Content uploaded by Gerald Jacobs
Author content
All content in this area was uploaded by Gerald Jacobs
Content may be subject to copyright.
Available via license: CC BY-NC-SA 4.0
Content may be subject to copyright.
Human Cone Pigment Expressed in Transgenic Mice Yields
Altered Vision
Gerald H. Jacobs,
1
John C. Fenwick,
1
Jack B. Calderone,
1
and Samir S. Deeb
2
1
Neuroscience Research Institute and Department of Psychology, University of California, Santa Barbara, California
93106, and
2
Departments of Medicine and Genetics, University of Washington, Seattle, Washington 98195
Genetically driven alterations in the complement of retinal pho-
topigments are fundamental steps in the evolution of vision. We
sought to determine how a newly added photopigment might
impact vision by studying a transgenic mouse that expresses a
human cone photopigment. Electroretinogram (ERG) measure-
ments indicate that the added pigment works well, significantly
changing spectral sensitivity without deleteriously affecting the
operation of the native cone pigments. Visual capacities of the
transgenic mice were established in behavioral tests. The new
pigment was found to provide a significant expansion of the
spectral range over which mice can perceive light, thus under-
lining the immediate utility of acquiring a new photopigment.
The transgenic mouse also has the receptor basis for a novel
color vision capacity, but tests show that potential was not
realized. This failure likely reflects limitations in the organiza-
tional arrangement of the mouse retina.
Key words: cone photopigments; transgenic mice; visual sen-
sitivity; color vision; gene coexpression; retina
Photopigments have effective fixed spectral bandwidths across
which the efficiency of absorption varies significantly. Examina-
tion of the great variety of photopigments found in contemporary
species suggests that two evolutionary strategies have been used
to accommodate this restriction in absorption bandwidth to an
array of different visual demands. One is to spectrally position the
photopigment to optimize some visual capacities. A classic exam-
ple is the correlation between the spectral positioning of rod
pigments found in deep sea fishes and the spectral distribution of
light in their marine environments (Douglas et al., 1998). A
second strategy is to add new photopigment types. Two general
advantages may accrue from adding new photopigments: (1) the
available spectral window can be significantly expanded, and (2) if
there are appropriate nervous system connections the signals
originating from different types of photopigment may yield color
vision or other wavelength-specific behaviors. The addition of a
third type of cone pigment and the emergence of trichromatic
color vision in primates from a background of mammalian di-
chromacy provides a well known example of the utility of this
second strategy (Jacobs, 1993).
Although the genetic mechanisms underlying shifts in pho-
topigment spectra and the addition of new pigments have been
much studied in recent years (Nathans, 1987; Deeb and Motulsky,
1996; Neitz and Neitz, 1998), there is little understanding of how
the presence of novel photopigments can yield new visual capac-
ities. In one group of animals that have been studied, the New
World monkeys, it appears that newly added photopigments were
probably immediately translated into expanded visual sensitivity
and additional color vision capacities (Jacobs, 1998). Other cases
could presumably also require some modifications of neural ar-
chitecture for maximum exploitation of the new potential for
capturing light. As a step toward allowing an experimental exam-
ination of these issues, in a recent investigation a gene encoding
a human cone photopigment was introduced into the mouse
genome (Shaaban et al., 1998). The human transgene was abun-
dantly expressed in mouse cones and the pigment was found to be
efficient at transducing light. We have now examined the manner
in which the activity of this new photopigment is reflected in
signals in the outer retina and have conducted behavioral exper-
iments to determine to what extent the mouse visual system is
able to capitalize on the presence of a newly acquired
photopigment.
MATERIALS AND METHODS
Subjects. Male mice, both transgenic animals and wild-type controls
(C57/BL6), were studied. Details of the methods used to produce the
transgenic mice and to establish expression of the foreign photopigment
have been published previously (Shaaban et al., 1998). In brief, mice
transgenic for a human long-wavelength (L) photopigment gene were
generated by microinjection of fertilized mouse eggs. The pattern of
inheritance of the transgene was consistent with autosomal integration of
the gene. The experiments reported here were all conducted on descen-
dants of a mouse having two copies of the transgene. Tissue-specific
expression of the transgene was confirmed by performing reverse tran-
scription and PCR amplification on RNA obtained from whole eyes,
brain, and liver. Immunocytochemical analysis showed that the human
L-pigment was coexpressed with native pigment(s) in the outer segments
of ;80% of all mouse cones.
Electroretinogram measurements. There were two sets of experiments.
Electroretinogram (ERG) measurements were made to examine outer
retinal signals initiated by cone activity, and behavioral tests of vi sual
discrimination were conducted to provide a specific examination of the
contributions of the human L-cone pigment to mouse vi sion.
ERG spectral measurements were made using a flicker photometric
procedure that is f ully described elsewhere (Jacobs et al., 1996). Briefly,
light pulses derived from a three-beam optical system were presented to
the eye in Maxwellian view (59° circular spot). The optical system was
mounted on an adjustable platform that could be positioned so that the
beam entered the eye along the optic axis. At its focal point, the beam
from the optical system was smaller than the size of the fully dilated pupil
of the mouse; the latter is estimated to have a diameter of 2 mm
(Remtalla and Hallett, 1985). The photometer test light was from a
monochromator (10 nm half-energy passband). Its intensity was con-
trolled through rotation of a circular, 3.0 log unit neutral-density wedge
Received Dec. 9, 1998; revised Feb. 5, 1999; accepted Feb. 8, 1999.
This work was supported by Grants EY02052 (G.H.J.) and EY08395 (S.S.D.) from
the National Eye Institute. We thank Kris Krogh and Alexander Nugent for
technical assistance.
Correspondence should be addressed to Dr. Gerald H. Jacobs, Neuroscience
Research Institute, University of C alifornia, Santa Barbara, CA 93106.
Copyright © 1999 Society for Neuroscience 0270-6474/99/193258-08$05.00/0
The Journal of Neuroscience, April 15, 1999, 19(8):3258–3265
positioned in the light pathway. The photometer reference light and an
additional adaptation light constituted the other two beams. These orig-
inated from tungsten–halide lamps, and their content was varied through
the use of neutral-density step filters and interference filters (Optical
Thin Films, 10 nm half band). All three light sources were underrun at 11
V from regulated DC power supplies. High-speed mechanical shutters
were used to control the timing of test and reference lights. Light
intensities were measured in the plane of pupil using a supplier-
calibrated photodiode (Pin-10 DF, United Detector Technology).
To record ERGs, mice were anesthetized with an intramuscular injec-
tion of a mixture of xylaz ine hydrochloride (6.7 mg / kg) and ketamine
hydrochloride (67 mg/kg). The pupil of the test eye was dilated by topical
application of a mixture of 0.2% phenylephrine hydrochloride and 0.02%
cyclopentolate hydrochloride (Olsson et al., 1992). The mouse was posi-
tioned on a heating pad in a head restraint to allow alignment of the eye
with the optical system. ERGs were differentially recorded from a pair of
stainless-steel ring electrodes. One ring was against the cornea, and the
other was positioned farther back on the conjunctiva. Each ring con-
tacted the eye through a layer of artificial tears. A ground electrode was
placed in the mouth. All recordings were made in an illuminated room
(ambient illuminance at the test eye 5300 lux). ERGs were generated by
an interleaved train of square-wave pulses from the test and reference
lights, each modulated with a 25% duty cycle at 12.5 Hz. T he effective-
ness of test and reference lights were equated by adjusting the intensity
of the test light until the ERG it produced was equivalent to that
produced by the constant reference light. The signal processing proce-
dure has been described elsewhere (Jacobs et al., 1996). ERG photomet-
ric equations were made by iteratively adjusting the intensity of the test
light while recording the averaged responses to the last 50 of a total of 70
stimulus cycles. The wedge density values at the points of equation were
recorded to a precision of 0.01 log unit. Equations were made for each
stimulus on two separate occasions during an experiment, and these
values were subsequently averaged.
Visual discrimination. Visual capacities were determined using a three-
alternative, forced-choice discrimination. The test apparatus has been
described previously (Jacobs, 1983, 1984). The mouse viewed three
circular test panels (diameter and center– center di stance 52.5 cm)
positioned in a line along one wall of a small test chamber. T he panels
were transilluminated by lights originating from an optical system located
outside the chamber. The system has two sources. One is a tungsten –
halide lamp that was used to diffusely and equally illuminate each of the
panels (background lights). The other source is an Instruments SA
(Model H-10) grating monochromator (half-energy passband of 16 nm)
with a 75 W xenon lamp. An automated mirror system was used to direct
the light from this source (the test light) to any one of the three panels
through three diffusing ports. Depending on the experiment being
conducted, the test light either was added to the background light or
it completely replaced the background light. The interior of the test
chamber was diffusely illuminated by a ceiling-mounted fluorescent tube
(100 lux).
Through a shaping procedure mice were trained to detect the panel on
which the test light appeared. They signaled their choice by touching the
panel. Correct choices were reinforced by deliver y of a highly palatable
fluid (Soymilk, West Soy Plus Plain) that was automatically pumped in
increments of 0.028 ml from feeder tubes mounted directly above each
panel. Over successive test trials the location of the test light was
randomly alternated across the three panels. The nature of the difference
between lights on the positive and negative panels was systematically
varied to allow examination of several visual capacities as described
below. Each test trial was signaled by the occurrence of a cueing tone; the
tone terminated when the animal responded or after 15 sec without a
response. Intertrial duration was 6 sec. A penalty time was used such that
the onset of a test trial was delayed by a period of 5 sec after any
adventitious between-trial responses. A noncorrection procedure was
used. All aspects of stimulus presentation, reinforcement delivery, and
response monitoring were computer-controlled. The mice were tested
daily and fed standard lab food after each test session in an amount
sufficient to hold their weight at a constant level.
The mice were free to move about the test chamber during the
experiment. This makes difficult a precise specification of the stimuli.
The floor of the test chamber is adjustable in height, and it was set so that
the centers of the test panels were at approximately the height of a mouse
eye when the animal was in a normal standing posture. Trained subjects
were observed with a video camera in an attempt to estimate thei r
average positioning relative to the test panels during the performance of
actual discriminations. We concluded that, depending on the position of
the mouse, the effective stimulus could vary in angular si ze over a range
from ;14 to 60°, the average probably being closer to the latter value.
Our experience with rodent subjects in discrimination situations like this
is that they can and sometimes do develop distinctly idiosyncratic view-
ing behaviors. Nevertheless, the reinforcement contingencies are such
that they would be expected to lead subjects to adopt viewing strategies
that maximize their performance. The relatively small variation in per-
formance observed across subjects (see Fig. 4) suggests that this in fact
happened.
RESULTS
Cone photopigments in the transgenic mouse
The L -pigment of the transgenic mouse is one of the two com-
mon polymorphic versions of the human L -pigment (Winderickx
et al., 1992; Neitz et al., 1993). As studied in artificial expression
systems, measurements of this pigment have yielded two different
estimates of its
l
MAX
: 552 and 556 nm (Merbs and Nathans, 1993;
Asenjo et al., 1994). To obtain the best estimate of the spectral
position of that pigment as expressed in mouse cones, we mea-
sured the long-wavelength limb of the spectral sensitivity function
using ERG flicker photometry. A 630 nm reference light (1.1 3
10
15
quanta zsec
21
zsr
21
) was used to minimize contributions
from native mouse pigments. At that wavelength, the sensitivity
of the mouse middle-wavelength (M) cone pigment has declined
to a value that is ,2% of its peak sensitivity. For a similar reason
the test lights were restricted to long wavelengths only (from 580
to 680 nm in steps of 10 nm). Figure 1 shows spectral sensitivity
so measured for six transgenic mice. Note the small size of the
inter-animal variation. The photopigment absorption f unction
(Fig. 1, continuous line) that best accounts for this array of
sensitivity values has a
l
MAX
of 556 nm. That peak value is
hereafter taken as the best estimate of the spectral position of the
L-pigment in the transgenic mouse. Figure 2 summarizes the
spectra of the three cone photopigments in this transgenic mouse.
In addition to the L-pigment, there are two native cone pigments,
Figure 1. The long-wavelength portion of the spectral sensitivity func-
tion of the transgenic mouse. The data points are average sensitivity
values for six animals (61 SD) obtained from ERG flicker photometric
measurements. The data obtained from individual animals were not
normalized before the calculation of the deviations and that same con-
vention holds for other figures in this report. The curve i s that for a
photopigment absorption spectrum having peak sensitivit y at 556 nm. It
was obtained by shifting a photopigment nomogram curve (Dawi s, 1981)
along a wavenumber axis in successive steps of 1 nm until the best
least-squares fit to the data array was obtained.
Jacobs et al. •Human Cone Pigment Alters Vision in Transgenic Mice J. Neurosci., April 15, 1999, 19(8):3258–3265 3259
one with the peak absorption (
l
MAX
) in the ultraviolet (UV) at
;360 nm and an M-pigment with a
l
MAX
of ;509–512 nm
(Jacobs et al., 1991; Lyubarsky et al., 1998).
M/L spectral sensitivity
ERG measurements of cone spectral sensitivity were made in 11
transgenic mice. Sensitivity was determined over the wavelength
range from 450 to 650 nm at steps of 10 nm. The pulse rate of the
photometer was 12.5 Hz, and the reference light was achromatic
(2450 K). In Figure 3, the solid circles are mean sensitivity values
(61 SD) for the group. The variation between animals was quite
small (mean SD value for the 21 test wavelengths 50.123 log
unit). The shape of the sensitivity curve clearly indicates com-
bined contributions from signals originating in the native
M-pigment and the transgenic L-pigment. To provide an index of
that signal combination, we assumed that contributions from the
two pigments to the ERG flicker response are linearly summed.
Photopigment absorption spectra having peak values of 512 and
556 nm were added in varying proportions (in relative steps of
1%) to determine the combination providing the best fit to the
data array. The curve in Figure 3 is the best fitting combination:
14% (512) 186% (556).
Photopic spectral sensitivity f unctions were measured in be-
havioral experiments for six transgenic animals. An increment–
threshold procedure was used in which the three panels were
steadily illuminated with an achromatic light (color tempera-
ture 55350 K), and monochromatic light was added to one of
these panels during the test trial. Over trials the intensity of the
test light was varied in steps of 0.3 log units descending from a
level at which the animal showed high levels of discrimination
(80% correct or greater) down to an intensity that produced
chance performance. The test light was varied in 10 nm steps
from 450 to 630 nm. Performance measures were accumulated
over daily test sessions until a total of at least 100 test trials had
been run at each wavelength/intensity combination. From these
cumulated values psychometric functions were constructed by
plotting mean percentage correct as a f unction of stimulus inten-
sity. These averaged data points were then fit to a logistic f unc-
tion having asymptotes of 100 and 33% correct with the variance
and mean as free parameters. The f unction providing the best
least-squares fit to the data set was determined. From these
functions, thresholds were subsequently computed as the stimulus
intensity required to yield performance corresponding to the
99% upper-confidence level.
The behavioral spectral sensitivity f unctions obtained when the
background lights were set to a luminance of 13.2 cd /m
2
are
summarized in Figure 4. The solid circles are mean values (61
SD) for six transgenic mice. The individual variation in the
increment threshold measurements was quite similar to that seen
for the ERG measurements (mean SD across all test wave-
lengths 50.14 log unit). The fitted f unction (continuous line) was
determined in the same manner as for the ERG spectral data, i.e.,
by seeking the best-fitting summative combination of pigment
absorption curves having respective peak values of 512 and 556
nm. As can be seen, the combination 512 (68%) 1556 (32%)
Figure 2. Schematic representation of the spectral sensitivity curves for
the three cone pigments of the transgenic mouse. The native pigments
(UV and M) have respective peak values of 360 and 512 nm. The peak of
the transgenic pigment (Human L) is 556 nm. The shapes of the curves
were generated from photopigment nomograms (Dawis, 1981). The spec-
tral positioning and shapes of the
b
peaks for M- and L -pigments are
those suggested by Palacios et al. (1998).
Figure 3. Cone spectral sensitivity f unctions for transgenic mice. T he
values (means for 12 animals 61 SD) were obtained from ERG flicker
photometric measurements. The sensitivity value plotted at 550 nm cor-
responds to a light intensity of 2.93 310
13
photons zsec
21
zsr
21
.The
curve is the best-fitting linear summation of two photopigment absorption
curves having respective peak values of 512 (15%) and 556 (85%).
Figure 4. Spectral sensitivity f unctions for six transgenic mice (mean
values 61 SD) and one wild-type mouse as determined in an increment–
threshold discrimination task. The sensitivity value for 500 nm corresponds
to a panel light intensity of 1.3 310
11
photons zsec
21
zmm
221
. The curve
drawn through the data for the wild-type mouse i s a photopigment
absorption curve having a 512 nm peak. The curve fit to the results from
the transgenic mice is the best-fit linear summation of two photopigment
absorption curves: 512 nm (68%) 1556 nm (32%).
3260 J. Neurosci., April 15, 1999, 19(8):3258–3265 Jacobs et al. •Human Cone Pigment Alters Vision in Transgenic Mice
provides a good account of the spectral sensitivity function.
Shown for comparison in Figure 4 is the spectral sensitivity
function (triangles) for a single wild-type mouse determined in
exactly the same manner as for the transgenic mice. The curve fit
to the latter data are the absorption spectrum for a single pigment
with a
l
MAX
of 512 nm. Comparison of the two curves makes
clear the very substantial increase in sensitivity in the long wave-
lengths provided by the L -pigment in the transgenic mouse.
In a second experiment, complete spectral sensitivity f unctions
(over a range from 450 to 620 nm) were additionally obtained for
four of the transgenic mice with the background light levels
increased to luminance of 26.9 cd /m
2
. Those f unctions (data not
shown) were in no way qualitatively different from those of Figure
4. At this higher background light level, the combination of the
two pigment absorption curves needed to best fit the averaged
spectral sensitivity f unction were very slightly different: (61%)
512 1(39%) 556. Finally, spectral sensitivity functions were also
measured over the range of 450 to 630 nm for two transgenic mice
with a dimmer background light (6.7 cd/m
2
). Again, the spectral
sensitivity f unctions were similar to those of Figure 4; the best
combined fit of the two cone pigments to this f unction was (75%)
512 1(25%) 556.
Sensitivity to UV light
Although our interest in these experiments was focused on the
effects of the added L-pigment, we also wanted to determine
whether the presence of this new pigment might exert some effect
on the operational integrity of the native pigments. The substan-
tial spectral overlap of the L-pigment with native M-pigment
makes such a determination difficult (Fig. 2). It is somewhat more
straightforward to do this in the case of sensitivity to the UV.
Accordingly, we measured spectral sensitivity down into the UV
range using both ERG and behavioral discrimination. Figure 5
(top) shows ERG spectral sensitivity f unctions obtained from a
transgenic and a control animal. There is clear evidence for
robust contribution to the spectral sensitivity f unction by the UV
cone in both animals. Figure 5 (bottom) also shows behaviorally
determined spectral sensitivity f unctions for two transgenic mice
and a single control animal. Although there is relatively much
lower sensitivity in the UV wavelengths for behavioral than for
ERG measurements, there is no obvious difference between the
transgenic and the wild-type animals. We return below to the
issue of the disparity in UV sensitivity seen in ERG and behav-
ioral tests.
A comparison was also made between the amplitudes of the
ERG cone-based signals in transgenic and wild-type mice. Am-
plitudes were measured using a stimulus flickering at 12.5 Hz at
three test wavelengths: 390, 500, and 660 nm. These three lights
were presented at the maximum intensity available from the
monochromator (1.87 310
13
, 2.79 310
14
, 4.77 310
14
quanta zsec
21
zsr
21
, respectively), with the responses averaged
for five separate presentations, each of which consisted of 50
flashes. The bar graph in Figure 6 summarizes these results for 10
transgenic and 6 control animals. Although there is considerable
individual variation, the transgenic and control mice showed no
significant differences in responsivity to 390 and 500 nm lights.
Not surprisingly, however, the presence of the L -pigment made
the transgenic mice much more responsive to the 660 nm test
light. Both the spectral sensitivity and the amplitudes recorded to
UV test lights would be expected to be significantly dependent on
activity generated by the mouse UV pigment. There seem to be
no clear differences in this regard for transgenic and control mice.
Spectral limits
It is clear from the above that the presence of the added L -cone
pigment significantly enhances sensitivity to long wavelength
lights in the transgenic mice. To document that effect, we deter-
mined the spectral limits for these animals. For this, mice were
required to discriminate the presence of a monochromatic light
added to a dim background light (0.41 cd /m
2
). The wavelength of
Figure 5. Full spectral sensitivity f unctions for wild-type (Œ) and trans-
genic (F) mice as determined with ERG flicker photometry (top, 1 animal
each) and behavioral increment–threshold measurements (bottom,2
transgenic mice, 1 wild-type mouse). The continuous curves are linear
summations of the photopigment spectra shown in Figure 2.
Figure 6. Mean ERG response amplitudes obtained from transgenic
(n510) and wild-type mice (n56) for three test wavelengths (error bars
represent 2 SEM). The stimuli were monochromatic lights flickering at
12.5 Hz.
Jacobs et al. •Human Cone Pigment Alters Vision in Transgenic Mice J. Neurosci., April 15, 1999, 19(8):3258–3265 3261
the monochromatic light was varied across the spectrum to allow
a determination of the limits of sensitivity. The monochromatic
lights were presented at a single intensity, the maximum available
at that wavelength from the source monochromator. The varia-
tion in intensity of the test light across the spectrum is shown at
the bottom of Figure 7. Two transgenic and two wild-type mice
were trained to discriminate the presence of the test light. The
test wavelength was varied in 10 nm steps over the following
ranges: 360– 410, 510 –580, and 630 – 680 nm. Once the mice were
fully trained, their performance on a total of 100 test trials was
accumulated at each of the wavelengths. The average perfor-
mance for each of the four animals is plotted in Figure 7. There
was no significant variation among these animals for any wave-
lengths shorter than 630 nm. However, at wavelengths longer than
that, the two types of mice diverged in performance. The trans-
genic mice (circles) were able to see much longer wavelength
lights than the control animals (triangles). For a criterion level of
performance (57% correct; Fig. 7, dashed line), the wild-type
mice failed at 645 nm, whereas the transgenic animals were
successf ul out to a wavelength of 676 nm. The presence of the
L-pigment has expanded the spectral window by at least 30 nm.
Effects of exposure to chromatic light
The M- and L -pigments in the transgenic mouse are well sepa-
rated in their spectral sensitivities (Fig. 2). This allows the pos-
sibility that spectral sensitivity may be selectively altered by
exposure to lights of different chromatic content. In previous
ERG measurements we showed that small shifts (;0.10 log unit)
in spectral sensitivity could be produced by intense chromatic
adaptation (Shaaban et al., 1998). The effects were of a size
consistent with passive pigment bleaching. Here we examined the
effects of chromatic adaptation on visual discrimination.
To accomplish this we measured increment thresholds on chro-
matic backgrounds. Specifically, 500 and 600 nm lights were used
both as test lights and background lights so that a total of four
thresholds were measured: two homochromatic and two hetero-
chromatic thresholds. The logic is that if these two lights have no
differential influence on spectral sensitivity, then on average the
heterochromatic thresholds will equal the homochromatic thresh-
olds; alternatively, if there is a differential influence, the hetero-
chromatic thresholds will be lower than the homochromatic
thresholds (Boynton et al., 1965). The four thresholds were mea-
sured using exactly the procedures described for assessing spec-
tral sensitivity (above). The intensity of the 600 nm background
light (109.6 cd/m
2
) was set to the brightest value that could be
used consistent with our still being able to measure a threshold
for a 600 nm test light. The intensity of 500 nm background light
was then adjusted according to the mouse photopic spectral
sensitivity f unction (Fig. 4) to make it approximately equally
effective to the 600 nm background light. Four transgenic mice
were tested. Because there were noticeable variations among
animals, a total of five separate threshold measurements were
made for each of the four test lights. To provide a comparative
baseline, exactly analogous tests were run on two wild-type mice
and on two human trichromats.
The results from these tests are summarized in Figure 8.
Plotted for each of eight subjects is an index of adaptation, here
defined as the difference in average (log) sensitivity between the
heterochromatic and homochromatic tests (error bars 51 SD).
Wild-type mice have only a single cone photopigment active over
the 500 to 600 nm portion of the spectrum. They should thus show
no differential chromatic adaptation, and they did not. To the
contrary, each of the transgenic mice did show evidence for
significant differential adaptation. For three of these animals the
magnitude of the effect was roughly similar; for the fourth animal
(result plotted leftmost in Fig. 8) the influence of different chro-
matic backgrounds was consistently larger. Adaptation effects for
the transgenic mice were in turn much smaller than that measured
for trichromatic humans comparably tested.
Wavelength discrimination
The presence of two photopigments that can be differentially
affected by chromatic adaptation raises the possibility that the
visual system of the transgenic mouse might be able to use signals
from M- and L-pigments to permit pure-wavelength discrimina-
tion, i.e., have color vision. We tested three transgenic mice to
determine whether they could make such a wavelength discrimi-
nation. For this, the test light was set to 500 nm, and the other two
Figure 7. Top, Spectral limits determinations for two transgenic (circles)
and two wild-type (triangles) mice. The plotted values represent the
average discrimination performance on 100 test trials at each tested
wavelength. The dashed line indicates threshold performance. Bottom,
The intensities of the test stimuli used as measured with the detector
(surface area 51cm
2
) placed against the stimulus panels. The ordinate
values are quanta zsec
21
zmm
22
310
12
).
Figure 8. Results from a chromatic adaptation test. The adaptation index
is based on a comparison of four thresholds (2 heterochromatic, 2 homo-
chromatic) measured in a discrimination task. See Results for details.
Values for the mice represent means (error bars represent 1 SD) for five
replications of the experiment. Two trichromatic human subjects were
tested in a single session.
3262 J. Neurosci., April 15, 1999, 19(8):3258–3265 Jacobs et al. •Human Cone Pigment Alters Vision in Transgenic Mice
panels were illuminated with 600 nm light. Unlike all of the
previous discrimination tests, in this experiment the test light was
not added to the background light but instead replaced it on each
trial. The intensity of the 600 nm light was the same as in the
previous experiment. A first step was to determine the intensity of
the 500 nm test light required to render it equally as bright as the
600 nm light. The way this was done is summarized in the inset to
Figure 9. Each of the three mice had previously participated in
several experiments in which they had been reinforced for picking
the brighter of three test lights. Consequently, at the point this
experiment was initiated they were well trained to select the
brighter of two stimuli. To determine a brightness equation, the
intensity of a bright 500 nm light was progressively dimmed in
steps of 0.2 log unit. When the 500 nm light was much brighter
than the 600 nm light, the animal selected it consistently, but as
the intensity was decreased the percentage of correct selections
declined and eventually reversed, i.e., the animal began to more
frequently select the 600 nm light because it was now, presumably,
the brighter of the two. The point at which performance passed
through the level of chance (33%; Fig. 9, dashed line) was taken as
the point of equal brightness between the two lights. Note that the
equation value was effectively the same for each of the three
animals, varying across subjects by ,0.1 log unit.
With the brightness equations established, each animal was
tested to determine whether it could learn to successf ully dis-
criminate between equally bright 500 and 600 nm lights. In each
daily test session, the two lights were presented at the calculated
point of equal brightness and on equal numbers of trials for values
that were both 0.2 log unit higher and lower. The average perfor-
mance for the equal brightness pairings for each test session is
shown at the bottom of Figure 9. These tests were continued until
it became apparent that the mice were unable to acquire this
discrimination. The total number of test trials accumulated for
the three subjects varied over the range from 8100 to 13,500. As
can be seen, across all that experience with the pair of test lights
there is no deviation from chance performance. To be certain
that this failure reflected the specific absence of a capacity for this
discrimination, not merely some general decline in performance,
a systematic difference (0.5 log unit) in the brightness of the two
lights was introduced at the end of the test period (shown to the
right of the vertical dashed line in Fig. 9). In the presence of this
systematic brightness cue, each of the three animals almost im-
mediately began to show successf ul discrimination. The results of
this experiment seem clear-cut: these transgenic mice are inca-
pable of making a pure-wavelength discrimination between 500
and 600 nm lights.
DISCUSSION
In recent years transgenic mice have been used to study basic
retinal physiology and to provide models for investigating retinal
disease. T ypically this has been done through expression of
mutant genes that influence rods (Lem and Makino, 1996). Our
experiments have involved a different approach in that a foreign
cone pigment gene was expressed in a transgenic animal for the
purpose of examining the extent to which a visual system can
incorporate and exploit a new source of environmental informa-
tion. The results make clear that photons absorbed by a newly
acquired photopigment expand significantly the spectral range
over which mice can see. An added class of photopigment theo-
retically could also underlie a new dimension of spectral experi-
ence, but that did not occur.
Cone photopigment arrangements and neural
organization in transgenic mice
The retinas of the transgenic mice contain three classes of cone
photopigment: the native UV and M-pigments and the transgenic
L-pigment (Fig. 2). Previous antibody labeling experiments
showed that the L-pigment is coexpressed with the UV and
M-pigments in many, although not all, cones of the transgenic
mouse (Shaaban et al., 1998). Because of the current ambiguity
about the distribution of cone pigments in the wild-type mouse, it
is not possible to be more precise about the cones of the trans-
genic mouse. The history of the issue is as follows. The first
opsin-antibody labeling experiments on the mouse retina de-
tected two classes of cone in the mouse retina that had a unique
topographic organization (Szel et al., 1992). Cones expressing
M-pigment were thought to be restricted to a region of the retina
located roughly superior to the horizontal meridian; cones ex-
pressing UV pigment were found throughout the retina, but were
at highest density in the ventral half of the retina. Later antibody
Figure 9. Test for wavelength discrimination in three trans-
genic mice (separate symbols). The inset at the top shows
results from the procedure that was used to establish indi-
vidual brightness equations for 500 and 600 nm lights (see
Results). Below are the average performance values re-
corded in daily test sessions in which each mouse was re-
quired to discriminate between equally bright 500 and 600
nm lights. The horizontal line shows the average performance
across the test period (vertical bar 52 SDs). The perfor-
mance to the right of the vertical dashed line was recorded
subsequent to the addition of a systematic brightness differ-
ence (0.5 log unit) between the stimulus pair.
Jacobs et al. •Human Cone Pigment Alters Vision in Transgenic Mice J. Neurosci., April 15, 1999, 19(8):3258–3265 3263
labeling experiments suggested that the mouse retina has a tran-
sitional zone between the superior and inferior retinal areas,
some 100–500
m
m in width, that contains cones coexpressing UV
and M-pigments (Rohlich et al., 1994). This implies that there are
three populations of cone in the retinas of wild-type mice as
defined by their photopigment complements (Table 1), but there
is considerable uncertainty about the relative representation of
these three cone types. That is because a recent antibody-labeling
experiment suggests that cones coexpressing UV and M-pigments
are much more widespread than the earlier report indicated
(Glosmann and Ahnelt, 1998), perhaps extending over much of
the entire retina. That contention receives support in results from
an electrophysiological experiment (Lyubarsky et al., 1998). In
sum, there are potentially six types of cone in the transgenic
mouse that have distinctly differing spectral sensitivities (Table
1), but their relative representation and spatial distributions are
not known.
Earlier measurements indicated that in transgenic mice of the
type used here L-pigment was expressed to a level that accounted
for ;77% of the total of M- and L-pigments (Shaaban et al.,
1998). The best summative fit of M and L contributions to the
averaged ERG spectral sensitivity curve (Fig. 3) required a sim-
ilar weighting (L, 86%), suggesting that the relative representa-
tion of the two pigments is straightforwardly reflected in the
amplitudes of ERG cone signals. The relationship is thus similar
to that seen in the human retina where the L / M weighting
required to best fit spectral sensitivity curves obtained from the
flicker ERG appears to index the relative numbers of L and M
cones (Jacobs and Neitz, 1993).
The relative influence from L -pigment signals was considerably
lessened in the behavioral measurements of spectral sensitivity
where the identical fitting procedure yielded a required L -cone
contribution of only 36% of the total of M 1L (Fig. 4). There are
numerous differences inherent in the measurement procedures
that could potentially influence the relative contributions of M-
and L-pigments to outer retinal signals and behavioral sensitivity:
for example, differences in light levels and state of adaptation.
One possible key to understanding the relative reweighting of M
and L influence is that UV cone signals were also represented
very differently in ERG and behavioral measurements. Specifi-
cally, cones containing UV pigment contribute relatively much
less to behavioral spectral sensitivity than they do to the ERG
spectral sensitivity f unctions (Fig. 5, compare top and bottom).
Because many cones in the transgenic mouse that contain UV
pigment also contain L -pigment, the loss of influence from the
two pigment types in the behavioral measures likely may simply
reflect a diminution in the influence of the signals from cones
containing UV pigment. Whatever the reason for this, and several
could be imagined, it is intriguing that apparently robust UV
cone signals do not contribute much to mouse vision in this test
situation.
Even with modest light levels, significant alterations in behav-
ioral M/L spectral sensitivity were produced by chromatic adap-
tation. These effects were substantially larger than those obtained
earlier for analogous ERG measurements made at much higher
light levels (Shaaban et al., 1998). A minimal implication of this
result is that signals originating from M- and L -pigments must be
represented, at least to some extent, in separable neural pathways
at locations beyond the ERG generators. One of the four trans-
genic mice tested showed significantly larger adaptation effects
than did the other three (Fig. 8). This animal was not unusual in
any other way; in particular, his spectral sensitivity functions were
not extreme in their relative representation of M- and L -pigment
signals.
Absence of M/L color vision in transgenic mice
The minimal requirements for vertebrate color vision include the
presence of more than one spectral mechanism and a means for
comparing the outputs from these spectral mechanisms some-
where in the nervous system. As far as is now known, the latter is
always accomplished through the presence of spectrally opponent
cells. From the six presumed receptor types in the transgenic
mouse retina (Table 1), there are eight separate pairings that
have sufficiently different spectral sensitivity to 500 and 600 nm
lights that they could potentially provide inputs that might allow
for color discrimination; in particular, of these, the M versus UV
1L receptors would be expected to provide strikingly different
relative sensitivities to the 500 and 600 nm lights. So a potential
receptor basis for M / L color vision exists in the transgenic
mouse. The details of the neural organization of the mouse retina
remain, somewhat surprisingly, relatively unstudied, but there are
two facts potentially relevant to the present case. First, many
mouse ganglion cells show antagonistic center/surround organi-
zations (Balkema and Pinto, 1982; Stone and Pinto, 1993). If
these centers and surrounds segregate different spectral inputs in
the transgenic mouse they could provide the information required
for the elaboration of M/L color vision. Second, a brief report
suggests an abundant presence (nearly 20% of all cells recorded)
of spectral opponency among mouse ganglion cells (Yamamoto
and Gouras, 1993). The opponency is described as involving
short- and middle-wavelength mechanisms. The former are pre-
sumably UV cones, and the latter could conceivably be either M
cones or rods. Both of these findings allow the possibility that the
mouse retina may have a neural organization that could be used
to produce novel color vision in the transgenic mouse.
It was against this background of research and our own obser-
vation that significant differential chromatic adaptation can be
produced in the transgenic mouse that we were encouraged to
determine whether these animals had acquired some new color
vision. A failure to demonstrate a sensory capacity in discrimi-
nation tests requires cautious interpretation. In particular, an
alternative test paradigm or training strategy might yield a dif-
ferent outcome. Nevertheless, the present results seem quite
convincing: the added L-pigment substantially expands the spec-
tral window of mouse, but it does not allow for any new chromatic
discriminations.
The fact that the added cone pigment did not translate into new
color vision in the transgenic mouse unexpectedly raises ques-
tions about color vision in the wild-type mouse. The reason is as
follows. The wild-type mouse might be predicted to have dichro-
matic color vision, mediated by neural comparison of signals from
Table 1. Range of cone types in wild-type and transgenic mice as
defined by photopigment complement
Wild type Transgenic
UV UV
UV 1L
UV 1MUV1M
UV 1M1L
M1L
MM
3264 J. Neurosci., April 15, 1999, 19(8):3258–3265 Jacobs et al. •Human Cone Pigment Alters Vision in Transgenic Mice
cones containing UV and M-pigments. However, these same
receptors and their neural connections should also have provided
the substrate for any added M/L color vision in the transgenic
mouse, e.g., by comparison of M versus UV 1LorM1UV
versus UV 1L (Table 1). The failure to see M versus L color
vision in the transgenic mouse thus raises a doubt about the
presence of any significant UV versus M color vision in the
wild-type mouse.
Receptor coexpression of photopigments
It has long been known that numerous species of fish and am-
phibia can construct photopigments from either of two chro-
mophores, frequently interchanging the two during lifestyle
changes such as migration or metamorphosis. These chro-
mophore changes yield shifts in the absorption spectra of pho-
topigments, and a consequence is that individual photoreceptors
may contain a pair of pigments having different spectra (Bow-
maker, 1991). There are recent indications that multiple pigments
can be similarly expressed in a single receptor in some species
through the presence of multiple opsins, each of which is com-
plexed to the same chromophore. For instance, microspectropho-
tometric measurements of cones in the retina of the guppy (Po-
ecilia reticulata) yielded spectra that appear to reflect the
combined presence of two photopigments that are peak-
separated by nearly 40 nm (Archer and Lythgoe, 1990), whereas
UV-sensitive cones in tiger salamanders (Ambystoma tigrinum)
may have as many as three active opsins that allow the photore-
ceptor to significantly absorb light all the way across the visible
spectrum (Makino and Dodd, 1996). Recent antibody-labeling
experiments suggest the presence of some cones containing two
spectrally discrete photopigments in a number of mammalian
species; the mouse (as described here), the guinea pig, and the
rabbit retina contain a population of cones that coexpress two
different photopigments (Rohlich et al., 1994). All of these exam-
ples suggest that the mice that are the focus of this investigation
provide a less artificial model than would have been supposed
even a few years ago. Our measurements show that coexpression
of pigments in single receptors can yield the immediate advantage
of an expanded spectral window, so the transgenic mouse may
provide a model of a normal step in the evolution of visual
systems. Whether that is true or not, the ability to obtain good
functional expression of a human photopigment in the mouse
opens the door to examination of the consequences of mutated
versions of human cone pigment genes in a well defined model
system.
REFERENCES
Archer SN, Lythgoe JN (1990) The visual pigment basis for cone poly-
morphism in the guppy, Poecilia reticulata. Vision Res 30:225–233.
Asenjo AB, Rim J, Oprian DD (1994) Molecular determinants of human
red/green color discrimination. Neuron 12:1131–1138.
Balkema Jr GW, Pinto L H (1982) Electrophysiology of retinal ganglion
cells in the mouse: a study of a normally pigmented mouse and a
congenic, hypopigmentation mutant, pearl. J Neurophysiol 48:968 –980.
Bowmaker JK (1991) Visual pigments, oil droplets and photoreceptors.
In: The perception of colour (Gouras P, ed), pp 108 –127. Boca Raton,
FL: CRC.
Boynton RM, Scheibner H, Yates T, Rinalducci E (1965) Theor y and
experiments concerning the heterochromatic threshold-reduction fac-
tor (HTRF). J Opt Soc Am A 55:1672–1685.
Dawis SM (1981) Polynomial expressions of pigment nomograms. Vi-
sion Res 21:1427–1430.
Deeb SS, Motulsky AG (1996) Molecular genetics of human color vision.
Behav Genet 26:195–207.
Douglas RH, Partridge JC, Marshall NJ (1998) The eyes of deep-sea
fish. I: Lens pigmentation, tapeta and visual pigments. Prog Retinal Eye
Res 17:597–636.
Glosmann M, Ahnelt PK (1998) Coexpression of M- and S-opsin ex-
tends over the entire inferior mouse retina. Invest Ophthalmol Vis Sci
39:S1059.
Jacobs GH (1983) Within-species variations in visual capacity among
squirrel monkeys (Saimiri sciureus): sensitivity differences. Vision Res
23:239–248.
Jacobs GH (1984) Within-species variations in visual capacity among
squirrel monkeys (Saimiri sciureus): color vision. Vision Res
24:1267–1277.
Jacobs GH (1993) The distribution and nature of colour vision among
the mammals. Biol Rev 68:413–471.
Jacobs GH (1998) A perspective on color vision in platyrrhine monkeys.
Vision Res 38:3307–3313.
Jacobs GH, Neitz J (1993) Electrophysiological estimates of individual
variation in the L / M cone ratio. In: Colour vision deficiencies X I
(Drum B, ed), pp 107–112. Dordrecht: Kluwer.
Jacobs GH, Neitz J, Deegan II JF (1991) Retinal receptors in rodents
maximally sensitive to ultraviolet light. Nature 353:655– 656.
Jacobs GH, Neitz J, Krogh K (1996) Electroretinogram flicker photom-
etry and its applications. J Opt Soc Am A 13:641–648.
Lem J, Makino CL (1996) Phototransduction in transgenic mice. C urr
Opin Neurobiol 6:453– 458.
Lyubarsky AL, Falsini B, Pennesi M E, Valentini P, Pugh Jr EN (1998)
UV- and midwave-sensitive cone-driven retinal responses of the mouse:
a possible phenotype for coexpression of cone photopigments. J Neu-
rosci 19:442–455.
Makino CL, Dodd RL (1996) Multiple visual pigments in a photorecep-
tor of the salamander retina. J Gen Physiol 108:27–34.
Merbs SL, Nathans J (1993) Role of hydrox yl-bearing amino acids in
differentially tuning the absorption spectra of the human red and green
cone pigments. Photochem Photobiol 58:706 –710.
Nathans J (1987) Molecular biology of visual pigments. Annu Rev Neu-
rosci 10:163–194.
Neitz M, Neitz J (1998) Molecular genetics and the biological basis of
color vision. In: Color vi sion–perspectives from different disciplines
(Backhaus WGK , K liegl R, Werner JS, eds), pp 101–119. Berlin:
Walter de Gruyter.
Neitz J, Neitz M, Jacobs GH (1993) More than three different cone
pigments among people with normal color vision. Vision Res
33:117–122.
Olsson JE, Gordon JW, Pawlyk BS, Roof D, Hayes A, Molday RS, Mukai
S, Cowley GS, Berson EL, Dryja T P (1992) Transgenic mice with a
rhodopsin mutation (Pro23His): a mouse model of autosomal dominant
retinitis pigmentosa. Neuron 9:815–830.
Palacios AG, Varela FJ, Srivastava R, Goldsmith TH (1998) Spectral
sensitivity of cones in the goldfish, Carassius auratus. Vision Res
38:2135–2146.
Remtulla S, Hallett PE (1985) A schematic eye for the mouse, and
comparisons with the rat. Vision Res 25:21–31.
Rohlich P, van Veen T, Szel A (1994) T wo different visual pigments in
one retinal cone cell. Neuron 13:1159 –1166.
Shaaban SA, Crognale M A, Calderone JB, Huang J, Jacobs GH, Deeb SS
(1998) Transgenic mice ex pressing a f unctional human photopigment.
Invest Ophthalmol Vis Sci 39:1036–1043.
Stone C, Pinto LH (1993) Response properties of ganglion cells in the
isolated mouse retina. Vis Neurosci 10:31–39.
Szel A, Rohlich P, Ca ffe AR, Juliusson B, Aguire G, Van Veen T (1992)
Unique separation of two spectral classes of cones in the mouse retina.
J Comp Neurol 325:327–342.
Winderickx J, Lindsey DT, Sanocki E, Teller DY, Motulsky AG, Deeb SS
(1992) Poly morphism in red photopigment underlies variation in co-
lour matching. Nature 356:431– 433.
Yamamoto S, Gouras P (1993) Color opponent neurons in mouse retina.
Soc Neurosci Abstr 19:1257.
Jacobs et al. •Human Cone Pigment Alters Vision in Transgenic Mice J. Neurosci., April 15, 1999, 19(8):3258–3265 3265
