Test of the paired-flash electroretinographic method in mice lacking b-waves

Article (PDF Available)inVisual Neuroscience 24(2):141-9 · March 2007with30 Reads
DOI: 10.1017/S0952523807070162 · Source: PubMed
Abstract
Previous studies of rod photoreceptors in vivo have employed a paired-flash electroretinographic (ERG) technique to determine rod response properties. To test whether absence versus presence of the ERG b-wave affects the photoreceptor response derived by the paired-flash method, we examined paired-flash-derived responses obtained from nob mice, a mutant strain with a defect in signal transduction between photoreceptors and ON bipolar cells that causes a lack of the b-wave. Normal littermates of the nob mice served as controls. The normalized amplitude-intensity relation of the derived response determined in nob mice at the near-peak time of 86 ms was similar to that determined for the controls. The full time course of the derived rod response was obtained for test flash strengths ranging from 0.11 to 17.38 scotopic cd s m(-2) (sc cd s m(-2)). Time-course data obtained from nob and control mice exhibited significant but generally modest differences. With saturating test flash strengths, half-recovery times for the derived response of nob versus control mice differed by approximately 60 ms or less about the combined (nob and control) average respective values. Time course data also were obtained before versus after intravitreal injection of L-2-amino-4-phosphonobutyrate (APB) (which blocks transmission from photoreceptors to depolarizing bipolar cells) and of cis 2,3-piperidine dicarboxylic acid (PDA) (which blocks transmission to OFF bipolar cells, and to horizontal, amacrine and ganglion cells). Neither APB nor PDA substantially affected derived responses obtained from nob or control mice. The results provide quantitative information on the effect of b-wave removal on the paired-flash-derived response in mouse. They argue against a substantial skewing effect of the b-wave on the paired-flash-derived response obtained in normal mice and are consistent with the notion that, to good approximation, this derived response represents the isolated flash response of the photoreceptors in both nob and normal mice.
Test of the paired-flash electroretinographic method
in mice lacking b-waves
JENNIFER J. KANG DERWENT,
1,2
SHANNON M. SASZIK,
3,4
HIDETAKA MAEDA,
3,5
DEBORAH M. LITTLE,
6
MACHELLE T. PARDUE,
7,8
LAURA J. FRISHMAN,
3
and DAVID R. PEPPERBERG
1
1
Lions of Illinois Eye Research Institute, Department of Ophthalmology and Visual Sciences, University of Illinois at Chicago,
College of Medicine, Chicago, Illinois
2
Department of Biomedical Engineering, Pritzker Institute of Biomedical Science and Engineering, Illinois Institute of Technology,
Chicago, Illinois
3
College of Optometry, University of Houston, Houston, Texas
4
Department of Ophthalmology, Northwestern University School of Medicine, Chicago, Illinois
5
Kobe University Medical School, Kobe, Japan
6
Department of Neurology and Rehabilitation, and Center for Cognitive Medicine, University of Illinois at Chicago,
College of Medicine, Chicago, Illinois
7
Atlanta VA Medical Center, Decatur, Georgia
8
Department of Ophthalmology, Emory University, Atlanta, Georgia
(Received September 27, 2006; Accepted February 14, 2007!
Abstract
Previous studies of rod photoreceptors in vivo have employed a paired-flash electroretinographic ~ERG! technique
to determine rod response properties. To test whether absence versus presence of the ERG b-wave affects the
photoreceptor response derived by the paired-flash method, we examined paired-flash-derived responses obtained
from nob mice, a mutant strain with a defect in signal transduction between photoreceptors and ON bipolar cells
that causes a lack of the b-wave. Normal littermates of the nob mice served as controls. The normalized
amplitude-intensity relation of the derived response determined in nob mice at the near-peak time of 86 ms was
similar to that determined for the controls. The full time course of the derived rod response was obtained for test
flash strengths ranging from 0.11 to 17.38 scotopic cd s m
2
~sc cd s m
2
!. Time-course data obtained from nob
and control mice exhibited significant but generally modest differences. With saturating test flash strengths,
half-recovery times for the derived response of nob versus control mice differed by ;60 ms or less about the
combined ~nob and control! average respective values. Time course data also were obtained before versus after
intravitreal injection of l-2-amino-4-phosphonobutyrate ~APB!~which blocks transmission from photoreceptors to
depolarizing bipolar cells! and of cis 2,3-piperidine dicarboxylic acid ~PDA!~which blocks transmission to OFF
bipolar cells, and to horizontal, amacrine and ganglion cells!. Neither APB nor PDA substantially affected derived
responses obtained from nob or control mice. The results provide quantitative information on the effect of b-wave
removal on the paired-flash-derived response in mouse. They argue against a substantial skewing effect of the
b-wave on the paired-flash-derived response obtained in normal mice and are consistent with the notion that, to
good approximation, this derived response represents the isolated flash response of the photoreceptors in both nob
and normal mice.
Keywords: Rod photoreceptor, Electroretinogram, Paired-flash ERG, nob mouse
Introduction
Paired-flash electroretinogram ~ERG! recording, in which the test
flash is followed at a defined time by a bright ~rod-saturating!
probe flash and the ERG a-wave response to the probe flash is
analyzed for amplitude, provides a measure of the rod response to
the test flash in the intact eye. Previous studies indicate that the full
time course of the test flash response derived using the paired-flash
technique represents, to good approximation, the in vivo isolated
rod response to the test stimulus ~Birch et al., 1995; Lyubarsky &
Pugh, 1996; Pepperberg et al., 1997; Robson & Frishman, 1999;
Hetling & Pepperberg, 1999; Friedburg et al., 2001!. However, as
the amplitude of the b-wave is relatively large at test flash strengths
that elicit an a-wave, and because the polarity of the b-wave is
opposite to that of the a-wave, the b-wave of the normal ERG
Address correspondence and reprint requests to: David R. Pepperberg,
Department of Ophthalmology and Visual Sciences, University of Illinois
at Chicago, 1855 W. Taylor St., Chicago, IL 60612, USA. E-mail:
davipepp@uic.edu
Visual Neuroscience ~2007!, 24, 141–149. Printed in the USA.
Copyright © 2007 Cambridge University Press 0952-5238007 $25.00
DOI: 10.10170S0952523807070162
141
influences the shape of the a-wave and thus is expected, to some
extent, to influence paired-flash determinations of the rod response.
Previous studies have described a naturally occurring, X-linked
recessive mutation in the mouse that is associated with the absence
of the ERG b-wave ~Pardue et al., 1998; Candille et al., 1999, Wu
et al., 2004a!. Available data indicate that nob phenotype is due to
absence of signal transmission from rod photoreceptors to rod
depolarizing ~ON! bipolar cells, although histological examination
of the eye tissues of this mutant has revealed normal retinal
structure ~Pardue et al., 1998, 2001!. Specifically, the nob defect
maps to a region within the nyx gene ~Gregg et al., 2003!,
associated with CSNB1 in human patients ~Bech-Hansen et al.,
2000; Pusch et al., 2000!. Nyx encodes a novel leucine-rich pro-
tein, termed nyctalopin, of as yet unknown function ~Bech-Hansen
et al., 2000; Pusch et al., 2000!.
Indications that a synaptic abnormality underlies the absence of
the b-wave in nob mice make nob a good animal model to test the
degree to which the test paired-flash derived response represents
the rod photoreceptor isolated response. To test the degree of
approximation of the isolated rod photoresponse provided by the
paired-flash ERG method, we have used nob mice and normal
littermate controls to compare the derived responses obtained from
paired-flash determinations.
Materials and methods
All animal procedures were in accordance with protocols approved
by the University of Illinois at Chicago and the University of
Houston, and with the principles embodied in the statement on the
use of animals in ophthalmic and vision research adopted by the
Association for Research in Vision and Ophthalmology. The ex-
periments were conducted on male nob mice and littermate con-
trols of ages 3– 6 months. ~The nob mutation was originally
identified on a BALB0cByJ background and was subsequently
crossed to C57BL06J mice; Pardue et al., 1998.! Because of inter-
mediate expression of nob in some carrier females, presumably due
to X inactivation, only male mice were used in these experiments.
Because nob is an X-linked recessive trait, affected nob mice and
normal littermates were obtained by mating affected males with
carrier females ~nob0!, resulting in male offspring that were ei-
ther affected or unaffected. Animals were screened for the nob mu-
tation by using a light-adapted ERG intensity series. Mice with any
indication of a positive b-wave were classified as normal, while
those with a flat or negative b-wave were classified as nob.
Except for experiments involving intravitreal injections and
related control measurements ~see below!, procedures used for
ERG recordings were similar to those described by Kang Derwent
et al. ~2002!. Briefly, animals that had been dark-adapted overnight
were anesthetized by an intraperitoneal injection of ketamine and
xylazine ~150 and 10 mg ~g body weight!
1
, respectively!. Boosts
of anesthetic ~approximately 106 of the initial dose! were delivered
subcutaneously at approximately 20 min intervals beginning
;40 min after the initial dose. The pupil of the eye to be tested was
dilated with 2.5% phenylephrine HCl and 1% tropicamide ~My-
driacyl!, and the cornea was anesthetized with 0.5% proparacaine
HCl. The body temperature was monitored by a rectal temperature
probe ~Model 555 temperature probe; Yellow Springs Instruments,
Yellow Springs, OH! and controlled to maintain a body tempera-
ture of about 37.58C to 38.58C ~Model TR-100; Fine Science
Tools, Inc., Foster City, CA!. The surface of the cornea was
periodically lubricated with methylcellulose solution ~Ultra Tears;
Alcon, Inc., Fort Wor th, TX! and moistened by the addition of
distilled water as previously described ~Kang Derwent et al.,
2002!. A stainless steel wire positioned on the cornea served as the
recording electrode. The ground electrode and reference electrode
were subdermal needle electrodes inserted at the nape of the neck
and within the cheek, respectively. The signal was amplified
1000-fold at a bandpass of 0.1–3000 Hz by a differential AC
amplifier ~Model CP511, Grass Instrument Co., West Warwick,
RI!. Recordings were saved on a computer using DT VEE soft-
ware ~Data Translation, Inc., Marlboro, MA! at a sampling rate of
10 kHz, except in 1 s recordings, which employed a sampling rate
of 1 kHz. A sampling rate of 1 kHz has been shown to be
sufficiently high to capture the relevant components of the ERG
~Robson & Frishman, 2004!. Test and probe flashes delivered from
separate flashguns to the inner surface of a hemispheric dome
coated with Kodak White Reflectance Coating ~part number 6080!
provided full-field stimuli ~Hetling & Pepperberg, 1999; Silva
et al., 2001; Kang Derwent et al., 2002!. Flash strengths in scotopic
candela s m
2
~sc cd s m
2
! were determined with the use of a
calibrated photometer ~Model IL1700, International Light, Pea-
body, MA!.
The experiments involving intravitreal injection of pharmaco-
logical agents used an apparatus and procedures that have previ-
ously been described ~Saszik et al., 2002!. Animals were anesthetized
initially with an intraperitoneal injection of ketamine ~70 and 7 mg
~g body weight!
1
, respectively!, and anesthesia was maintained
with ketamine and xylazine ~72 and 5 mg ~g body weight!
1
,
respectively; delivery every 45 min via a subcutaneous needle
fixed in the flank!. Pupils were dilated to 3 mm in diameter with
topical phenylephrine ~2.5%! and atropine ~0.5%!. Rectal temper-
ature was monitored, and maintained at 378Cto388C with an
electrically heated blanket ~CWE, Inc., Ardmore, PA!. The ani-
mal’s head was stabilized with a metal clamp over the nose, and a
mouth bar with a hole for the upper front teeth. The mouth bar
served as the ground electrode. ERGs were recorded differentially
between DTL fiber electrodes ~Dawson et al., 1979! placed on the
two eyes. Each electrode was moistened with 1.2% methylcellu-
lose in 1.2% saline, and on the stimulated eye the electrode was
covered with a contact lens, heat-formed from 0.2-mm clear
ACLAR film ~Honeywell, Morristown, NJ!. The cornea of the
non-stimulated eye was covered completely with an opaque con-
tact lens formed from 0.7 mm rigid black PVC sheet. The signal
was amplified 1000-fold and filtered using a bandpass of DC-
300 Hz, a range sufficient to capture the light-evoked signal, by a
Tektronix 5A22 amplifier ~Beaver ton, OR!, and after being digi-
tized at 1 kHz with a resolution of 2 µV, was sent to a computer for
averaging, display and storage. Ganzfeld illumination was pro-
duced by rear illumination of a translucent white diffusing screen,
35 mm in diameter, positioned close to the eye being tested and not
visible to the other ~covered! eye. The full-field flashed stimuli
were provided either by light from blue light-emitting diode lamps
~LEDs! with peak power output at 462 nm ~40 nm half-height
bandwidth! or, for higher energy flashes, with light from a small
xenon flash tube. The LEDs and the flash tube were positioned at
one end of a metal cylinder, which had a matte white internal
surface and whose other end was closed by the diffusing screen.
The diffusing screen absorbed radiation 380 nm. The stimulus
energy of the xenon tube, which was driven by an approximately
constant current, was adjusted by altering flash duration ~8to
128 µs!. For the LEDs, the driving current and flash duration were
adjusted to yield the required luminance. Flash strengths ~sc cd s
m
2
! again were determined using a calibrated photometer ~Inter-
national Light, Model IL1700!.
142 J.J. Kang Derwent et al.
Procedures used for intravitreal injection were similar to
those described ~Saszik et al., 2002!. Under a dissecting micro-
scope ~10!, the eye was punctured just behind the limbus with a
27-gauge needle, and a glass pipette needle ~tip ;20 mm! was
inser ted through the hole. The following pharmacological
agents were injected ~ 1.0–1.5 µl! using a Hamilton micro-
syringe ~Hamilton Company, Reno, NV!: APB ~l-2-amino-4-
phosphonobutyrate, 1–2 mM!, and PDA ~cis-2,3-piperidine-
dicarboxylic acid, 4.5–5.3 mM!. The vitreal volume of the adult
mouse eye was assumed to be 20 µl ~Saszik et al., 2002!, and doses
were based on those used in previous studies in cat ~Robson &
Frishman, 1995; Kang Derwent & Linsenmeier, 2001! and pri-
mates ~Bush & Sieving, 1994; Robson et al., 2003!. These drugs
did not consistently increase or decrease the amplitude of the
leading edge of the a-wave. Only data obtained after the ERG
response had stabilized after an injection, which generally oc-
curred within an hour, were included in this study.
A paired-flash procedure similar to that previously described
~Hetling & Pepperberg, 1999; Kang Derwent et al., 2002; Pepper-
berg, 2006! was used to derive the rod response to a test flash of
given strength I
test
. In each paired-flash run, the test flash was
presented at time zero and a bright probe flash of fixed strength
was presented at later time t
probe
. The probe response was analyzed
for amplitude at a determination time t
det
~6 ms in all experiments!
following probe flash presentation. A~t !, the amplitude of the
derived rod response at time t after the test flash, is then given by
the following equation:
A~t ! A
mo
A
m
~t !~1!
where t t
probe
t
det
, A
mo
is the response to the probe flash
delivered alone ~probe-alone response!, and A
m
~t ! is the probe
response amplitude determined in a paired-flash trial. Within a
series of trials, probe presentation times t
probe
were varied in a
random pattern, and the resulting probe responses were collected
and analyzed. The amplitude-response function at a given post-
test-flash time was determined with use of a fixed test-probe
interval t
probe
of 80 ms. The full time course of the rod response to
a fixed test flash was obtained by varying the test-probe interval
t
probe
. In all cases a 1- to 1.5-min period of dark adaptation
separated consecutive runs that involved probe flash presentation.
Results
Amplitude-intensity relation and time course
of the derived response
Fig. 1 shows responses elicited by a single flash ~test flash! from
a normal control mouse ~Fig. 1A! and a nob mouse ~Fig. 1B!. The
left- and right-hand sides of each panel show representative re-
sponses recorded on a fast and slow time scale, respectively. In the
control mouse, the a-wave amplitude increased with the strength of
the test flash, and the ERG contained a prominent b-wave com-
ponent and oscillatory potentials ~see, e.g., Pugh et al., 1998;
Frishman, 2006!. The a-wave recorded from the nob mouse also
increased with the test flash strength. Here, however, the ERG
contained little or no b-wave or oscillatory potentials, and resem-
bled the response obtained from the normal mammalian eye fol-
lowing treatment with glutamate analogs to remove all postreceptoral
ERG components ~e.g., Wakabayashi et al., 1988; Robson &
Frishman, 1995; Kang Derwent & Linsenmeier, 2001!. For reasons
noted previously ~Hetling & Pepperberg, 1999; Silva et al., 2001;
Kang Derwent et al., 2002; also see Lyubarsky et al., 1999;
Krishna et al., 2002!, the cone photoreceptors of the mouse are
unlikely to contribute substantially to the response recorded here.
The positive-going component evident in the waveforms illus-
trated on the longer time scale ~right panels of Figs. 1A and 1B! is
a developing c-wave ~Wu et al., 2004b!.
Fig. 2 shows instantaneous amplitude-intensity functions deter-
mined from paired-flash trials conducted on normal and nob mice.
Here the test flash strength I
test
was varied among trials and the
test-probe interval t
probe
was fixed at 80 ms, a period determined in
previous photocurrent and paired-flash ERG studies to correspond
with a near-peak time in the weak-flash response of mouse rods
~e.g., Xu et al., 1997; Hetling & Pepperberg, 1999; Calvert et al.,
2001; Krispel et al., 2003!. Fig. 2A shows probe responses re-
corded in these experiments from normal and nob mice. Waveform
PA is the probe-alone response obtained in darkness; labeled
responses are probe responses obtained in paired-flash trials with
the indicated test flash strength. These responses were analyzed for
amplitude at t
det
6 ms to yield, through eq. ~1!, amplitudes of the
derived response at t 86 ms ~ t
probe
t
det
!. Panel B shows
results obtained from control mice and nob mice, respectively.
Within each panel, the illustrated data represent values of A~86!0
A
mo
, the derived response at 86 ms normalized to the probe-alone
response A
mo,
plotted against the logarithm of the test flash strength
~log I
test
!. Each set of data was analyzed through the exponential
relation
A~86!0A
mo
1 exp~k
86
I
test
!~2!
where A~86!0A
mo
is the normalized derived response amplitude at
86 ms, and k
86
is a sensitivity parameter with units of inverse flash
strength ~Hetling & Pepperberg, 1999; Kang Derwent et al., 2002!.
The results obtained are shown by the dashed curves in Fig. 2B.
For the control mice, this analysis yielded k
86
4.79 ~sc cd s m
2
!
1
Fig. 1. Single-flash responses obtained from a normal, control mouse ~A!
and a nob mouse ~B!. In each panel, waveforms shown at the left ~short
time scale! are responses to flash strengths of 0.11, 0.98, 2.57, 4.37, 17.4,
27.5, and 348 sc cd s m
2
. Those shown at the right ~long time scale! are
responses to flash strengths of 40.0, 68.4, 102, 170, and 348 sc cd s m
2
.
Each waveform represents one recorded response. Scale bars on the left for
time and amplitude describe all left-hand waveforms ~i.e., those of both A
and B!. Scale bars on the right describe all ~A and B! right-hand wave-
forms.
Test of the paired-flash ERG method 143
and a least-squares fit R
2
0.989; for the nob mice, k
86
5.62 ~sc
cdsm
2
!
1
and R
2
0.993. The data were also analyzed through
the Hill equation ~i.e., a Naka-Rushton relation with variable
exponent! n:
A~86!0A
mo
I
test
n
0~I
test
n
I
test,0.5
n
!~3!
where I
test,0.5
is the test flash strength that produced half-saturation
of the derived response. The results, shown by solid curves in
Fig. 2B, indicated similar values of I
test,0.5
for the data obtained
from nob and control mice ~0.11 and 0.13 sc cd s m
2
, respec-
tively!, and exponent ~n! values of 1.22 and 1.48 for the nob and
control data. As determined by application of repeated-measures
ANOVA, there was no significant difference in normalized ampli-
tude data obtained from the investigated groups of normal and nob
mice ~F 1, P 0.878!.
The full time course of the derived response was obtained by
fixing the test flash strength and varying the test-probe interval.
Fig. 3A shows representative probe and probe-alone waveforms
obtained from normal and nob mice at a test flash strength of
2.57 sc cd s m
2
. Probe responses obtained with the shor ter
test-probe intervals were relatively small, indicating near-saturation
of the derived response; as the test-probe interval increased, the
probe response recovered to the maximal size corresponding with
the probe-alone response. A similar recovery can be seen in the nob
mouse. Fig. 3B shows the overall time course determined in
normal and nob mice with different test flash strengths. For the
results obtained at a given test flash strength, a repeated-measures
ANOVA was performed to compare the derived response data
obtained with controls versus nob mice. Results of this ANOVA
analysis indicated that there was a significant difference at all
three of the investigated test flash strengths, with the normalized
amplitudes for nob typically exceeding those for the controls at a
given post-flash time. At the lowest test flash strength ~0.11 sc cd
sm
2
!, this analysis yielded F 72 ~P 0.0001!. With the 2.57
and the 17.38 sc cd s m
2
test flashes, the analysis yielded F 500
~P 0.0001! and F 360 ~P 0.0001!, respectively. The
saturating derived responses obtained with the higher test flash
strengths can also be characterized by the time at which the
response’s falling phase passed through a criterion amplitude equal
Fig. 2. Normalized amplitude-intensity function obtained at a test-probe interval of 80 ms ~i.e., at 86 ms after the test flash!. A: Probe
responses obtained from a normal mouse ~left! and a nob mouse ~right!. Labels indicate test flash strengths in sc cd s m
2
. Response
PA is the probe-alone response. Each illustrated waveform is a single response. B: Normalized derived responses obtained from five
normal mice ~left! and six nob mice ~right!. Dashed and solid curves illustrate, respectively, fitting of the exponential equation ~eq. 2!
and Hill equation ~eq. 3! to the data.
144 J.J. Kang Derwent et al.
to 0.5A
mo
~i.e., to one-half of the maximal excursion!. Post-test-
flash times corresponding with this criterion normalized amplitude
were determined by interpolation between the data points. For nob
mice, post-test flash times determined in this manner to the 2.57
and 17.38 sc cd s m
2
stimuli were approximately 600 and
1050 ms, respectively; for control mice, the respective approxi-
mate values were 480 and 980 ms, respectively.
Effects of treatment with APB and PDA
If postreceptoral ERG components such as the b-wave and oscil-
latory potentials, or other signals from the OFF pathway ~e.g.,
Robson et al., 2003!, contributed significantly to the probe-flash-
induced ERG response determined at 6 ms after the probe flash,
these postreceptoral components would skew the paired-flash de-
terminations of the rod response to the test flash. To investigate the
possible contribution of postreceptoral responses to the probe
response obtained from nob mice, we obtained derived responses
before versus after the intravitreal injection of APB, an agent
known to block ON bipolar responses and subsequent responses in
the ON pathway ~Slaughter & Miller, 1981! and PDA, an agent
known to block responses from OFF bipolar, horizontal, amacrine,
and ganglion cells ~Bush & Sieving, 1994!. Because APB blocks
ON bipolar cell responses, we expected the light responses fol-
lowing APB treatment to be similar to those of nob mice to the
same stimuli. In the initial phase of the experiment, we determined
the nominal ~pre-treatment! time course of the derived response to
a fixed test flash of near-saturating strength ~0.45 sc cd s m
2
!
~see Fig. 2!. A single aliquot ~1 µl! of APB was then delivered
intravitreally, and the mouse was maintained in darkness for a
period of ;1 to 1.5 h. The derived response to the 0.45 sc cd s m
2
test flash was then again determined. Fig. 4A shows probe and
probe-alone responses obtained before and after APB from a
normal and nob mouse. Shown in each set are the probe-alone
response and those obtained in paired flash trials corresponding
with two times after a 0.45 sc cd s m
2
test flash: 200 ms, and at
a time of intermediate recovery of the probe response ~500 ms for
normal; 400 ms for nob!. In the normal, APB essentially abolished
the b-wave and oscillatory potentials, leaving only a small positive-
going potential immediately after the a-wave peak. However, APB
had no effect on the rising phase of the a-wave, and also had no
significant effect on the normalized recovery ~;50%! represented
by the probe response at 500 ms. In nob mice, the administration
of APB had little if any effect on the waveform ~Fig. 4A!. That is,
Fig. 3. Full time course of the derived flash response. A: Test flash strength of 2.57 sc cd s m
2
. Data obtained from a control mouse
~left! and a nob mouse ~right!. Labels identify the test-probe interval in ms. B: Normalized derived responses A~t !0A
mo
in response
to three different test flash strengths. Data indicate mean 6 SDs for results obtained from five normal and six nob mice at each test
flash strength.
Test of the paired-flash ERG method 145
both the pre- and post-APB waveforms ~labeled PA, 200 and 400!
lacked a b-wave and oscillatory potentials, and the recovery at the
interflash interval of 400 ms was not substantially affected by APB
treatment.
Fig. 4B shows the overall time course of the derived response
determined in normal and nob mice with a 0.45 sc cd s m
2
test
flash presented before versus after APB treatment. Here, open
symbols show the data obtained from control mice before ~squares!
and after ~circles! the treatment with APB; filled symbols show the
data obtained from nob mice before ~triangles! and after ~dia-
monds! treatment. Repeated-measures ANOVA was used to com-
pare the results obtained from control versus nob mice under a
given experimental condition ~pre- vs. post-APB treatment! and
indicated the absence of a significant difference ~F 1, P 0.910
for controls; F 1, P 0.912 for nobs!. However, there was a
significant difference among the aggregate data ~i.e., combined
pre- and post-APB data! obtained from controls versus nobs ~F
9.2, P 0.009!, with average amplitudes of the derived response
for controls exceeding those of nobs at mid- and late post-test-flash
times. Prior to APB treatment, the falling phase of the derived
response for controls and nobs exhibited a criterion amplitude of
0.5A
mo
at post-test-flash times of 430 and 350 ms, respectively, as
determined by interpolation between data points ~cf. text accom-
panying Fig. 3B!; following APB treatment, the respective crite-
rion post-test-flash times were 400 ms and 350 ms, respectively.
Figs. 5A and 5B show results obtained with cis 2,3-piperidine
dicarboxylic acid ~PDA!. Here a protocol similar to that described
earlier ~Figs. 3 and 4! was used to determine the course of the
derived response for nob and normal mice. Fig. 5 illustrates the
overall time course determined before and after PDA treatment. In
Fig. 5B, open squares and filled triangles show, respectively,
pre-PDA results obtained from control and nob mice; data obtained
from control and nob mice after PDA treatment are shown, respec-
tively, by open circles and filled diamonds. For both the nob and
normal mice, the injection of PDA did not substantially alter the
time course of the derived response. Prior to PDA treatment,
Fig. 4. Treatment with APB. A: Probe responses obtained from a normal
mouse ~upper! and nob mouse ~lower!, before ~left! and after ~right! the
intravitreal injection of APB ~1 µl!. The test flash strength was 0.45
sc cd s m
2
in all cases. Post-APB responses were recorded approximately
60 min after APB treatment. B: Time course of the normalized derived
response A~t !0A
mo
determined before and after APB treatment. Data
obtained from six control mice and three nob mice, including those of ~A!.
Data indicate means 6 SDs.
Fig. 5. Treatment with PDA. A: Representative probe responses obtained
before and after the intravitreal injection of PDA. The test flash strength
was 0.45 sc cd s m
2
in all cases. Results obtained from normal ~upper! and
nob ~lower! mice before and after application of PDA ~1 µl!. Post-PDA
waveforms were obtained beginning approximately 60 min after injection.
B: Time course of the derived response determined before and after PDA
treatment. Data obtained from one control mouse and two nob mice,
including those of ~A!. Data indicate means 6 SDs.
146 J.J. Kang Derwent et al.
half-recovery times for the control and nob derived responses were
440 and 460 ms, respectively; following treatment, both half-
recovery times were 450 ms.
Fig. 6 compares the raw ERG response and the paired-flash-
derived response determined in a nob mouse with use of a 0.45
sc cd s m
2
test flash. These data were obtained before ~Fig. 5A!
and after ~Fig. 5B! treatment with PDA. Here the peak amplitude
of the derived response has been normalized to the peak amplitude
of the raw waveform. The comparison illustrated in Figs. 6A and
6B is generally similar to that shown in Fig. 1 of Hetling &
Pepperberg ~1999!, where, at higher test flash strengths, the de-
rived response grew at a slightly faster rate than the leading edge
of the a-wave of the single-flash response waveform. The present
Fig. 6 data show that prior to and following PDA treatment, the
main difference between the single-flash response and the derived
response is the falling phase component of the derived response.
Fig. 6. Comparison of the single-flash ERG waveform with the paired-flash derived response obtained from a nob mouse before and
after PDA treatment. Results obtained in a single experiment ~one of those described in Fig. 5!. The illustrated waveforms and the
paired-flash data were obtained with test flash duration of 8 ms. A: Single-flash waveform and paired-flash-derived response ~mean 6
SD of two measurements at each time point! obtained with a 0.45 sc cd s m
2
test flash before PDA injection. Inset: Fitting of
eq. ~4! to the panel A data, yielding g 1.69, a 0.0021 ms
2
and t
v
247 ms. B: Data obtained following PDA injection. Same
format as that of A. Inset: Fitting of eq. ~4! to the panel B data, yielding g 1.57, a 0.0014 ms
2
and t
v
222 ms.
Test of the paired-flash ERG method 147
This main difference can be attributed to the presence of the c-wave
in the recorded waveform, which contributes strongly to the up-
swing of the response. The insets of Figs. 6A and 6B reproduce the
pre- and post-PDA derived responses shown in the respective main
panels, and illustrate the fit of a nested exponential equation shown
previously to approximate the kinetics of the derived rod flash
response ~Hetling & Pepperberg, 1999; their eq. ~6!!
A~t !0A
mo
1 exp@k
86
I
test
u~t !# ~4a!
u~t ! g$1 exp@a~t t
d
!
2
#%exp~t0t
v
!. ~4b!
Consistent with the similarity of the recovery-phase kinetics of
the pre- and post-PDA derived responses shown in Fig. 5, fitting of
eq. ~4! to the experimental data yielded similar values of the
exponential time constant t
v
~247 and 222 ms, respectively, in the
insets of Fig. 6A and Fig. 6B!.
Discussion
In this study we have examined paired-flash-derived responses
from nob mice and normal littermate controls. The results yield
three main findings. First, the instantaneous amplitude-intensity
function determined at a near-peak time ~86 ms! in the nob
response is similar to that obtained from control mice. For exam-
ple, half-saturation of the Naka-Rushton relation fitted to the data
occurs at 0.11 sc cd s m
2
for nob mice and 0.13 sc cd s m
2
for
controls ~Fig. 2 and accompanying text!. Second, the full time
courses of the derived response obtained from nob versus control
mice exhibit only modest albeit significant differences from one
another. Among the test flash strengths investigated in the Fig. 3
experiments, the difference in recovery-phase kinetics of the de-
rived response was greatest at 2.57 sc cd s m
2
, where the
post-test-flash times required to attain a criterion amplitude of
0.5A
mo
were about 480 and 600 ms, respectively, for control and
nob mice. At the 2.57 sc cd s m
2
test flash strength, as well as at
the 0.11 and 17.38 sc cd s m
2
flash strength, recoveries of the
control and nob derived responses were of generally similar shape.
Based on previous paired-flash ERG and photocurrent studies of
mouse rods ~Lyubarsky & Pugh, 1996; Hetling & Pepperberg,
1999; Chen et al., 2000!, an increase from 480 to 600 ms in the
half-recovery time of the response to a rod-saturating stimulus
corresponds, in normal mice, to an increase of roughly two-fold in
stimulus strength ~e.g., Fig. 8B of Hetling & Pepperberg, 1999!.
The modest differences in half-recovery time of the nob versus
control derived response were not replicated in the apparatus used
for the pharmacological experiments; the reasons for this discrep-
ancy are not clear, because the experimental conditions were quite
similar. Third, the results obtained with APB and PDA indicate a
similarity of the data obtained from a given mouse ~nob or control!
before and after pharmacological treatment. Previous investigators
have used APB to block ON bipolar cells and PDA to block all
post-receptoral pathways ~Sieving et al., 1994; Robson & Frish-
man, 1995, 1996; Kang Derwent & Linsenmeier, 2001!. However,
previous studies of nob have not performed treatments to test
whether there are any additional ERG components suppressible by
these drugs. In the nob mouse, which has a defect in the rod-
mediated pathway, we find that APB and PDA produce relatively
little change in the dark-adapted ERG waveform.
These findings support the conclusion that the derived response
determined in normal as well as in nob mice is largely or entirely
a photoreceptor response. That is, paired-flash ERG determination
of the full time course of the response in the presence of the
b-wave in normal mice approximates the photocurrent response
developed in the rods. Furthermore, in nob mice, the cellular
abnormality that underlies b-wave deficiency does not substan-
tially affect the rod phototransduction process.
A noteworthy point concerns the rapid upswing immediately
after the a-wave peak in the voltage response of nob mice to a
bright flash, yielding a “nose” in the response ~Figs. 1B and 2A!.
This rapid upswing has been observed in voltage traces recorded
from cat, monkey, and rat, and persists after blocking postsynaptic
responses with pharmacological agents such as aspartate, APB or
PDA ~Wakabayashi et al., 1988; Reiser et al., 1996; Kang Derwent
& Linsenmeier, 2001! and has been interpreted as due to a photo-
receptor inner segment, voltage-dependent I
h
current of the type
described by Bader et al. ~1982!~cf. Schneeweis & Schnapf, 2000;
Dong & Hare, 2000; Kang Derwent & Linsenmeier, 2001!. The
present results indicate the persistence of this rapid upswing in
responses obtained from both normal and nob mice following
treatment with APB ~Fig. 4A! or with PDA ~Fig. 5A!, and are
consistent with a basis of the upswing in an I
h
-type conductance of
the rod inner segment.
Acknowledgments
This research was supported by NIH grants EY05494, EY016094, EY01792,
EY06671, EY07551 and MH075791; by the Department of Veterans
Affairs; by an unrestricted grant from Research to Prevent Blindness, Inc
~New York, NY!; by a grant from the Daniel F. and Ada L. Rice Founda-
tion ~Skokie, IL!; and by a grant from the Macular Degeneration Research
Program of the American Health Assistance Foundation ~Clarksburg, MD!.
D.R.P. is a Senior Scientific Investigator of Research to Prevent Blindness.
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Test of the paired-flash ERG method 149
    • "We have therefore examined for a wide range of stimulus energies the correspondence between the simulations and ERGs recorded both from normal mice after intravitreal injection of APB as well as from nob mice.Fig. 11 shows a series of mouse ERGs evoked by stimuli with a range of energies of about 5000 to 1 (from data obtained by Kang Derwent et al., 2007). On the left are recordings from a wild-type mouse after intravitreal injection of APB while the recordings on the right were obtained from a nob mouse. "
    [Show abstract] [Hide abstract] ABSTRACT: The a-wave of the electroretinogram (ERG) reflects the response of photoreceptors to light, but what determines the exact waveform of the recorded voltage is not entirely understood. We have now simulated the trans-retinal voltage generated by the photocurrent of dark-adapted mammalian rods, using an electrical model based on the in vitro measurements of Hagins et al. (1970) and Arden (1976) in rat retinas. Our simulations indicate that in addition to the voltage produced by extracellular flow of photocurrent from rod outer to inner segments, a substantial fraction of the recorded a-wave is generated by current that flows in the outer nuclear layer (ONL) to hyperpolarize the rod axon and synaptic terminal. This current includes a transient capacitive component that contributes an initial negative "nose" to the trans-retinal voltage when the stimulus is strong. Recordings in various species of the a-wave, including the peak and initial recovery towards the baseline, are consistent with simulations showing an initial transient primarily related to capacitive currents in the ONL. Existence of these capacitive currents can explain why there is always a substantial residual transient a-wave when post-receptoral responses are pharmacologically inactivated in rodents and nonhuman primates, or severely genetically compromised in humans (e.g. complete congenital stationary night blindness) and nob mice. Our simulations and analysis of ERGs indicate that the timing of the leading edge and peak of dark-adapted a-waves evoked by strong stimuli could be used in a simple way to estimate rod sensitivity.
    Full-text · Article · Dec 2013
    • "However, the sensitivity of rod responses recorded in Ames is in closer agreement with the electroretinogram (half saturating flash strengths are ∼9 Rh* for single rod responses in Ames, ∼20 Rh* in Locke's and ∼10 Rh* for the electroretinogram; see Supplementary Materials and Hetling and Pepperberg, 1999). Furthermore, the isolated rod component of the electroretinogram shows a prominent contribution from the rod inner segment (Green and Kapousta-Bruneau, 1999; Nymark et al., 2005; Kang Derwent et al., 2007), complicating comparison with outer segment transduction currents. Ames and Locke's solutions are of course only two possibilities, and it is likely that in vivo conditions differ from both. "
    [Show abstract] [Hide abstract] ABSTRACT: Reliable signal transduction via G-protein-coupled receptors requires proper receptor inactivation. For example, signals originating from single rhodopsin molecules vary little from one to the next, requiring reproducible inactivation of rhodopsin by phosphorylation and arrestin binding. We determined how reduced concentrations of rhodopsin kinase (GRK1) and/or arrestin1 influenced the kinetics and variability of the single-photon responses of mouse rod photoreceptors. These experiments revealed that arrestin, in addition to its role in quenching the activity of rhodopsin, can tune the kinetics of rhodopsin phosphorylation by competing with GRK1. This competition influenced the variability of the active lifetime of rhodopsin. Biasing the competition in favor of GRK1 revealed that rhodopsin remained active through much of the single-photon response under the conditions of our experiments. This long-lasting rhodopsin activity can explain the characteristic time course of single-photon response variability. Indeed, explaining the late time-to-peak of the variance required an active lifetime of rhodopsin approximately twice that of the G-protein transducin. Competition between arrestins and kinases may be a general means of influencing signals mediated by G-protein-coupled receptors, particularly when activation of a few receptors produces signals of functional importance.
    Article · Sep 2009
    • "Effects of PDA on the dark-adapted a-wave of normal and Nob mouse also have been studied. In a recent study in this lab (Kang Derwent et al., 2007), effects of PDA were found to be insignificant when the leading edge of the saturated rod-driven a-wave was measured around 6 ms after the flash. However, for less saturated a-waves with slower onset and time to peak, reductions in a-wave amplitude measured at later times in the response were observed after PDA injection in Nob mice, and Control mice injected with APB (unpublished observations). "
    [Show abstract] [Hide abstract] ABSTRACT: The purpose of this study was to determine the contributions of postreceptoral neurons to the light-adapted ERG of the Nob mouse, a model for complete-type congenital stationary night blindness (CSNB1) that lacks a b-wave from depolarizing bipolar cells. Ganzfeld ERGs were recorded from anesthetized adult control mice, control mice injected intravitreally with L-2-amino-4-phosphonobutyric acid (Control APB mice) to remove On pathway activity, and Nob mice. ERGs also were recorded after PDA (cis-2,3-piperidine-dicarboxylic acid, 3-5mM) was injected to block transmission to hyperpolarizing (Off) bipolar and horizontal cells, and all third-order neurons. Stimuli were brief (<4ms, 0.4-2.5log sc td s) and long (200ms, 2.5-4.6log sc td) LED flashes (lambda(max)=513nm, on a rod suppressing background (2.6log sc td). Sinusoidal modulation of the LEDs (mean, 2.6log sc td; contrast, 100%; 3-36Hz) was used to study flicker ERGs. Brief-flash ERGs of Nob mice presented as long-lasting negative waves with a positive-going intrusion that started about 50ms after the flash and peaked around 120ms. Control APB mice had similar responses, and in both cases, PDA removed the positive-going intrusion. For long flashes, PDA removed a small, slow "d-wave" after light offset. With sinusoidal stimulation, the fundamental (F1) amplitude of control mice ERG peaked at 8Hz ( approximately 70microV). For Nob mice the peak was approximately 20microV at 6Hz before PDA and approximately 10muV at 3Hz or lower after PDA. F1 responses were present up to 21Hz in control and Nob eyes and 15Hz in Nob eyes after PDA. Between 3 and 6Hz, F1 phase was 170-210 degrees more delayed in Nob than control mice; phase was hardly altered by PDA. With vector analysis, a substantial postreceptoral input to the Nob flicker ERG was revealed. In control mice, the second harmonic (F2) response showed peaks of approximately 10mocrpV at 3Hz and 13Hz. Nob mice showed almost no F2. In summary, in this study it was found that in Nob mice, postreceptoral neurons from the Off pathway make a positive-going contribution to the light-adapted flash ERG, and contribute substantially to sinusoidal flicker ERG.
    Full-text · Article · Jul 2008
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