing the rod spectrum below 460 nm but has been scaled
linearly by 0.85 as a correction for the absence of
self-screening.
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As expected for rhodopsin, the action spectrum derived from the
dark-adapted a-wave data has a primary peak near 500 nm and a broad
secondary maximum in the UV. We fitted the spectrum with the pigment
template function (Eq. 13) adjusted for self-screening and corrected
for the media transmissivity as described by Equation 8. The spectrum
is reasonably well described by a retinal1 pigment with
max = 498 nm and an end-on optical density of 0.3 (dark gray, smooth curve). The template pigment curve does not extend much
below the pigment max, and in particular does not
include the UV region of the spectrum. To derive a representation of
the spectrum below 460 nm, we fitted the data with a fifth-order
polynomial (light gray curve), which was forced to agree with the
template curve up to 500 nm.
Cone b-wave action spectrum
Figure 6B presents the action spectrum for the
cone-driven b-wave derived from the analysis illustrated in Figure 5.
The spectrum, which represents data of seven mice, features two maxima:
one at 510 nm in the visible spectrum and a second at 355 nm in the UV; these maxima clearly reflect the well established UV- and
M-cone photopigments (Jacobs et al., 1991). The smoothed, dark gray
curves were generated with Lamb's (1995) template (Eqs. 8, 13) with
max values at 355 and 508 nm, respectively, and were fitted to the data by eye. Flash sensitivity near the UV maximum is
~3.5- to fivefold higher than at the midwave maximum, based on three
distinct but related criteria: (1) the relative ordinate positions of
the template curves in Figure 6B, (2) the average of
all data points near the two maxima, and (3) the ratio of sensitivity at nom = 340 nm to that at nom = 500 nm
for four mice that were each tested at both 340 and 500 nm. The
absolute sensitivities of the latter four mice at these two wavelengths
are reported in Table 1. Because the midwave maximum is located close
to the rod action spectrum peak, it is reasonable to assume that the M-cone pigment is very similar to rhodopsin in its spectral properties. Therefore, the height of the  -band of the rod spectrum
(Fig. 6A) provides an estimate for any contribution
of the M-cone pigment to the peak UV sensitivity of the cone-driven
b-wave; thus, in Figure 6B we approximated the M-cone
spectrum below ~450 nm with the rod spectrum. Judging from this
approximation, such contribution is unlikely to exceed 10% in the
spectral region below ~380 nm.
UV-sensitive cone pathway can be suppressed by
long-wavelength illumination
The max values of the pigments underlying the mouse
cone-driven b-waves are separated by ~150 nm. We expected that such
wide spectral separation would allow isolation of UV-cone-driven
signals by selective chromatic adaptation, i.e., isolation in
experiments in which M-cone signals were transiently suppressed with an
appropriately intense long-wavelength conditioning flash. Figure
7 shows the results of such an
experiment, designed as follows. A steady 520 nm background was applied
to suppress most of the rod activity. Then, an intense orange
conditioning flash ( > 530 nm) producing ~1% isomerization of
the rhodopsin, and a similar fractional isomerization of the M-cone
cone pigment, was delivered 2 sec after the background onset to
suppress M-cone signals. A relatively intense probe flash, either
broadband UV (330-390 nm) or nom = 500 nm, was
delivered at various times from 100 to 800 msec after the orange
conditioning flash to monitor sensitivity of signal pathways driven by
the UV- and M-cones, respectively. As expected, responsivity to the 500 nm probe flash was temporarily suppressed by the conditioning flash:
the response reappeared at 200 msec and reached 50% recovery in
350-400 msec. However, quite surprisingly, responsivity to the UV
probe flash was also completely suppressed, appearing only after 125 msec. Given the intensity of the probe flashes (Fig. 7, legend), it is
clear that the loss of responsivity to both probes is attributable to
signal saturation caused by the orange conditioning flash and not mere
loss of sensitivity.
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Figure 7.
Interaction of signals originating in the UV- and
M-cone pigments. Recovery of the cone b-wave after a bright orange ( > 530 nm; OG-530 colored glass filter) conditioning flash
(OCF) estimated to isomerize 1% of the M-cone
pigment. Numbers near the traces are the interflash
intervals in milliseconds; the trace labeled OCF is the
response to the conditioning flash alone (no probe flash).
Right-hand traces show responses to a nom = 500 nm probe flash producing 71,000 photons
µm2 at the cornea. Left-hand
traces show responses to a broadband UV probe flash (330-390
nm; Schott UG1 glass) producing ~43,000 "equivalent" 350 nm
photons µm2 at the cornea; the equivalency is
computed with respect to the UV pigment template shown in Figure 6 (Eq. 3). The probe flashes were the most intense the apparatus could
generate (Fig. 5A, abscissa). (To eliminate most of the
rod signal but keep the cones maximally sensitive, the entire
experiment was performed in the presence of a steady 520 nm background
producing = 750 photoisomerizations/sec per rod. To insure
absence of any UV light in the orange conditioning flash, the intensity
of the flash generated by the same flash unit with a combination of
Schott glass filters (UG1 + OG-530 + BG39) was measured with a
photodiode; the BG39 filter was added to block infrared light. The
photodiode failed to record any signal. This indicates that the OCF
produced <200 photons µm2 at the cornea in the
UV region of the spectrum.)
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In Figure 8, the time courses of recovery
of the responses to the UV and 500 nm probe flashes are summarized for
three animals. In each animal, the orange conditioning flash always
completely suppressed responsivity to the UV stimulus, although the
recovery of responsivity to the UV probe flash was always more rapid
than to the 500 nm flash. A calculation based on Equation 3
demonstrates that the orange conditioning flash used in the experiment
of Figures 7-8 should not produce a number of photoisomerizations of
the UV pigment sufficient even to generate a detectable response from the UV-sensitive pathway, much less drive this pathway into saturation. Thus, assuming that the absorbance of the UV pigment at long
wavelengths is described by Lamb's (1995) template 2 as fitted to the
data in Figure 6B, we calculated with Equation 3 that
the total light transmitted through the Schott OG530 filter is
equivalent to <200 quanta µm2 of 350 nm light
at the cornea. On the basis of the absolute sensitivity of the
cone-driven b-wave at 350 nm, ~8 nV (photon
µm2)1 (Fig. 5A, Table
1), this amount of 350 nm light is predicted to elicit a cone b-wave of
<2 µV; such a response would be practically undetectable and is
certainly far from saturation.
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Figure 8.
Recovery time course for cone b-wave responses
probed with 350 and 500 nm flashes. Recovery time course for the peak
amplitude of the response to a UV probe flash () and 500 nm probe
flash (), after exposure to an intense orange conditioning flash at
t = 0. Data points are mean ± SD for three
experiments identical to (and including) that of Figure 7, performed on
different mice. The response amplitudes are normalized by the amplitude
at 800 msec.
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Evidence for postreceptor functional distinctiveness of UV- and
M-cone signaling pathways
The experiments illustrated in Figures 7 and 8 cast doubt on the
use of any selective chromatic adaptation protocol for completely separating in the ERG signals originating in the UV- and M-cones. Nonetheless, there is reason to believe that the signaling pathways of
the two cone classes can be isolated by appropriate monochromatic flashes at the lowest flash strengths that elicit cone-driven responses. The action spectrum data of Figure 6B
provide evidence that in the UV region M-cone-driven responses should
be negligible near threshold. Additional evidence for isolation near
threshold is provided in Figure 4, where it can be seen that the lowest amplitude responses to the 340 and 500 nm flashes produce responses of
different shape: the response to the 340 nm flash has two to three
oscillatory bumps, whereas that to the 520 nm flash exhibits only a
single peak. The oscillatory potentials of the lowest amplitude traces
in Figure 4 may have been artificially smoothed by extensive on-line
averaging. To provide further evidence about the shapes of the
dim-flash responses in Figure 9 we
present responses from four additional animals. A pattern emerges: the
dim-flash, UV-driven responses characteristically exhibit a triple
oscillation, with the middle oscillatory peak coinciding with the peak
of the smoothed response. In contrast, the dim-flash responses to 500 nm light typically exhibit only a pair of oscillatory peaks, peaks that are not centered on the smoothed response maximum and have more variation in relative amplitude across animals than the UV-driven oscillations.
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Figure 9.
Dim-flash cone-driven b-waves for UV and midwave
light flashes. Noisy black traces are the responses of
four different mice (a-d) under cone-isolation
conditions to 340 nm (left) and 500 nm
(right) flashes that produce responses of amplitude
<20% of the maximum amplitude of the cone-driven b-wave; each trace
is the average of three to five individual records. Smooth,
thicker gray traces were obtained by filtering the responses
with a Gaussian filter of bandwidth 16.6 Hz, as in Figure 4.
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DISCUSSION |
Murine cone a-wave
On the basis of its rise time, its relative insensitivity to
steady light, and its magnitude, we tentatively identify the corneal-negative ERG component measured in the presence of
rod-saturating backgrounds (Fig. 2A,C) as a cone
a-wave, i.e., a field potential caused by suppression of cone
circulating current. The presumed saturated amplitude of the murine
cone a-wave was found to be ~20 µV, roughly consistent with
expectation. Specifically, based on a cone/rod density ratio
cone/rod = 0.03 (Carter-Dawson and LaVail, 1979a), amax,rod = 360 ± 132 µV
(Table 1), and the assumption that cones and rods have equal
circulating currents, the rod gauge (Eq. 18) predicts the saturating
cone a-wave to be 0.03 × 360 µV = 12 µV.
Rod and cone spectral sensitivities
The spectral sensitivities of murine rods and cones (Fig. 6)
derived from our ERG measurements are consistent with known facts about
murine photopigments (see introductory remarks). An interesting quantitative feature of the murine rod spectrum in situ
(Fig. 6A) is its relatively high sensitivity in the
-band (UV) spectral region, a sensitivity that ranges from 30 to
40% of that at the max; from this it is clear
that mice have effective rod vision in the UV region of the spectrum.
Absolute magnitudes of rod- and cone-driven b-waves: implications
for cell densities
Substantial evidence has now accumulated that the mammalian
scotopic b-wave is generated primarily by glutamate-modulated currents
of rod bipolar cells (e.g., see Robson and Frishman, 1995, 1996). The
rod b-wave maximum in mice under our recording conditions is
bmax,rod 400 µV, whether this number is
obtained by direct analysis of the saturation function of the scotopic b-wave (Pennesi et al., 1998; Pugh et al., 1998) or by subtraction of
the cone bmax from the total rod + cone b-wave
amplitude bmax,rod+cone (Table 1). Assuming a
rod/rod-bipolar (RB) ratio RB/rods = 1:20 to 1:50 (Dacheux and Raviola, 1986; Freed et al., 1987), the rod
gauge (Eq. 18) predicts the maximum rod bipolar "generator current"
to be JRB = 500-1250 pA.
There are many similarities between rod bipolars and cone on-bipolar
cellsincluding their radial retinal location and arborization and
their likely use of G-protein signaling cascades coupled to mGluR6
metabotropic receptors (Nakajima et al., 1993; Vardi et al., 1993).
Thus, on the assumption that the cone-driven b-wave originates in the
signal generator current of cone on-bipolars (COB), based on the
relative amplitudes of the rod b-wave maximum (400 µV) and
cone-driven b-wave maximum (200 µV) (Table 1), Equation 18
generates the prediction RB 2COB
JCOB. If the cone on-bipolar and rod bipolar
have the same magnitude maximum generator currents
(JRB JCOB),
then the spatial density COB of cone on-bipolars is
predicted to be one-third that of the bipolars,
RB, in mice.
Absolute sensitivities of rod- and cone-driven b-waves
The absolute sensitivity of the murine rod b-wave at the cornea is
50-100 µV (photon µm2)1; this
sensitivity can be derived from scotopic b-wave response versus
intensity data of several investigations, as summarized elsewhere
(Pennesi et al., 1998; Pugh et al., 1998). In contrast to that of the
rod-driven b-wave, the absolute sensitivity of the UV-cone-driven
b-wave at the cornea is ~8 nV (photon
µm2)1, ~10,000-fold lower than
that of the rod b-wave. How might such an enormous difference in
sensitivity arise? At least three factors are at play: (1) the
difference in individual rod- and cone-collecting areas "at the
cornea," (2) the intrinsic differences in sensitivities of rods and
cones, and (3) the difference in photoreceptor  bipolar convergence.
The first factor arises in the physical optics of rods and cones: the
cone-collecting area at the cornea is reduced by the Stiles-Crawford (SC) effect (Stiles and Crawford, 1933), whereby the
waveguiding properties of cones diminish the capture of light not
impinging axially on the cone inner segments (Snyder and Pask, 1973).
Because the mouse eye with fully dilated pupil has a large numerical
aperture (Remtulla and Hallett, 1985), light from the pupil margin can
strike the retina at angles as great as 30°. Assuming parameters
describing human cones (Snyder and Pask, 1973), we estimate that the SC
effect could reduce the effective area of the dilated mouse pupil for
cone-driven signals from its physical area of 3.1 mm2 to approximately one-third this value.
A second and major factor involved in the insensitivity of cone b-waves
relative to those of rods is the absolute sensitivities of the
photoreceptors themselves. From intracellular recordings, Schneeweis
and Schnapf (1995) estimated the absolute sensitivities of the
dim-flash response peaks of primate rods and cones to be ~1 mV/
and 5 µV/, respectively, giving a likely output gain ratio of 200.
A third factor reducing the light sensitivity at the cornea of
cone-driven b-wave responses relative to those of rods is the photoreceptor bipolar convergence ratios. The convergence ratio for
rods RBs is likely 20-30 times greater than that for cones cone on-bipolars (Dacheux and Raviola, 1986; Freed et al., 1987). This
convergence factor can be thought of in terms of the total surface area
at the retina available for collecting photons, which is ~20-30
times greater for rods than cones.
In summary, the product of the three factors considered, 3 × 200 × 20 = 12,000, appears more than sufficient to account
for the 10,000-fold ratio of rod- to cone-driven b-waves at the cornea. Because the estimates of the individual factors must be considered rough, however, differential "synaptic gain" between photoreceptors and bipolars cannot be ruled out.
The nature of the interaction of signals originating in murine UV-
and M-cone photopigments
The most surprising finding of this investigation is reported in
Figures 7 and 8: a long-wave conditioning flash can completely suppress
responsitivity of the UV-cone-driven signaling pathway. (The
cone-signal interaction hypothesis discussed in this section underscores a weakness in current cone nomenclature. In keeping with
past usage, we will continued to identify cones that express primarily the UV pigment as "UV-cones" and cones that
express primarily the M pigment as "M-cones.") Given that photons
captured by the UV photopigment cannot be the cause, what might
underlie the complete loss of responsivity to UV flashes seen in
Figures 7-8? Clearly, photons captured by a visual pigment with
absorption in the spectral band of the orange conditioning flash must
be forcing the pathway for UV-cone-driven responses into saturation, at
or before the level of the UV-sensitive b-wave generator. It follows
that photons captured by the M-cone photopigment must be the cause of
the loss of sensitivity to UV flashes, and thus that the M pigment and
UV pigment signaling pathways must be linked. One hypothesis for the
loss of UV responsivity is that M-cone signals block all UV-cone
signals at some site of convergence at or beyond the cone bipolar
synapse. Because M-cones are located exclusively in the dorsal half of
the mouse retina and most of the UV-cones are in the ventral half (Szel
et al., 1992), it is unlikely that the signals from M-cones could
completely suppress UV-cone signaling at any site of
convergence in the retina. An alternative hypothesis that can explain
the results in Figures 7-8 is that the M-cone pigment is expressed to
some degree in all the UV-cones. This latter hypothesis is not without
precedent: in suction electrode recordings from isolated salamander
cones, Makino and Dodd (1996) found that cones with peak sensitivity in
the UV have action spectra reflecting expression of three distinct opsins. Some evidence for the coexpression of the M pigment in the
UV-cones of mice was provided by the immunocytochemical investigation of Rohlich et al. (1994), who reported that in the transitional zone of
the retina between dorsal and ventral retina, there are many cones
labeled by antibodies to both S- (UV) and M-cone pigments. Even more
pertinent to our results are the very recent results of Gloesman and
Ahnelt (1998), who have found with antibodies to the UV- and M-cone
pigments double-labeling of all but a small minority of UV-labeled
cones in the ventral retina.
On the basis of the hypothesis that the cones expressing
primarily the UV pigment also express some M pigment, our data yield a
rough upper limit to the amount of M pigment present, as follows. The
time course of recovery of the response to the 500 nm flash is seen in
Figure 8 to be shifted by ~90 msec to the right of the recovery of
the response to the UV flash (consider, for example, the lateral shift
between the curves at the times of 20% recovery). Given that the
"dominant time constant" c of mammalian cone
recovery from saturating flashes has been estimated to be ~25 msec
(T. Kraft, personal communication), and the assumption that the
b-wave recovery tracks the cone recovery, the leftward shift of
T = 90 msec of the recovery to the UV flash dictates
that the UV-cones contain no more than
exp(T/c) = 0.03 of the M-cone
pigment [for analysis of the concept of a dominant recovery time
constant, see Pepperberg et al. (1996) and Nikonov et al.
(1998)]. In closing we note that coexpression of even 3% of M
pigment in the UV-cones provides a satisfactory explanation of the
ability of intense white flashes from flash units or apparatuses that
filter out UV light to saturate murine cone-driven b-wave signals.
| |
FOOTNOTES |
Received July 13, 1998; revised Oct. 19, 1998; accepted Oct. 21, 1998.
This work was supported by National Institutes of Health Grant
EY-02660, and the Penn Therapeutic Initiative for Retinitis Pigmentosa.
B.F. was supported by a fellowship from the Fulbright Foundation.
Correspondence should be addressed to Dr. E. N. Pugh Jr,
University of Pennsylvania, Department of Psychology, 3815 Walnut Street, Philadelphia, PA 19104-6196.
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Top
Abstract
Introduction
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