Electroretinographic evidence for altered phototransduction gain

and slowed recovery from photobleaches in albino mice

with a MET450 variant in RPE65

Steven Nusinowitza,*, Lina Nguyena, Roxanna Radua, Zahra Kashania,Debora Farbera, Michael Dancigerb

aJules Stein Eye Institute, UCLA Medical Center, 100 Stein Plaza, Los Angeles, CA 90095, USAbLoyola Marymount University, Los Angeles, CA, USA

Received 16 January 2003; accepted in revised form 30 April 2003

Abstract

Our purpose was to investigate the physiological phenotype of albino mice with a variation in the Rpe65 gene encoding either methionine

or leucine at amino acid #450. Full-field electroretinograms (ERGs) were recorded from C57BL/6J-c2J albino mice with MET450 and

BALB/cByJ albino mice with LEU450. Recordings from pigmented mice (C57BL/6J) served as controls. Rod ERG a-waves were fitted with

a model to estimate parameters of activation. Recovery of function following a photobleach was studied by monitoring the return to pre-

bleach a- or b-wave amplitudes of the dark-adapted electroretinogram. The parameter, S; derived from the fit of the rod model, was

significantly higher for albino mice compared to pigmented controls. Between the albino mice, S was highest for BALB/cByJ compared to

C57BL/6J-c2J. The parameters td and RmP3 were not different across the three strains. The difference in S between the BALB/cByJ and

C57BL/6J-c2J albino strains is interpreted to reflect differences in intrinsic phototransduction gain. Recovery from a photobleach was also

slower for the C57BL/6J-c2J albino mice compared with BALB/cByJ albino mice, consistent with prior studies showing slowed rhodopsin

regeneration in mice with the RPE65-METH450 variant. ERG recordings show that C57BL/6J-c2J albino mice with the MET450 variant of

the RPE65 protein have a lower gain of activation and slower recovery from photobleach than do the BALB/cByJ albino mice with LEU450.

Both the slower recovery from photobleach and lower gain of activation characteristic of the C57BL/6J-c2J strain may contribute to the

mechanism by which it is protected from light-induced photoreceptor death relative to BALB/c.

q 2003 Elsevier Ltd. All rights reserved.

Keywords: photoreceptor transduction; light damage; electroretinography; RPE65

1. Introduction

In studies of albino mice exposed to prolonged light,

C57BL/6J-c2J (c2J) mice showed a significantly greater

resistance to light-induced photoreceptor damage than did

BALB/cByJ (BALB/c) and other albino strains of mice

(LaVail et al., 1987a,b). The factors affording protection

against light-induced photoreceptor damage to c2J albino

mice have not been well understood. Recently, a quantitat-

ive genetics study was carried out on the progeny of a

backcross between F1 (c2J £ BALB/c) and c2J mice using

the thickness of the outer nuclear layer of the retina as

the quantitative trait reflecting retinal damage after

prolonged light exposure. The strongest and most highly

significant quantitative trait locus accounted for approxi-

mately 50% of the light protective effect and localized to

distal mouse chromosome 3 where the Rpe65 gene maps.

Sequencing the gene revealed one difference between c2J

and BALB/c mice at codon 450 – c2J had an ATG

(methionine) and BALB/c had a CTG (leucine) at this

location. All other animals reported in the genome databases

(dog, human, rat, cow, chicken and tiger salamander)

showed leucine at this codon (Danciger et al., 2000).

Normal function of the RPE65 protein is essential for the

regeneration of rhodopsin which is necessary for the visual

cycle. The absence of RPE65 results in photoreceptors that

0014-4835/$ - see front matter q 2003 Elsevier Ltd. All rights reserved.

DOI:10.1016/S0014-4835(03)00217-3

Experimental Eye Research 77 (2003) 627–638

www.elsevier.com/locate/yexer

* Corresponding author. Steven Nusinowitz, Jules Stein Eye Institute,

UCLA Medical Center, 100 Stein Plaza, Los Angeles, CA 90095, USA.

E-mail address: nusinowitz@jsei.ucla.edu (S. Nusinowitz).

are devoid of rhodopsin (Redmond et al., 1998). In humans

and mice, the absence of rhodopsin leads to severe retinal

abnormalities (Thompson et al., 2000). c2J mice with the

MET450 variant in RPE65, however, have a normal

complement of rhodopsin in the fully dark-adapted state.

A methionine for leucine substitution at codon 450 is a

conservative amino acid change that does not affect the

normal functioning of the RPE65 protein. However, in the

presence of H2O2, hydroxyl radicals, and other oxidizing

agents (that would increase in the presence of constant

light), the methionine residue of RPE65 could be converted

to methionine sulfoxide and cause the protein to loose

function. Since RPE65 is involved in the recycling of

chromophore in the RPE from all trans to 11-cis retinal, this

would have the effect of slowing down or shutting down the

visual cascade by inhibiting the recovery of rhodopsin. In

fact, it is this slowed rhodopsin regeneration following light

exposure that has been correlated with protection against

light-induced retinal damage (Wenzel et al., 2001).

The main purpose of this study was to investigate (1)

whether there is an electroretinographic (ERG) phenotype

that distinguishes c2J from BALB/c mice, and (2) whether

these recordings would reveal clues that could provide

further insights into the mechanisms by which c2J mice are

protected from light induced photoreceptor damage. We

reasoned, based on the prior biochemical analysis (Wenzel

et al., 2001), that c2J mice with the MET450 variant in

RPE65 would show slower recovery of vision from intense

light exposure than BALB/c mice carrying the LEU450

variant. A second goal of this study was to examine whether

there might also be differences in the activation steps of

phototransduction between the c2J and BALB/c strains.

2. Methods and materials

The experiments described here were carried out on 14-

to 16-week old c2J and BALB/c mice weighing 23–25 g

C57BL/6J (B6) pigmented mice of the same age served as

controls. All mice were maintained in a normal 12 hr on and

12 hr off light cycle and were placed on common shelves in

the vivarium. These precautions ensured that all mice were

exposed to approximately the same light levels over the

course of the experiment. The ambient light intensity in the

vivarium during the light phase was 5– 10 ft c. All

experimental procedures were carried out in compliance

with the guidelines on animal experimentation set forth by

the National Institute of Health and by the Association for

Research in Vision and Ophthalmology (ARVO).

Following overnight dark-adaptation, mice were

anesthetized with an intraperitonal injection of normal

saline solution containing ketamine (15 mg g21) and

xylazine (7 mg g21 body weight). ERGs were recorded

from the corneal surface of one eye after pupil dilation (1%

atropine sulfate) using a gold loop corneal electrode

together with a mouth reference and tail ground electrode.

A drop of methylcellulose (2·5%) was placed on the corneal

surface to ensure electrical contact and to maintain corneal

integrity. Responses were amplified (Grass CP511 AC

amplifier, £ 10 000; 3 dB down at 2 and 10 000 Hz) and

digitized using an I/O board (National Instruments, PCI-

1200) in a personal computer. Signal processing was

performed with custom software (National Instruments,

LabWindows/CVI). Mouse body temperature was main-

tained at a constant 388C using a heated water pad.

2.1. Basic testing protocol

The right eye of each mouse was positioned orthogonal

to an opening in a large Ganzfeld dome (LKC Technol-

ogies). The interior surface of the dome was painted with a

highly reflective white matte paint (Eastman Kodak

Corporation, #6080). A flash head affixed to the outside of

the dome at 908 to the viewing porthole illuminated the

interior surface with brief flashes of light.

Activation step. A series of high intensity flashes was

generated with a Xenon gas flash tube (Novatron Inc. of

Dallas) which was driven by a 1600 W power supply. All

stimuli were calibrated with a Radiometer/Photometer

System (EG and G, Gamma Scientific, Model 550) and

are expressed in scotopic trolands (td). A detector (silicone

multiprobe with flat radiometric filter) was placed at the

plane of the mouse pupil. The light falling on the detector’s

surface was measured and integrated over time. Retinal

illuminance (in scotopic troland-seconds) was calculated

using the method of Wyszecki and Stiles (1982). (Although

the meaning of the troland is the same with regard to both

the mouse and human eyes, the effective retinal illuminance

of a particular stimulus will be significantly higher in the

mouse for the same pupil area because of the smaller size of

the mouse eye.)

Dark-adapted ERGs were recorded to blue (Kodak

Wratten 47B) light flashes up to the maximum intensity of

3·32 log scot td sec. At the highest intensities, the a-wave of

the ERG was clearly saturated. Unlike human dark-adapted

a-waves that have a significant cone contribution that must be

subtracted from the individual waveforms (Hood and Birch,

1994), dark-adapted a-waves from mice are entirely rod-

driven (Goto et al., 1995; Hetling and Pepperberg, 1999;

Lyubarsky et al., 1999; Toda et al., 1999; Kang Derwent et al.,

2002). A test of cone intrusion using the brightest blue flash

on a rod-saturating background did not reveal a significant a-

wave response (see cone response in Fig. 1). The leading

edge of the a-wave of the ERG was fitted with a compu-

tational model to provide estimates of photoreceptor activity.

Based on the Lamb and Pugh (1992) model of phototrans-

duction, we define the leading edge of the a-wave by

P3ði; tÞ ø {1 2 exp½2iSðt 2 tdÞ2�}Rmp3; for t . td; ð1Þ

where P3 is the sum of the responses of the individual rods

(Hood and Birch, 1993, 1994). The amplitude, P3; is

S. Nusinowitz et al. / Experimental Eye Research 77 (2003) 627–638628

a function offlash energy, i; and time, t; after flash onset. S is a

sensitivity (or gain) parameter that scales flash energy i; and

Rmp3 is the maximum saturated photovoltage. The latter is

thought to be proportional to the number of cGMP-gated

channels closed during signal transduction (Hood and Birch,

1990a,b, 1994; Cideciyan and Jacobson, 1993; Breton et al.,

1994). The parameter, td; is a brief delay before response

onset that reflects the delay inherent in a number of

biochemical steps in the activation process. For each

mouse, Eq. (1) was fitted simultaneously to the averaged

responses to all but the highest intensity using a least-squares

minimization procedure (Matlab, Mathworks, Natick, MA,

USA). (We excluded the response to the highest intensity

because this response in the albino mice, was often smaller

than that for the preceding intensity but with a similar rise

time. Excluding this response gave better fits.) This

procedure provided a single estimate for the parameters td;

S and Rmp3 for the ensemble of responses. Hereafter, all

references to the parameters td; S; and RmP3 will be those

determined from the ensemble fit.

Recovery from photobleach. The kinetics of recovery

from a photobleach was studied using two methods. In the

first method (Method I), recovery was assessed using a

paired-flash technique. With this technique a brief con-

ditioning flash was presented to produce a photobleach, and

a second probe flash, presented at varying times after the

conditioning flash, was used to assess recovery. Recovery

was inferred from the amplitude of the a-wave to the

probe flash. In these experiments, the bleaching flash

was achromatic with a retinal illuminance of 3·61 or

3·97 log scot td sec. The probe flash was blue (W47B) with

a retinal illuminance of 3·32 log scot td sec and was

selected so as to produce a rapid saturation of the a-wave.

The interflash interval was varied between 1- and 700-sec

and was under precise computer control. A recovery period

of at least 4 min was allowed between each paired flash

presentation. For each trial, the amplitude of the a-wave at a

fixed time following flash onset was calculated. This value

was normalized to the baseline a-wave amplitude at the

same time (obtained from the response to the probe flash

alone) to obtain R=Rmax; the relative amplitude of the

response. The R=Rmax; vs. time response curve can be

described in terms of the exponential relation

R=Rmax ¼ 1 2 exp½2ðt 2 TCÞ=tr�; ð2Þ

where TC is the critical delay prior to the initiation of

recovery, tr is the recovery time constant for the given

conditioning flash intensity, and t is the elapsed time

between the first bleaching flash and second probe flash.

(For a detailed discussion of this technique, see Birch et al.

(1995), Lyubarsky and Pugh (1996), Pepperberg et al.

(1996), and Kang Derwent et al., (2002)).

In the second method of assessing recovery (Method II),

mice were exposed to 1000 lux of illumination for 30 sec

(Goto et al., 1995; Sieving et al., 2001). The adapting

illumination was presented inside the dome stimulator with

an additional lamp housing attached to its side. An

electronic shutter (Uniblitz, Inc) precisely controlled the

exposure duration to this adapting light. The 30 sec light

exposure bleached 48% of the available rhodopsin in the B6

Fig. 1. Representative rod ERG a-waves to a range of flash intensities for B6 (left panel), BALB/c (middle panel), and c2J mice (right panel). Retinal

illuminance was varied in 0·3 log unit steps from 1·03 to 3·32 log scot td sec for all three representative mice. Flash onset is at time 0·0 and flash duration is

approximately 600 msec. The irregular wavy curves are the raw data and the smooth dashed curves are the fit of Eq. (1) obtained by estimation of a single set of

parameters for each series of responses (the ensemble fit). The cone response was obtained with the highest flash intensity on a rod-saturating background (see

text for details). In fitting, Eq. (1), each record was truncated at the point were the b-wave begins to intrude, indicated by the reversal of direction of the tracing.

Parameter estimates (td (msec), log S (sec22(td sec)21) and log lRmP3(mV)l) derived from the fit of Eq. (1) are 3·2, 2·29, and 2·43 for the B6 mouse, 3·0, 2·68,

and 2·47 for the BALB/c mouse, and 3·2, 2·56, and 2·49 for the c2J mouse. The numbers in the left panel are flash intensities in log scot td sec.

S. Nusinowitz et al. / Experimental Eye Research 77 (2003) 627–638 629

pigmented controls, 82·5% in the c2J albino mice, 90·5% in

the BALB/c albino mice (Table 1). After returning the

mouse to the dark, the time course of rod recovery was

examined by monitoring the growth of the rod ERG b-wave

to a dim achromatic probe flash (20·91 log scot td sec).

ERGs were recorded at 5 min intervals for 60 min.

2.2. Rhodopsin determinations

Measurement of rhodopsin content before and after a

photobleach was done using previously described methods

(Katz et al., 1991; Sieving et al., 2001). Mice were dark-

adapted overnight and then anesthetized as described above.

One eye was enuculeated prior to exposure to light and the

rhodopsin content of this eye served as baseline. The other

eye was then exposed to the bleaching light. The anterior

segment of each eye was dissected out with removal of the

lens and the vitreous, followed by a quick freezing in liquid

nitrogen. The eyecups were homogenized in 1 ml hom-

ogenization buffer (100 mM sodium phosphate, 2% sucrose

and 1·4% Emulphogene in a 2 ml glass–glass homogenizer)

and centrifuged to remove unsolubilized material. A 500 ml

aliquot of the supernatant was transferred to a quartz cuvette

and 50 ml of freshly prepared 100 mM hydroxylamine in

100 mM sodium phosphate, pH 7·4 was added with mixing.

The absorbance of the solution (200–600 nm) was deter-

mined before and after bleaching to obtain difference

spectra. Spectra were zeroed at 600 nm, and the rhodopsin

content of each eye was calculated from the absorbance

difference at 494 nm, using a molar extinction coefficient of

42 700.

2.3. Statistical comparisons and tests

There were two statistical comparisons. One involved

comparisons between B6 pigmented mice and each of

BALB/c and c2J albino mice. These comparisons, particu-

larly between B6 and c2J, were made to evaluate the effects

of ocular pigmentation. The main comparison, however,

was between the BALB/c and c2J albino mouse strains. In

each instance Student’s t-tests were used to determine if

difference were of statistical significance.

3. Results

3.1. Activation steps

Rod a-waves for representative B6, c2J and BALB/c

mice are shown in Fig. 1 for a series of seven flash

intensities. The smooth dashed curves are the fit of Eq. (1)

obtained by estimating a single set of parameters for the

series of responses. In fitting Eq. (1), each record was

truncated at the point were the b-wave begins to intrude

(indicated by the reversal of direction of the tracing), and the

responses to all but the highest intensity were included in

the fit. Flash onset is at time 0·0 and flash duration was

approximately 600 msec.

The parameter td was found not to be statistically

different amongst the three groups of mice. Individual t-tests

comparing B6 vs. BALB/c and c2J mice, and between

BALB/c and c2J mice, did not reach statistical significance.

The mean td (^1 S.D.) for B6, BALB/c and c2J were 3·12

(0·66), 3·19 ( ^ 0·18) and 3·01 (^ 0·39), respectively. The

mean (^1 S.D.) for td across all three groups was 3·14

(^0·46) msec, which is close to the value previously

reported for mice (Lyubarsky and Pugh, 1996) and is

virtually identical to that reported for humans (Hood and

Birch, 1994). When estimating the parameters S and RmP3;

td was held constant at the mean value for each group of

mice, and only S and RmP3 were allowed to vary. The

parameters log S (sec22(td sec)21) and log RmP3 (mV) from

the fit of Eq. (1) are summarized in Fig. 2 (top panel) for

each mouse tested. Here, the values of log S and log RmP3

are expressed as the differences from the mean of the B6

control mice ðn ¼ 9Þ: A value of 0·0 for each parameter

corresponds to the mean for that parameter obtained from

the B6 mice (a departure score of 0·0). The filled circles are

the data for the pigmented mice and the open circles and

squares are for BALB/c ðn ¼ 10Þ and c2J ðn ¼ 10Þ mice,

respectively. The vertical and horizontal lines show the

mean departure score ^1 S.D. for the parameter log S

(vertical lines) and log RmP3 (horizontal lines).

Almost without exception, RmP3 for the c2J and the

BALB/c mice were within the limits defined by the B6

pigmented mice. Individual t-tests comparing RmP3 for B6

vs. BALB/c and c2J, and between BALB/c and c2J, did not

reach statistical significance. The mean log RmP3 (^1 S.D.)

Table 1

Rhodopsin content (in pmoles) before and after a photobleach by mouse strain

Mouse strain N Coat Amino acida Dark-adaptedb Post-bleachc Bleach (%)

C57BL/6J 2 Pigmented MET 608·9 (^33·1) 316·2 (^82·8) 48·0

C57BL/6J-c2J 2 Albino MET 456·7 (^16·5) 82·0 (^16·5) 82·5

BALB/cByJ 2 Albino LEU 491·8 (^1·0) 46·8 (^1·0) 90·5

a Amino acid at codon 450 of the Rpe65 gene.b 12 hr of dark-adaptation.c The bleach was 1000 lux for 30 sec.

S. Nusinowitz et al. / Experimental Eye Research 77 (2003) 627–638630

for B6, BALB/c and c2J were 2·36 (^ 0·17), 2·35 (^ 0·13)

and 2·40 (0·09), respectively. These results imply that the

total number of rods and/or the total number of cGMP-gated

channels in the rod outer-segment contributing to the

generation of a response is the same across the three groups

of mice.

In contrast, the parameter S was significantly different

across the three strains of mice. On average, S was 0·57 and

0·37 log units higher than B6 for the BALB/c and c2J mice,

respectively (t-test had Ps ¼ 0·004 and 0·0002). In addition,

S for the BALB/c mice was on average 0·2 log units higher

compared to the c2J albinos (t ¼ 2·89; df ¼ 18, P ¼ 0·011).

It is also of interest to note that the time-to-peak of the a-

wave responses are slightly different across the three strains

of mice. For example, in Fig. 1, the peak of the a-wave for

the BALB/c mice occurs at a slightly faster time compared

to either the c2J or the B6 mice. These timing differences

persist at each of the flash intensities. To quantify these

differences, we determined the time-to-peak of the a-wave

(measured from baseline to the point where the a-wave

reverses direction) for each intensity and mouse strain. The

results are shown in Fig. 2 (bottom panel). Clearly, the

albino strains have faster rise times than the pigmented

strain. In addition, between the albino strains, the c2J mice

are slightly slower than the BALB/c mice. These differences

in time-to-peak are suggestive of different photoresponse

rise times and are consistent with the analysis of the

parameter S shown in the upper panel.

3.2. Recovery from photobleach

Recovery of the rod a-wave following high-intensity

conditioning flashes (Method 1) is shown in Fig. 3 for

representative B6 (left panel), BALB/c (middle panel), and

c2J (right panel) mice. Each curve shows the a-wave

response to the probe flash for progressively longer inter-

stimulus intervals between the bright first conditioning

(bleaching) flash and the second probe flash. For the data

shown in Fig. 3, the conditioning flash was achromatic with

a retinal illuminance of 3·61 log scot td sec and the probe

flash was blue (W47B) with a retinal illuminance of

3·32 log scot td sec. The response to the probe alone (with-

out a prior conditioning flash) is indicated by the trace

labeled baseline. For the representative B6 mouse shown in

Fig. 3 (left panel), the baseline a-wave amplitude is 237 mV.

a-wave responses to the probe flash following the bright

conditioning flash are shown for inter-stimulus intervals

(ISIs) of 1, 10, 30, and 60 sec. Note that an a-wave to the

probe flash is non-detectable at 1 sec but that there is a rapid

growth in a-wave amplitude with progressively longer ISIs.

At 10 and 30 sec ISIs, a-wave amplitudes for the B6

pigmented mouse are 15 and 67% of baseline, respectively.

With an ISI of 60 sec, the a-wave amplitude to the probe

flash is almost completely recovered to baseline.

In contrast, after 60 sec of recovery, the a-wave

amplitude for the BALB/c mice had returned to approxi-

mately 50% of baseline and the a-wave for the c2J mice to

only 30% of baseline. These results suggest that while

recovery from a photobleach is slowed for albino mice

relative to pigmented control mice, c2J albino mice are

slower to recover than BALB/c albino mice.

Fig. 4 shows the normalized a-wave amplitudes for the

entire range of ISIs for the mice shown in Fig. 3.

The response function was fitted with Eq. (2) to obtain TC;

Fig. 2. (Top Panel) Departures from B6 estimates for log S and log RmP3:

Changes in log S and log Rmp3 are expressed as the differences (D log S and

0·0 DlogRmP3) from the log of the mean of the B6 controls. A value of 0·0

corresponds to the mean of the B6 mice (a departure score of 0·0). The filled

circles are the individual B6 data, and the open circles and squares are for

the BALB/c and c2J mice, respectively. The horizontal dashed lines give

the mean and the upper and lower boundary limits (defined as 1·96 standard

deviations from the mean) for the B6 D RmP3 and the vertical solid lines

give the mean and the upper and lower boundary limits for D log S: The

mean (^1 S.D.) log RmP3 and log S for the B6 mice are 2·36 (^0·17) and

2·4 (^0·22). (Bottom Panel) Time-to-peak of RmP3 for the seven different

stimulus intensities. Time-to-peak was measured from baseline to the peak

of the a-wave.

S. Nusinowitz et al. / Experimental Eye Research 77 (2003) 627–638 631

the critical delay prior to the initiation of recovery and tr the

recovery time constant. Analysis of the data through Eq. (2)

(free variation of TC and tr) indicates that TC is 2·9, 7·2, and

13·2 sec for the B6, BALB/c, and c2J mice shown in Fig. 3,

and the corresponding recovery time constants were 39·2,

78·5, and 126·6 sec.

Group recovery data are summarized in Fig. 5. Fig. 5 (left

panel) shows the results for the conditions just described, The

filled symbols are the mean response ðn ¼ 5Þ for the B6 mice,

the open circles are for the BALB/c albino mice ðn ¼ 5Þ and

the open squares are for the c2J albino mice ðn ¼ 6Þ:Only the

first 200 msec of the recovery curves are shown to emphasize

the details during the early phase of recovery. There is

considerable variability in the grouped mouse data but there

is a clear indication of slowed recovery for the albino mice.

An analysis of the grouped data with Eq. (2) indicates that the

TC values are 1·02, 5·28, and 7·34 sec for the B6, BALB/c,

and c2J mice, respectively. The corresponding recovery time

constants are 37·0, 61·8, and 117·8 sec. TC was not

significantly different comparing c2J and BALB/c mice

(t ¼ 1·08; df ¼ 9, P ¼ 0·31). However, TC was marginally

significant comparing B6 and BALB/c mice (t ¼ 2·03;

df ¼ 9, P ¼ 0·07) and was of greater significance comparing

c2J and B6 mice (t ¼ 2·47; df ¼ 8, P ¼ 0·039). The recovery

time constant, tr; was significantly different comparing B6

and c2J mice (t ¼ 3·63; df ¼ 9, P ¼ 0·006) and between c2J

mice and BALB/c mice (t ¼ 2·3; df ¼ 9, P ¼ 0·047). The

difference between tr for B6 and BALB/c mice was marginal

(t ¼ 2·02; df ¼ 8, P ¼ 0·078).

The pattern of results just described was replicated for

the higher conditioning flash intensity (3·97 log scot td sec)

shown in Fig. 5 (right panel), although the number of mice

in each group was smaller. For this conditioning flash

intensity, TC was 1·00, 6·7, and 16·6 sec for the B6 ðn ¼ 4Þ;

Fig. 3. Responses to a short-wavelength flash at various ISIs (inter-stimulus intervals) following an achromatic conditioning flash for a B6 (left panel), BALB/c

(middle panel), and a c2J mouse (right panel). The record labeled as baseline is the response to the saturating short-wavelength flash without prior presentation

of a bright conditioning flash. Probe flash responses are shown for 1, 10, 30, and 60 sec inter-stimulus intervals.

Fig. 4. Normalized a-wave amplitudes for a range of ISIs for the mice shown in Fig. 3. The response function was fitted with Eq. (2) to obtain TC; the critical

delay prior to the initiation of recovery and tr; the recovery time constant. Analysis of the data through Eq. (2) (free variation of TC and tr) indicates that TC is

2·9, 7·2, and 13·2 sec for the B6, BALB/c, and c2J mice, respectively, and the corresponding recovery time constants were 39·2, 78·5, and 126·6 sec.

S. Nusinowitz et al. / Experimental Eye Research 77 (2003) 627–638632

BALB/c ðn ¼ 2Þ; and c2J ðn ¼ 2Þ mice, respectively, and

the corresponding recovery time constants were 48·9, 167·6,

and 213·4 sec.

The above analysis demonstrates a slowed recovery of

a-wave amplitude following a photobleach for the albinos

compared to the pigmented mice, and slower recovery for

C2J compared to BALB/c. However, prior research has

also reported a persisting desensitization of rod photo-

receptors in pigmented mice despite a complete or near-

complete recovery of the rod a-wave following an adapting

illumination (Kang Derwent et al., 2002). This persistent

desensitization was, in part, characterized by a delayed

rising phase of the response to the probe flash during

recovery. The delay in the rise time is also apparent in Fig.

3, particularly for the B6 mice, and is indicated by the shift

in the response curves to the right on the time axis. To

explore this phenomenon further we replotted each of the

probe flash responses scaled to the same amplitude as the

probe response alone (without prior conditioning flash).

Representative data are shown in Fig. 6 for the two

conditioning flash intensities. In each figure the response to

the probe flash alone is indicated by the heavy black

tracing. The remaining records are for inter-stimulus

intervals where the a-wave amplitude was near to, or,

completely, recovered. Consistent with prior research, our

mice also demonstrated a delay in the rising phase of the

response but with otherwise normal kinetics. To quantify

this delay, we determined the time-shift for the scaled

response at which the amplitude had achieved 50% that of

the probe alone. For the B6, BALB/c, and c2J mice, the

mean delay at 50% recovery was 1·5 (^0·3), 1·2 (^0·1),

and 1·2 (^0·5) msec, respectively. The delays are similar

to the values previously reported under different conditions

(Kang Derwent et al., 2002). The corresponding time delay

for the higher conditioning flash intensity (Fig. 6, bottom

row) are 1·1 (^0·2), 1·3 (^0·2), and 1·5 (^0·3) msec,

respectively. (Note that the higher conditioning flash

intensity produced about the same level of rhodopsin

bleach in the pigmented mouse as did the lower

conditioning flash intensity in the albino mice.)

The recovery data from a substantially higher photo-

bleach (Method 2) are shown in Figs. 7 and 8. In this

experiment, mice were exposed to 1000 lux of illumination

for 30 sec, bleaching 48% of the available rhodopsin in the

B6 pigmented controls, 82·5% in the c2J albino mice, 90·5%

in the BALB/c albino mice (Table 1). The b-wave to a dim

probe flash was recorded at 5 min intervals for up to 1 hr

after mice were returned to the dark. In Fig. 7, the recovery

of the b-wave is shown for representative c2J and the

BALB/c albino mice. Group data (n ¼ 7 for each strain)

from this experiment are summarized in Fig. 8. Here b-wave

amplitudes, normalized to pre-bleach baseline levels, are

plotted against time after the photobleach. Although neither

strain of mouse recovered completely to baseline levels, the

c2J strain of albino mice were clearly slower to recover than

the BALB/c albino strain of mice. After 60 min of dark-

adaptation, BALB/c mice had returned to approximately

40% of baseline, while the c2J mice had returned to only

about 20% of baseline. (t-tests comparing the mean b-wave

amplitude reached statistical significance for times greater

than 30 min. At times longer than 50 min, t-tests had

associated P values of ,0·01.)

Fig. 5. (Left panel) Normalized group mean a-wave amplitudes for a range of ISIs. Analysis of the group mean data gave TC of 1·02, 5·3, and 7·34 sec for the

B6, BALB/c, and c2J mice, respectively, and the corresponding recovery time constants were 37·0, 61·8, and 117·8 sec. (Right Panel) Normalized a-wave

amplitudes for a higher conditioning flash intensity. Analysis of the group mean data gave TC of 1·00, 6·7, and 16·6 sec for the B6, BALB/c, and c2J mice,

respectively, and the corresponding recovery time constants were 48·9, 167·6, and 213·4 sec. Only the first 200 msec of averaged recovery curves are shown.

S. Nusinowitz et al. / Experimental Eye Research 77 (2003) 627–638 633

Fig. 6. Representative probe flash responses scaled to the same amplitude as the probe response alone (without prior conditioning flash). The data in the top and

bottom rows are for the 3·61 and 3·97 log scot td sec conditioning flash intensities, respectively. In each figure, the response to the probe flash alone is indicated

by the heavy black tracing. The remaining records are for inter-stimulus intervals where the a-wave amplitude was near to, or completely, recovered.

Fig. 7. Representative rod ERG b-waves to a dim probe flash (20·91 log scot td sec) at different times following exposure to 1000 lux illumination for 30 sec

for BALB/c (left panel) and c2J (right panel) albino mice. Each record is the average of 10 responses to a flash presented at 1 Hz.

S. Nusinowitz et al. / Experimental Eye Research 77 (2003) 627–638634

4. Discussion

In studies of albino mice exposed to prolonged light, the

c2J strain has been shown to have a significantly greater

resistance to light-induced photoreceptor damage than the

BALB/c strain (LaVail et al, 1987a,b). In a previous

quantitative genetic study involving these two strains, we

found that a methionine variant at codon 450 of RPE65

present in c2J mice (BALB/c mice have leucine at this

codon) cosegregated with a substantial portion of the light

damage protection exhibited by c2J (Danciger et al., 2000)

The probability that this correlation occurred by chance was

,1 in 1019. Said another way, the presence of this MET450

variant of RPE65 (vs. the presence of LEU450) was

associated with nearly 50% less retinal degeneration at an

extremely high significance (LOD score of 19·3) in progeny

of a backcross between BALB/c and c2J mice. In the present

study, the main goal was to investigate whether the retinas

of mice with this amino acid variation in RPE65 had altered

physiological responses to light as measured by in vivo

electrorentinographic recordings. To this end, we recorded

ERGs from c2J albino mice with methionine at amino acid

#450 and BALB/c albino mice with leucine at amino acid

#450. Our intent was to provide physiological correlates for

prior biochemical analyses (Wenzel et al., 2001) and to

provide further insights into the potential mechanisms by

which c2J mice are protected from light-induced retinal

damage relative to BALB/c mice.

To evaluate the activation steps of rod phototransduction,

we recorded the a-wave to an intensity series of light

flashes. The leading edge of the a-wave was fitted with a rod

model to provide estimates for td; the time before response

onset, S, a sensitivity (or gain) parameter, and RmP3; the

maximum saturated photovoltage. The parameter td; was

found not to be different among the pigmented and albino

mice. In addition, the maximum saturated amplitude, RmP3;

was also found not to be different across the three strains of

mice. RmP3; is generally thought to be proportional to the

number of cGMP-gated channels in the rod outer-segment

membrane that are closed during the activation phase of

phototransduction (Hood and Birch, 1990a,b; Cideciyan

et al., 1993; Breten et al., 1994). Thus, any variable that

limits the number of cGMP-gated channels in the rod outer-

segment (for example, shorter and/or disorganized outer

segments, reduced number of photoreceptors, or regional

loss of photoreceptors) will reduce the maximal photo-

receptor response. (The magnitude of RmP3 alone would not

allow one to distinguish among the various possible

mechanisms of cell loss.) The similarity of RmP3 supports

the hypothesis that the total number of gated channels in the

rod outer-segment membrane that are closed with bright

flash stimulation, is similar across the three strains of mice.

This finding is consistent with our own unpublished data,

and that of others (LaVail et al., 1987b) that shows that the

thickness of the outer-nuclear layer of the retina of the two

albino strains of this study at 3–6 months of age is not

significantly different.

In contrast, the parameter S was found to be significantly

higher for the albino mice compared to the pigmented

controls. More importantly, among the albino strains, S was

significantly higher for BALB/c than for c2J mice.

S is generally thought to be a sensitivity, or gain,

parameter that scales flash energy (Hood and Birch, 1993,

1994). Factors that might alter the estimate of S include

pigment content, photoreceptor alignment, and pre-retinal

screening by ocular media and lens, all factors that might

alter quantal catch. In addition, differences in intrinsic gain

at one or more of the biochemical steps involved in

phototransduction would also be expected to alter the

estimate of S. (For reviews of the mechanisms of

phototransduction, see Pugh and Cobbs (1986), Stryer

(1986), and Pugh and Lamb (1993).)

Differences in ocular pigmentation are likely responsible

for a large portion of the differences in S that we observed

between the pigmented and albino mice. Ocular melanin is

known to absorb light, acting like a neutral density filter that

reduces light scattering within the eye. The higher estimate

for S for the albino mice is likely the result of increased

quantal catch caused by the absence of melanin. c2J mice

originally obtained from the Jackson Laboratory (Bar

Harbor, ME) are genetically identical (coisogenic) to B6

mice, except that they are homozygous for a mutation in the

tyrosinase gene (c/c) making them albino. The differences in

estimate of S between the B6 and c2J mice could be

interpreted to mean that the nominal neutral density of ocular

melanin is on the order of 0·37 log units. This is the same

order of magnitude that was observed when retinal

Fig. 8. Mean (^1 S.D.) normalized rod ERG b-wave amplitudes to a dim

probe flash (20·91 log scot td sec) measured at 5 min intervals following a

80–90% photobleach for BALB/c (open circles) and c2J (open squares)

albino mice.

S. Nusinowitz et al. / Experimental Eye Research 77 (2003) 627–638 635

illuminance was adjusted to give the same ‘effective’

illuminance for pigmented and albino mice (Rapp and

Williams, 1980). Further, as shown in Fig. 9, when flash

intensities were selected so that the effective retinal

illuminance was similar for the B6 pigmented and the C2J

albino mice, the a-wave records were in reasonably good

agreement.

In a comparison among the two albino mouse strains, we

have also found a small but significantly higher estimate for

S for BALB/c compared to c2J mice. This significant

difference (a difference of approximately 37%, Fig. 2)

cannot be explained by factors that alter quantal catch.

Measurement of rhodospin content after 12 hr of dark-

adaption revealed that the total amount of available

rhodopsin in BALB/c albino mice and in c2J albino mice

are similar, although there appears to be slightly more

rhodopsin in BALB/c mice (approximately 5% difference:

see Table 1) compared to c2J. The similarity of dark-

adapted rhodopsin content in mouse strains with and

without the MET450 variant has been previously reported

(Wenzel et al., 2001). Differences in other factors that could

affect quantal catch, such as pre-retinal filtering by melanin

and/or photoreceptor alignment, would not be expected to

account for much of the remaining difference in S. Thus, all

else being equal, the total number of photons captured per

flash would be expected to be similar, seemingly ruling out

the possibility that a simple quantal catch hypothesis could

account for the differences in S between the two albino

strains. As a result, we cannot preclude the possibility of

differences intrinsic to rod photoreceptors themselves,

namely differences in the gain at one or more of the steps

in the phototransduction pathway. Whether there are

differences in the phototransduction machinery among

different normal strains of mice is not currently known.

Recovery from a photobleach was also found to be

different among the three strains of mice. We evaluated

recovery using two methods – a paired flash technique in

which recovery of the a-wave was probed at varying times

following a bright conditioning flash that bleached a

relatively small amount of the available rhodopsin, and a

second method that probed the recovery of the b-wave

following a more extensive bleach.

The pigmented B6 mice were always faster to recover

from a photobleach than either of the albino strains. With

the paired-flash technique, the time constant of recovery of

the a-wave was on average 2–3 times faster for the

pigmented strain than for the albino strains. The faster

recovery of vision in B6 pigmented mice is largely, if not

entirely, the result of a weaker rhodopsin bleach caused by

the filtering of light by melanin (Table 1). Increasing the

conditioning flash intensity so that the effective retinal

illuminance and the level of the photobleach was similar for

the B6 and c2J mice, slowed the recovery for the B6

pigmented mice, as expected on the basis of quantal catch,

but did not result in the superimposition of the response

curves for the two strains of mice (compare B6 and c2J mice

at the higher and lower conditioning flash intensities,

respectively, in Fig. 5a and b). The c2J mice were much

slower than would have been predicted on the basis of

quantal catch alone. We can only speculate on the reason for

this failure. It is possible that the sequential presentation of

the bright conditioning flashes in the albino mice produced a

stronger and lingering adaptation effect than in the

pigmented mice, or perhaps even produced a low level of

retinal damage in the light sensitive albino mice.

Most importantly, however, between the two albino

strains, the c2J albino was slower to recover vision

following a photobleach than was the BALB/c albino.

After 1 h of dark-adaption following an extensive bleach,

the b-wave in c2J mice recovered to approximately 50% of

the level of the b-wave in BALB/c mice (Fig. 8). A similar

order of magnitude difference between the albino strains

was observed in the paired-flash technique to assess

recovery (Fig. 5). This finding cannot be explained by a

weaker bleach in the BALB/c albino mice compared to c2J

albino mice. The rhodopsin measurements before and after

exposure to the adapting light indicated that the extent of

bleach was comparable between the two albino strains

(Table 1), with a trend, in fact, in the direction of a weaker

bleach for c2J mice. A quantal catch explanation for the

differences in recovery would have predicted that the c2J

albino mice might have a faster, not slower, recovery of

function because of the slightly weaker bleach. In a

biochemical study, Wenzel et al. (2001) demonstrated a

slowed regeneration of rhodopsin following a bleach for

mice carrying the MET450 variant in RPE65. Here, we

extend those findings by providing a physiological correlate

to the biochemical observations.

Fig. 9. A comparison of a-wave recordings for B6, BALB/c, and c2J mice

under conditions of approximately equal effective retinal illuminance. Each

record is the mean of three responses. Stimulus intensity for the B6 mice is

2·89 log scot td sec and for the albino mice is 2·53 log scot td sec, a

difference of 0·36 log units. The optical density of melanin estimated from

the comparison of log S from the fit of the rod model for B6 pigmented and

c2J albino mice is 0·37 (see text for details).

S. Nusinowitz et al. / Experimental Eye Research 77 (2003) 627–638636

Prior research has reported a persisting desensitization of

rod photoreceptors following complete or near-complete

recovery of the rod a-wave following an adapting illumina-

tion (Kang Derwent et al., 2002). We examined our data to

see whether there was also evidence for this type of

lingering desensitization, particularly among our albino

strains. As shown in Fig. 6, the rising phase of the response

to the probe in the paired-flash experiments was also

delayed during recovery even though the amplitude of the

probe response had almost completely reached baseline

levels (see, for example, the B6 data in Fig. 6). To quantify

this effect, the delay was estimated for the response where

the probe amplitude had recovered to approximately 50% of

baseline. For the B6, BALB/c, and c2J mice, the mean delay

at 50% recovery was 1·5 (^0·3), 1·2 (^0·1), and 1·2 (^0·5)

msec, respectively. This timing delay is comparable to that

previously reported (Kang Derwent et al., 2002). The higher

conditioning flash intensity that resulted in a higher

rhodopsin bleach did not change this timing delay

significantly, but the number of mice in each group was

small. (Note that the higher conditioning flash intensity

produced about the same level of rhodopsin bleach in the

pigmented mouse as did the lower conditioning flash

intensity for the albino mice.) Despite the delay in rise

time, however, the response curves appear to have normal

kinetics over a range of inter-stimulus intervals where the

amplitude was near baseline.

It has been hypothesized (Kennedy et al., 2001; Kang

Derwent et al., 2002) that this lingering desensitization

derives from a process that reduces the efficiency of signal

transmission downstream from rhodopsin, as for example,

that which might be caused by the depletion of activatable

transducin (Sokolov et al., 2001). The similarity of the

magnitude of the delay would suggest that differences in

phototransduction kinetics across the three strains of mice

during the activation phase derives from rhodopsin

activation and recovery, rather than from components of

the phototransduction cascade downstream from rhodospin.

The reader is referred to an elegant study by Kang Derwent

et al. (2002) for a more complete treatment of this specific

phenomona.

The severity of light-induced retinal damage is modu-

lated by the properties of light exposure, including the

duration, intensity, and wavelength composition of the light

(Noell et al., 1966; Lanum, 1978; Lerman, 1990). Other

factors that have been implicated include age, body

temperature, ocular pigmentation, light exposure history,

and levels of stress (Noell et al., 1966; Noell and Albrecht,

1971; Lanum, 1978; Noell, 1979; O’Steen, 1980; Rapp and

Williams, 1980; O’Steen and Donnelly, 1982; Penn et al.,

1985; Lerman, 1990). We previously demonstrated a very

strong and highly significant association between the

MET450 variant of RPE65 and protection of the retina

from damage due to prolonged light exposure (Danciger

et al., 2000). From this information, and examples of the

behavior of key MET residues in other proteins during

oxidative stress, we hypothesized that the MET450 in

RPE65 is more susceptible to oxidation than LEU450

(present in the RPE65 of BALB/c) during prolonged light

exposure. This, in turn, decreases RPE65 activity (RPE65 is

involved in production of the 11-cis retinal chromophore of

rhodopsin) which slows down rhodopsin regeneration and

phototransduction and consequently protects the photo-

receptors. Wenzel et al. (2001) showed that mice with

MET450 have a slower regeneration of rhodopsin compared

to those with LEU450 after intense light exposure and that

these findings were correlated with protection from light

damage. Our findings are consistent with the rhodopsin

regeneration data because they show that c2J retinas have a

slower recovery of the rod ERG a- and b-waves after a

bright flash relative to those of BALB/c. In addition, our

analysis of the leading edge of the a-wave to bright flash

stimulation suggests that c2J albino mice have also a

decreased intrinsic gain of phototransduction which may

also slow down the visual cycle.

To summarize, the main focus of this paper was to

investigate whether there are physiological differences

detectable by the ERG between two albino strains of

mice, one with MET450 (c2J) and one with LEU450

(BALB/c). Analysis of data obtained from the albino strains

suggests that there are intrinsic differences in rod photo-

receptor gain (c2J mice have a lower gain) as well as

differences in the kinetics of recovery from photobleaches

(c2J mice are slower to recover). This may suggest that c2J

photoreceptors undergo fewer cycles of phototransduction

with fewer cycles of hyperpolarization/depolarization than

those of BALB/c under the oxidizing conditions of

prolonged constant light. It should be made clear that we

have not specifically demonstrated the relationship between

these findings and susceptibility to light damage. However,

it has already been documented that the rate of rhodopsin

regeneration correlates with the sensitivity to light damage

(Grimm et al., 2000; Wenzel et al., 2001), and that there is a

type of light damage that is dependent upon phototransduc-

tion, at least in the presence of the alpha subunit of

transducin (Hao et al., 2002). Therefore, our data suggesting

fewer cycles of hyperpolarization/depolarization in c2J vs.

BALB/c photoreceptors evidenced by slower recovery from

a photobleach and lower gain of activation support our

original hypothesis that the MET450 variant of RPE65

protects against light-induced damage to the retinal

photoreceptors at least in part by slowing down the visual

cycle. More importantly, the results of this study provide

physiological clues to the understanding of why one albino

mouse strain is more resistant to light damage than another.

Acknowledgements

Supported by a grant from The Foundation Fighting

Blindness, The Foundation Fighting Blindness, 11435

Cronhill Drive, Owings Mills, MD 21117-2220, USA.

S. Nusinowitz et al. / Experimental Eye Research 77 (2003) 627–638 637

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