(Investigative Ophthalmology and Visual Science. 1999;40:3262-3267.)
© 1999
by The Association for Research in Vision and Ophthalmology, Inc.
A Point Mutation (W70A) in the Rod PDE
Gene Desensitizing and Delaying Murine Rod Photoreceptors
Daniel J. Salchow1,2,
Peter Gouras1,
Kentaro Doi1,
Stephen P. Goff3,
Eberhard Schwinger2 and
Stephen H. Tsang3,4
1 From the Edward S. Harkness Eye Institute, Department of Ophthalmology, Columbia University, New York, New York;
2 Institut für Humangenetik, Medizinische Universität zu Lübeck, Lübeck, Germany;
3 Howard Hughes Medical Institute Research Laboratories, Columbia University, New York, New York; and
4 Jules Stein Eye Institute, UCLA School of Medicine, Los Angeles, California.
 |
Abstract
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PURPOSE. To examine the corneal electroretinogram (ERG) of transgenic mice (W70A
mice) carrying a point mutation (W70A) in the gene encoding for the
-subunit of rod cGMP phosphodiesterase (PDE).
METHODS. The ERG of W70A mice was compared with that of normal mice. Cone
responses were separated from rod responses by light adaptation,
whereas rod sensitivity was assessed by threshold stimulation with dim
light. Spectral sensitivity curves of the ERG were obtained using a
constant response criterion.
RESULTS. The ERG of the W70A mouse has a desensitized, delayed rod b-wave at
threshold, and a prolonged rod b-wave at higher flash intensities. The
a-wave is absent even at maximal stimulation. The cone ERG of the W70A
mouse is indistinguishable from that of normal mice. The spectral
sensitivity of the W70A mouse is maximal in the UV spectrum, in
contrast to the normal mouse, which is most sensitive in the green
region of the spectrum. This supports the interpretation of the results
as normal cone and abnormal rod function in the W70A mouse.
CONCLUSIONS. The W70A mouse represents new model of stationary nyctalopia that can
be recognized by its unusual ERG features.
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Introduction
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When light reaches rod photoreceptors, rhodopsin is photoexcited
and activates a G-protein (transducin), which activates rod
phosphodiesterase (PDE) by removing the inhibitory -subunits from
the PDE core, so that it can hydrolyze cyclic guanosine monophosphate
(cGMP). The lowered cytoplasmic cGMP concentration closes cGMP-gated
cationic channels in the plasma membrane, causing the rod to
hyperpolarize, which is the adequate stimulus for second-order neurons
in the retina.
We have used targeted gene disruption to eliminate the expression of
the rod PDE, which leads to photoreceptor degeneration in these
mice.1
The introduction of a transgene with a point
mutation (alanine substitutes for tryptophan at position 70 [W70A] in
the 87 amino acids comprising the PDE molecule) created a distinct
phenotype.2
The retina of the W70A mouse does not
degenerate, inasmuch as electroretinograms (ERGs) of 13-month-old W70A
mice had the same appearance as those of young ones, and histology
revealed no abnormalities at 13 months of age.2
However,
the electrophysiology of rod photoreceptors is affected in the W70A
mouse. In single rod recordings, the response to light was desensitized
and delayed and the recovery of the response was prolonged. In previous
experiments, we found the W70A mouse desensitized but could not
identify a delay in the ERG corresponding to the delay in the single
rod responses.2
We have now identified what we consider to
be a delayed rod response in the ERG of the W70A mouse, bringing the
ERG into closer agreement with the single photoreceptor
electrophysiology. In this report, we characterize in vivo retinal
function of the W70A mouse and compare it with disorders found in
humans,3
4
5
6
7
8
9
10
and in murine11
12
13
models.
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Methods
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The creation of mice lacking PDE and of the W70A mouse has been
described in detail elsewhere,1
2
as have been the methods
used for genotyping and protein analysis.
ERGs were obtained from mice anesthetized with a mixture of ketamine
(50 mg/kg) and xylazine (10 mg/kg) administered intramuscularly. The
pupils were dilated with 1% phenylepinephrine and 1% cyclopentolate.
The mouse was placed on a heated stage calibrated to maintain the
temperature of the body around 37°C. A 30-gauge-needle reference
electrode was placed subcutaneously (SC) on the forehead and a similar
ground electrode on the trunk. A saline-moistened cotton wick electrode
contacted the cornea. The stimuli were obtained from a stroboscope that
was removed from its housing and mounted in a metal box with a circular
aperture, 3 cm in diameter and placed 9 cm from the center of the
pupil. This produces a field of approximately 20°; it is assumed that
most of the light stimulus is derived from scattered light. In support
of this assumption, ERGs obtained from normal and W70A mice, using a
full field dome surrounding the head of the mouse, yielded
qualitatively similar responses.13
Neutral density and
spectral filters could be placed in front of the aperture of the
stroboscope to change the intensity and the wavelength of the flash.
The following absorption filters were used: Kodak Wratten gelatin
filters 36, 50, 48, 75, 74, 21, and 29 (Eastman Kodak, Rochester,
NY) and Corning glass filters 5113 and 5970 (Corning Glass,
Corning, NY). The transmission of each filter was measured with
a spectrophotometer (Beckman Instruments, Palo Alto, CA), and the
wavelength of peak transmission: 410, 458, 471, 488, 538, 593, 633,
360, and 380 nm, respectively, was used for plotting spectral
sensitivity curves. The maximum flash intensity (in
µW/cm2) at the cornea delivered through each
spectral filter was measured with a digital photometer (J16; Tektronix
Instruments, Beaverton, OR), after removing infrared radiation
with an appropriate filter. The detector, 1 cm in diameter, was placed
at the level of the cornea so that the stroboscope light covered the
detecting area completely. The maximal light intensity of the white
light (unfiltered) flash was 0.8 x 103
µW/cm2 at the level of the cornea. The duration
of the flash was approximately 20 µsec, as stipulated by the
manufacturer of the stroboscope (Grass Instruments, Quincy, MA).
ERG responses were detected with an oscilloscope and an evoked
response–detecting computer in parallel (CA 1000; Nicolet Instruments,
Madison, WI), which averaged responses at a digitization rate of 1 MHz.
The bandpass of the input amplifier was 1 to 250 Hz. The mice were
dark-adapted overnight before testing. Stimulation was begun at 4.8
logarithmic units below maximum intensity of the stroboscope and
responses were averaged to one flash every second. At high flash
intensities, each flash was presented every 20 seconds, which was found
long enough to exclude interference of one flash to the next. To
determine the spectral sensitivity, we recorded responses to different
intensities at each wavelength, from threshold to suprathreshold levels
of stimulation. We determined the relative number of quanta per flash
to produce a constant criterion response at each wavelength and plotted
the reciprocal of these values on a logarithmic scale versus wavelength
on a linear scale as a spectral sensitivity function.
We also examined the light-adapted ERG by exposing the eye to a beam
focused on the pupil in Maxwellian view, which illuminated the entire
eye of the mouse. The beam was obtained from a slit lamp, entering the
eye slightly off the optical axis, to keep the mirror of the slit lamp
from blocking the strobe light. The brightness of this field, as seen
through the pupil of a human observer, was at maximum
107 candela (cd)/m2. The
level of brightness of the adapting field was changed by altering the
voltage across the bulb of the slitlamp.
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Results
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Figure 1
shows ERGs obtained from one representative normal and one W70A mouse.
The W70A mouse is less sensitive to light; its ERG shows a prolonged
b-wave with strong stimulation (upper traces) and lacks an initial
negative a-wave. Arrows highlight a slow positive wave that gradually
merges with the b-wave at strong stimulation. Figure 2
gives the b-wave amplitude and the b-wave implicit time as functions of
the light intensity; these data are combined from W70A mice derived
from different founders (line 1 and line 2). For the b-wave amplitude
(Fig. 2A) , the curve of the W70A mice is shifted to the right on the
abscissa (light intensity) by approximately two logarithmic units,
indicating desensitization of the W70A mouse. With maximal stimulation,
the amplitude of the b-wave of the W70A mouse approaches that of the
normal mouse. Figure 2B
shows the implicit time (time to peak after
stimulation) of the b-wave and also shows the implicit time of the late
positive wave of the W70A mouse. The implicit time of the b-wave of the
W70A mouse is relatively short at all light intensities, including
threshold. In contrast, the implicit time of the b-wave recorded from
normal mice is short at high light intensities but becomes longer in
response to dim stimuli, which is most accentuated at threshold. In the
W70A mouse, the implicit time of the late positive wave is much longer
but approaches that of normal mice in response to high-intensity
stimulation.
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Figure 1. (A) Dark-adapted ERGs of a normal mouse
(left) and a W70A mouse (right) at
different flash intensities, which are indicated by the neutral density
filtering in logarithmic units at the left of each
trace. The vertical line indicates 100 µV, except for
the bottom four traces on the left and the bottom six on
the right, where it represents 50 and 25 µV for the
responses with greater amplification (2x and 4x). The duration of
each trace is 300 msec. The arrows point out a slow
positive wave seen in the ERG response of the W70A mouse.
(B) Responses from a similar pair of mice at a slower time
base (700 msec per trace) to reveal the extreme slowness of the late
positive b-wave like response in the W70A mouse
(arrows).
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Figure 2. (A) B-wave amplitude plotted against the logarithm of flash
intensity for normal (n = 15) and W70A mice
(n = 9). Error bars, ±SEM. (B) B-wave
implicit time plotted against the logarithm of flash intensity for
normal and W70A mice. The implicit time of the slow positive wave, seen
only in the W70A mouse, is also shown. These data represent the average
of 15 normal mice, including 7 mice heterozygous for the W70A mutation,
which are indistinguishable from normal mice, and 9 W70A mice. The
implicit time of the slow response represents results from 6 mice.
Error bars, ±SEM.
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Figure 3
demonstrates that the ERG signature of the W70A mouse was consistent in
both transgenic lines, and Figure 4A
shows the effect of light adaptation on the ERG of normal and W70A
mice. In the dark-adapted state virtually no a-wave is detectable in
the ERG of the W70A mouse, whereas it is conspicuous in that of the
normal mouse. In the light-adapted state, the a-wave in the normal ERG
disappears, and the ERG of the W70A mouse is indistinguishable from
that of the normal mouse (Figs. 4A
, 4B)
.
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Figure 3. ERG responses to the same flash intensity (0.9 log units of neutral
density filtering) obtained from mice of different transgenic W70A
lines. Arrows indicate the late positive wave typical
for the ERG of the W70A mouse.
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Figure 4. (A) ERG responses of a normal (left) and a
W70A mouse (right) in the dark (top trace)
and at increasing levels of steady background light, (lower 2
traces). The strength of the adapting light is shown at the
left. The vertical line indicates 125
µV for the left and 63 µV for the right
column of responses. Each trace is 300 msec in duration.
(B) Amplitude of the a-wave (top) and b-wave
(bottom) plotted against the strength of the background
as the logarithm of candelas per square meter. This data represents the
average of five normal and five W70A mice. Error bars, ± SE.
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We examined the ERG to different parts of the spectrum, including the
UV region. Figure 5A
shows ERG responses to UV (360 nm), blue (450 nm), and yellow (575 nm)
stimuli from threshold to maximal levels for normal (above) and W70A
(below) mice. The late positive wave characteristic of the W70A
response is apparent at all wavelengths. The difference to the normal
ERG can be seen in spectral sensitivity functions (Fig. 5B)
. The
spectral sensitivity of the dark-adapted W70A mouse is 1 to 2
logarithmic units below that of the normal mouse at all wavelengths
except in the UV region, where it reaches similar levels. The maximum
sensitivity of the normal mouse is in the greenish region, whereas that
of the W70A mouse is in the UV region of the spectrum. The b-wave
implicit time at threshold in the normal ERG is between 130 and 145
msec from 400 to 650 nm; in the UV it is shorter. The implicit time of
the threshold b-wave of the W70A mouse is shorter at all wavelengths,
but the difference between normal and W70A mice is least in the UV
region.
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Figure 5. (A) ERG responses of a normal (top) and a
W70A mouse (bottom) to flashes from different parts of
the spectrum, i.e., at wavelengths of 360 (left), 450
(middle), and 575 (right) nm. Flash
intensities are indicated by the neutral density filtering at the
left of each trace. The vertical line at
the top right represents 125 µV. Each trace is 700
msec in duration. The arrows point out the slow b-wave
response in the W70A mouse. (B) Spectral sensitivity
functions (top) of normal (closed
circles) and W70A (open circles) mice. The
ordinate represents the logarithm of the reciprocal of
the relative number of quanta in a flash to elicit a constant response
of 50 µV. The abscissa represents the wavelength of
the flash in nanometers. The implicit time of the near-threshold
response is plotted against the wavelength of the flash
(bottom). These data are the average of five normal and
five W70A mice. Error bars, ±SE.
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All experiments were conducted on two different transgenic lines of
W70A mice, and the results have been found reproducible. No differences
could be detected in the ERGs of different lines of W70A mice.
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Discussion
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This report describes a unique pattern in the ERG of a transgenic
mouse, the W70A mouse. This mouse model is less sensitive and its
b-wave implicit time is shorter than that of normal mice. With strong
stimuli, the implicit times of the b-waves of normal and W70A mice
become similar. In the light-adapted state, the ERG of the W70A mouse
is indistinguishable from that of the normal mouse. This picture can be
explained by assuming that cones and not rods photoreceptors determine
b-wave thresholds in the W70A mouse, because cone b-wave implicit times
in the mouse are shorter than those of rods.14
Interestingly, the spectral sensitivity curve of dark-adapted W70A mice
is virtually identical with that of light-adapted normal
mice.15
This supports our conclusion that threshold
responses are predominantly cone-mediated in the W70A mouse.
The ERG of the W70A mouse furthermore has an unusual waveform. With
strong stimuli, there is virtually no a-wave and the b-wave is more
prolonged than the b-wave of the normal mouse. With weaker stimuli a
late b-wave-like response is detectable in the ERG of the W70A mouse,
which was not appreciated previously.2
This late,
insensitive response has an implicit time at threshold that is about
three times longer than that of the normal mouse. This is the same
order of magnitude at which single rod responses of the W70A mouse are
delayed at threshold.2
The abnormal response can be
explained by such delay combined with the insensitivity of rod
photoreceptors in the W70A mouse. As flash intensity increases, the
delayed rod response adds on to the cone response, producing a
prolonged b-wave. The lack of an a-wave also is explainable by the
insensitivity of W70A rods. A relatively strong rod response is
required before an a-wave becomes detectable, which the rods of the
W70A are incapable of producing at the flash intensities available. The
biochemical reasons for the delay and desensitization of the rod
photoresponse have already been discussed.2
In brief, the
W70A mutation impairs PDE activation and deactivation, resulting in
decreased sensitivity and slowed termination of the photoresponse.
The W70A mutation appears to leave the cones, including the UV ones,
unaffected, which is further support for the finding that rods and
cones use different forms of PDE.16
17
Because the rods
of W70A mice are desensitized, the spectral sensitivity of this mouse
is maximal in the UV region of the spectrum. In contrast, dark-adapted
normal mice are most sensitive to the greenish part of the
spectrum,18
19
where rhodopsin absorbs maximally. Normal
mice have a relatively high sensitivity in the UV region of the
spectrum. Our results show that in this region, the b-wave implicit
time at threshold becomes shorter, as if mediated in part by cones. The
relatively high sensitivity of murine UV cones in the light-adapted
state has been reported previously, and the presence of UV cones has
been demonstrated,15
which is supported by our results.
Recently rod responses have been detected in the Nougaret form of
stationary nyctalopia in man.20
In this case there is a
point mutation in the 
-subunit of transducin. Transducin binds
PDE to catalyze the rod photoresponse. This mutation in transducin
also desensitizes rod photoreceptors by about two logarithmic units,
but there is no delay in the threshold rod ERG because rod a- and
b-waves of normal waveform are elicitable. This appears to be a gain of
function mutation, which leaves the rods constitutively light-adapted.
In the W70A mutant there is a loss of function mutation; the defect is
only seen in the homozygous state, it is therefore recessive.
To summarize, the findings in the ERG of the W70A mouse—desensitized
rods in young and old mice, normal functioning cones, absence of
retinal degeneration—are characteristic for congenital stationary
nyctalopia. Thus, the W70A mouse exhibits a new form of stationary
nyctalopia. The unique ERG signature of this mouse should make it easy
to detect, if it were to occur in humans. It is also useful in
providing another example of a genetic defect in a protein involved in
rod phototransduction that does not lead to photoreceptor degeneration.
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Acknowledgements
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The authors thank Monica Mendelsohn and members of the Gouras and
Goff laboratories for support and discussion of this study.
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Footnotes
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Supported by National Institutes of Health Grant R01 EY 11510, Pro
Retina Deutschland e.V. (DJS), Research to Prevent Blindness (SHT),
Fight for Sight (SHT), and National Eye Institute (SHT).
Submitted for publication October 9, 1998; revised June 8, 1999; accepted June 24, 1999.
Commercial relationships policy: N.
Corresponding author: Daniel J. Salchow, c/o Peter Gouras, Edward S.
Harkness Eye Institute, Department of Ophthalmology, Columbia
University, 630 W. 168th Street, New York, NY 10032. E-mail: pg10{at}columbia.edu
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Top
Abstract
Introduction
