The method of visual stimulation and ERG recording has been
described previously.
14 16 Briefly, the stimuli were
presented on a computer-controlled monitor (BARCO CCID 121) driven at
100 Hz by a VSG 2/3 graphics card (Cambridge Research System). The
spectral characteristics of the monitor phosphors were measured with a
spectroradiometer (Instrument Systems). The luminance output
was calibrated using the internal luminance measuring device of the
BARCO monitor. The VSG software automatically performed the gamma
correction. The monitor subtended 124 by 108° at the 10-cm viewing
distance. We used 30-Hz square wave modulation of the red, green, and
blue phosphors with predefined Michelson contrasts. The time-averaged
luminance of the monitor was 66 candela (cd)/m
2 (40 cd/m
2 for the green phosphor, 20
cd/m
2 for the red phosphor, and 6
cd/m
2 for the blue phosphor). The time-averaged
chromaticity in CIE (1964) large field coordinates were
x = 0.3329 and
y = 0.3181. The
excitations in all cone types by the monitor phosphors were calculated
by multiplying the phosphor emission spectra with the psychophysically
based fundamentals.
17 The modulation of cone excitation
was quantified by the Michelson cone contrast and defined stimulus
strength for each cone type separately. The S-cones were not modulated
(i.e., the S-cone contrast was 0% in all conditions). In the majority
of normal subjects, we measured ERG responses to 32 different stimuli:
Eight conditions of different L- to M-cone contrast ratios (1:1; −1:1;
1:2; 0:1; 2:1; −2:1; −1:2: 1:0; negative ratios indicate
counter-phase modulation) with four contrasts at each condition (100%,
75%, 50%, and 25% of the maximally possible cone contrast). In the
RP patients, we limited the number of measurements to the four most
important conditions of L- to M-cone contrast ratios (1:1, 1:0, 0:1,
and −1:1), which allowed the simultaneous measurements of reliable
amplitudes and of response phases of cone-driven ERGs. The different
conditions were presented in a quasi-random order. Owing to the broad
emission spectra of the blue and green phosphors, the possible cone
contrasts were limited, see
Fig. 1 in Usui et al.
16 The
maximal cone contrast in the L-cone–isolating condition (M- and
S-cones were both silently substituted, i.e., their contrasts were 0%)
was 24.7% and 31.2% for the M-cone–isolating condition (double
silent substitution for L- and S-cones).
We assumed that the cone photopigment absorption spectra in RP patients
were identical with those of the controls. However, a decrease in
photopigment density would cause a change in the absorption spectra
and, thus, in cone contrasts. Our calculations have shown that halving
the density in all cone types would result in only a moderate change in
cone contrasts.
ERG recordings were obtained from one eye for all subjects. The pupils
of the normal subjects were dilated with 0.5% tropicamide, those of
the patients with both 0.5% tropicamide and 5% phenylephrine. The
eyes were kept light-adapted for at least 10 minutes before the ERG
recording. Corneal ERG responses were measured with DTL fiber
electrodes (UniMed Electrode Supplies), which were positioned on the
conjunctiva directly beneath the cornea and attached with the two ends
at the lateral and nasal canthus. The reference and skin electrodes
(gold cup electrodes) were attached to the ipsilateral temple and the
forehead, respectively. The signals were amplified and filtered between
1 and 300 Hz (Grass Instruments) and sampled at 1000 Hz with a National
Instruments AT-MIO-16DE-10 data acquisition card. ERG responses to 12
runs, each lasting 4 seconds, were averaged in each measurement.