A . Jenkins, M.W. Hankins Brain Research 887 2000 230 –237
231
retina Rutilus rutilus recorded through a 24-h diurnal tion. Cells were impaled with no photic stimuli and
cycle, reported no significant modulation of the HC light initially characterized using a single paired red and green
response amplitude, but noted significant changes in the flash of moderate intensity 650 nm, 531 nm, I
56.7
max 22
light response kinetics [9]. L-HCs in the roach, like most mW cm
, 450–500 ms. These responses provided the cyprinids receive a dominant LWS red cone input, with a
data for the principal amplitude and kinetic analysis with a weak MWS green cone input [2]. This raises the question
minimal light exposure SA, n569; LA, n579. The as to whether long-term light history or circadian diurnal
responses of the cells were then additionally characterized regulation might drive the changes in cone-driven L-HC
using a range of spectral or spatial stimuli. These responses kinetics. Interestingly, long-term light-history appears to
provided information confirming the relative spectral sen- affect the temporal response of the human photopic cone
sitivity, and allowed us to assess the extent of the HC electroretinogram [7]. Thus it was shown that the b-wave
receptive fields of individual cells. After the experiments, implicit time of the photopic cone ERG exhibits a pro-
detailed measurements of the individual light evoked nounced sinusoidal oscillation, the period of oscillation
responses S-potential were performed in accordance to being |24 h with peak temporal response in the middle of
those outlined in Fig. 1. This involved simple amplitude the day. This raises the possibility that the temporal
measurement of the principal components, together with a response of cone pathways is modulated according to
range of kinetic measurements on the rate of hyperpolari- long-term light-history.
zation and depolarization. An assessment of receptive field We have therefore examined the effect of long-term dark
size was made using two methods. Firstly, the amplitude of adaptation on the light responses of L-HCs cells in the
the annulus response for each cell was extrapolated to an roach retina. Some of this work has been reported in
equivalent amplitude response spot area Fig. 1c. Second- abstract form [5].
ly, the ratio of the amplitude of the annulus and spot annulus spot was calculated for each cell to give a
measure of the relative strength of surround response.
2. Methods
Experiments were performed on the isolated freshwater 3. Results
cyprinid Rutilus rutilus roach retina. The fish were maintained in outdoor holding tanks under natural day–
3.1. The amplitude of the light response and the night ambient lighting. The experiments were performed
receptive field properties during the middle 3 h of subjective day. The fish were
dark-adapted according to one of two regimes. In the case We examined the general light responsiveness of the
of short dark adaptation SA the fish were placed in L-HCs cells using a number of approaches. First we
absolute darkness for 10 min, in long dark adaptation LA examined the HC response amplitudes to the initial single
22
they were placed in darkness for 3 h. After this period they red stimulus 650 nm, 4.1 mm o.d., 6.7 mW cm
. We were killed T50 and the retinae isolated under IR
found no significant difference in the amplitudes in the two illumination. After the preadaptation both groups of retinae
groups; thus the response of the SA cells was 19.6360.86 experienced the same conditions throughout the recording
mV and that for the LA cells was 20.2760.80 mV mean61 period. The retinae were placed photoreceptor side upward
S.E.M.. For a population of cells, we examined the V in a moist oxygenated plexiglass recording chamber. The
log I relation and found no significant differences in chamber was semi-enclosed to allow electrode penetration
amplitude at any of the intensities Fig. 2a. This was also from above. The experimental recording period then lasted
confirmed by measuring the 1 2V stimulus intensities
max
from T55 min to T520 min. Recordings were made from for the individual cells, and the mean intensity for SA
luminosity-type horizontal cells L-HCs using KCl-filled retinae 21.260.2 log units was not significantly different
glass micropipettes 80–100 MV, and conventional re- from the LA retinae 21.360.27 log units. We also
cording methods. Cell recordings were digitized and examined the dark resting membrane potential E for the
m
recorded Axon Instruments for detailed post-experimental two groups, and again found no significant difference in
analysis. Additional experiments Fig. 6 utilized the the distribution or mean level SA, 236.761.5 mV; LA,
superfused roach retina preparation [8], where the retina 238.161.5 mV. The relative spectral sensitivity of the
was clamped photoreceptor side up in a plexiglass chamber cells was assessed at a range of stimulus wavelengths, the
and the retina perfused from the photoreceptor surface with results showed no significant difference between the SA
cyprinid ringer. and LA cells Fig. 2b. In the case of six cells, the spectral
The light stimuli were generated using a twin-channel sensitivity was assessed using the photopic stimuli pre-
Maxwellian optical system. This provided a range of sented upon a rod saturating 500 nm background and this
spectral 450, 486, 531, 570, 620 and 650 nm and spatial also failed to reveal differences between the two conditions
stimuli spot: 0.56–4.1 mm o.d. and annulus 0.9 mm i.d., not illustrated. In addition, we examined the V log I and
4.1 mm o.d. that were projected from below the prepara- spectral sensitivity curves for L-HCs obtained from a
232 A
Fig. 1. Measurement of HC light response components. a Schematic of the light response waveform of an L-HC to a |500-ms stimulus. Amplitude measurements were made relative to dark resting membrane potential of the steady state light response at 450 ms. ON-relaxation amplitude was measured
from the light peak to the steady state level. The OFF-transient peak was measured relative to E . Hyperpolarizing and depolarizing rates of change were
m
measured within the pseudo-linear range 25–75 of the light response S. b Time to reach 2.5, 5, 10, 50 and 66 of the steady light response was also measured for each cell. c Method used to determine the equivalent area of the annulus response. The amplitude of the annulus light response of each cell
A was translated to an equivalent area response EA by extrapolation from its response spot size curve.
group of retinae obtained under infra-red illumination in response to a single red-flash stimulus 650 nm, normal-
the middle of the night. The data from the night-HCs were ized the response to the steady-state light response level
not significantly different from the SA and LA retinae for each cell and averaged these waveforms. The results
Fig. 2a,b. are shown in Fig. 4b, and establish that there are signifi-
Analysis of the cell responses to a range of spot and cant changes in the waveform, most clearly apparent in the
annulus stimuli were used to assess the general extent of speed of hyperpolarization at light-ON. SA cells were
HC receptive field. The results show there are highly significantly faster than those recorded in LA retinae. Cells
significant P,0.001 differences in the mean extent and from the LA retinae were characterized by a slower rate of
distribution of receptive fields according to their long-term hyperpolarization, reflected by a long delay in reaching
preadaptation history Fig. 3. The annulus spot ratios maximal amplitude. Furthermore, these differences in ON-
were significantly larger in the LA retinae, consistent with kinetics were also apparent when cells were recorded in
an increased sensitivity to the peripheral stimulus. In the presence of a rod-saturating 500 nm background.
addition, when we examined the equivalent isoluminant Typical single light responses for SA and LA cells are
area see methods for the annulus stimulus 0.9 mm i.d., given in Fig. 4a.
2
4.1 mm o.d. we found this value to be 1.9560.35 mm in Using the waveform parameters outlined in Fig. 1, we
2
the SA cells and 5.6860.59 mm in the LA cells n531, a performed a detailed analysis of the light responses
significant difference at P,0.001, t-test. recorded in the two groups of retinae. The latencies were
examined at a number of -response increments. The data 3.2. Changes in the L-HC light response S-potential
revealed that the onset of hyperpolarization was signifi- waveform
cantly delayed in the LA cells compared to that of the SA group Fig. 5a. The latency threshold 2.5 for the SA
Whilst we saw no difference in the amplitude of the cells was 13.6260.41 ms, whilst in the LA group it was
light response, examination of the waveform suggested 21.4860.33 ms mean61 S.E.M.. We also examined the
that the kinetics were modified following prolonged dark rate of hyperpolarization for the two groups of cells, by
adaptation. To examine this change we took the cells first measuring the slope in the pseudo-linear portion of the
A . Jenkins, M.W. Hankins Brain Research 887 2000 230 –237
233
Fig. 3. Frequency distributions of the annulus spot ratios for the SA and LA cells. In this case the cell responses to a spot o.d. 4.1 mm and
Fig. 2. a V log I response curves. Mean steady-state HC light response annulus i.d. 0.9 mm, o.d. 4.1 mm were used to calculate an annulus
amplitude mV is plotted as a function of stimulus intensity. SA h and
spot ratio for each cell. Note that in the LA cells the distribution and LA
j are plotted together with the standard errors of means 61 mean ratio is shifted to higher values, corresponding to an increased
S.E.M.. Data are also shown for L-HCs recorded from retinae obtained in sensitivity to the peripheral annulus stimuli. The mean ratio for the SA
the middle of the night under infra-red ? ? ?. The data were statistically group was 0.5560.03 n533, whilst that for the LA group was 0.860.02
tested t-test and Bonferroni correction and none of the differences n530. This difference was found to be significant at the P,0.001 level.
between SA and LA were found to be significant at the P,0.05 level The data are consistent with an increased effective HC-receptive field in
n519. Stimulus 650 nm, spot 4.1 mm diameter o.d., Log 0.2.193 the LA condition.
13 22
21
10 quanta cm
s . b Relative spectral sensitivity. The amplitude of
the light response mV is plotted as a function of stimulus wavelength
additional waveform components, including a light-ON
l, nm. Data are shown for the spectral sensitivity for the SA h and LA
j. Data are also shown for L-HCs recorded from retinae obtained
relaxation roll-back before the response settles to the
in the middle of the night under IR ? ? ?. We found no significant
steady state 450 ms, and or a light-OFF transient de-
differences in the SA and LA spectral response at any of the wavelengths
polarizing overshoot. Where present, we measured the
tested t-test, Bonferroni correction. Stimulus spot: 4.1 mm, nSA537,
22
amplitudes of these components in accordance with the
nLA525 I 6.7 mW cm
.
max
definition outlined in Fig. 1. When we examined the mean amplitudes of both these components in the two groups of
light response 25–75. This was performed both on the preadaptation conditions, we found no significant differ-
raw data mV ms and normalized data ms. Both sets ences in their relative expression Table 1. Furthermore
of data show that there is a significant difference P, the percentage of cells that expressed either of these
0.001 in the rate of polarization Fig. 5b. Time-constants components did not vary according to the light-history.
t were also calculated for the light-ON using the time points between threshold 2.5 and 66 response. These
3.3. Horizontal cell responses in the superfused retinal show that the mean
t for the SA cells was 32.462.39 ms preparations
n562, whilst that for the LA cells was 53.463.03 ms n561 and again these results were significant at the
We performed a number of experiments utilizing SA and P,0.001 level. In contrast, measurements of the rate of
LA retinae recorded in superfused retinal preparations repolarization at light-OFF showed no significant differ-
n525. We found that in general the responses recorded ences between the SA and LA cells Fig. 5c. Therefore
from L-HCs in superfused retinae were slower. However, the change in response kinetics appears to be confined to
the relative difference between SA and LA retinae was the ON rather than the OFF response of the HC light
maintained, with SA cells displaying characteristically response.
faster responses at light-ON Fig. 6a. We also examined The light responses of individual HCs can include
the effect of exogenous dopamine DA in these prepara-
234 A
Fig. 5. Kinetics of the HC light response. a Delay in the onset of the light response. In this graph the time taken to reach 2.5, 5, 10, 50 and
66 of the steady-state amplitude are shown 61 S.E.M.. The differ- Fig. 4. HC light response waveform. a The S-potential light response
ences between the SA and LA cells were all significant at the P,0.001 for a typical cell from the SA group ——— and the LA group ? ? ?.
level. The latency to 2.5 threshold was 13.6260.41 ms for the SA cells The waveforms are normalized to the steady state light level. Stimuli 650
and 21.4860.33 ms for the LA cells mean61 S.E.M.. b The rate of nm, spot 4.1 mm. b the averaged normalized waveform range 61
hyperpolarization at light-ON. A comparison of the absolute mV ms and S.E.M. error spreads plotted for the total cell populations in the study.
normalized ms rates for the SA and LA cells, these differences were Note the clear shift in the kinetics of the light-ON hyperpolarization in
both significant at the P,0.001 level. c The rates, absolute and the LA group combined with a significant delay to reach steady state
normalized, for the depolarization at light-OFF, showing no significant nSA574, nLA587.
difference between the SA and LA cell populations.
tions, and in four out of six cells studied we found that 10 our experiments with the SA and LA retinae in superfused
mM DA selectively and reversibly affected the light-ON preparations Fig. 6.
kinetics in L-HCs recorded from LA retinae Fig. 6b. When we examined the steady-state amplitudes of the
light responses of L-HCs and their V log I functions, we found no difference in the cells recorded from the two
conditions Fig. 2a. Furthermore, there was no significant
4. Discussion change in the relative spectral sensitivity Fig. 2b, such as
