Targeting small bistratified blue-ON cells
Small bistratified, blue-ON cells were targeted in vitro by their relatively large cell bodies comparable to that of the parasol ganglion cells (Dacey, 1993
; Dacey, et al., 1994
) observed either by epiillumination after vital staining with acridine orange or by infrared illumination of the unstained retina. Blue-ON cells were easily distinguished from parasol cells by sustained OFF responses to 500 ms light steps using the red or green LED primary and sustained ON responses to the blue LED primary. Parasol cells and other non-opponent cell types (Crook et al., 2008
) showed transient responses that varied in amplitude but did not change in sign in response to the LED primary lights. A large bistratified blue-ON cell (Dacey and Packer, 2003
) would have had responses similar to a small bistratified blue-ON but these cells show smaller cell body size and have significantly larger receptive fields than those mapped for the small bistratified blue-ON cells. Altogether we include data collected from 89 blue-ON cell recordings. For a subset of these cells we mapped S and LM receptive fields, compared S and LM sensitivity and ran a series of pharmacology experiments. The effect of the inhibitory receptor antagonists, picrotoxin and strychnine, was tested on 17 cells, L-AP-4 was tested on 22 cells either alone or in combination with the inhibitory antagonists, and for 3 cells that had been tested with picrotoxin, strychnine and L-AP4, HEPES was also applied. Rod inputs were characterized in an additional 16 cells. Finally, data from cells recorded intracellularly (n = 6) were also included.
Identification of a rod-mediated ON input to blue-ON cells
One of the main goals of this study was to measure the relative weights and spatial dimensions of S-ON and LM-OFF input to the small bistratified blue-ON cell. However the stimuli used to isolate the S vs LM cone inputs described in the Methods can also provide strong stimulation of rods. Conflicting reports describe a distinctive excitatory rod input (Virsu et al., 1987
) or an apparent lack of rod input for cells of the blue-ON pathway (Lee et al., 1997
). At the start of an experiment the macaque in vitro preparation was maintained in a dark adapted state and when first recording from a blue-ON cell we often observed strong ON responses to both S and LM cone isolating stimuli in identified small bistratified cells at retinal illuminances (<101
photoisomerizations/cone/s) below the threshold for cone-mediated responses in our preparation (Dacey et al., 2005
). Since additivity of rod and cone signals at mesopic levels in ganglion cells has been well documented (Gouras and Link, 1966
; Gouras, 1967
; Enroth-Cugell et al., 1977
; Lee et al., 1997
) it thus became critical to provide a preliminary characterization of this apparent rod input and, crucially, to determine the illuminance at which rod signals in our in vitro preparation could potentially interact with and effect our measurement of cone signal properties. The response of a blue-ON cell to a 1500 ms blue or green primary LED light step shows a dramatic change in temporal response as a transition is made from cone dominated to a rod dominated response over a 4 log unit illuminance range () that spans the mesopic level (~0–3 log10
photopic trolands). At mid photopic levels (~103
photoisomerizations/cone/s) a sustained ON response to the blue primary and an OFF response to the green primary are observed and these responses are presumably driven primarily by an S-ON response to the blue and an LM-OFF response to the green primary (, top traces). Latency from blue stimulus onset (or green stimulus offset) to first spike is ~40–60 ms (, top traces). As retinal illuminance is reduced however from ~104
photoisomerizations/cone/s the OFF response to the green primary is replaced by an ON response (n = 16). Thus at ~5–10 photoisomerizations/cone/s both the blue and green stimuli elicit similar, sustained ON responses (, bottom traces). At this light level the rod quantal catch dominates and the latency from stimulus onset to spike threshold increases to ~150–200 ms (, bottom traces). At intermediate illuminances (, middle traces; 103
photoisomerizations/cone/s) the rod-ON and LM-OFF responses are not easily distinguished and there appears to be some mixing of the two signals. The large latency difference between the rod and cone dominated responses was also manifest in the temporal frequency response to sinusoidal modulation at high vs low retinal illuminances (). At low illuminances the rod mediated response peaked at 2 Hz, rolling off sharply between 4–7 Hz compared to the cone mediated response peak at ~10 Hz, rolling off between 40 and 50 Hz. Response latency derived from linear fits to response phase was about 5 times slower (177 ± 36 ms, mean ± sd; n = 10) than cone latencies (40 ± 3 ms, mean ± sd; n = 20). Finally at the low retinal illuminances eliciting a sustained ON response to both the blue and green LED primaries, bath application of L-AP4, the mGluR6 receptor agonist, completely eliminated the ON light response (n = 2) consistent with an origin of this signal from the depolarizing rod bipolar cell (). These results provide evidence for a strong rod mediated ON input to the small bistratified blue-ON cells. Below cone threshold the light response is converted from an S-ON, LM-OFF response to a rod-ON response. From cone threshold to rod saturation (between 102
photoisomerizations/cone/s) there is potential for interaction of the rod-ON input with the S and LM cone inputs. Given the large difference between the rod and cone signal dynamics the nature of this interaction would be dependent on the stimulus temporal frequency as well as the illuminance level. At 104
photoisomerizations/cone/s and above we assume rods are in saturation; we believe this is a safe assumption as psychophysical experiments show that saturation of primate scotopic vision begins when a background light reaches around 100 scotopic trolands (Hayhoe et al., 1976
); this is ~400 photoisomerizations/cone/s, and is consistent with recordings from individual primate rods (Baylor et al., 1984
). We have employed stimuli well above this level for all subsequent measurements of cone responses in the small bistratified blue-ON cells.
Figure 3 Blue-ON cells have rod-ON responses at low retinal illuminances. A, Blue-ON responses to 1500 ms blue and green primary LED light steps measured across a 4 log unit illuminance range with the integrated irradiance (photons/s/μm2) of the blue primary (more ...)
Weighting of S-ON vs LM-OFF input to the blue-ON cells
The balance and linearity of S ON vs LM OFF inputs to the blue-ON cell was assessed with two different protocols. As a first rough test cone-isolating pulses were modulated at 16% contrast in the following combinations; S, L+M (LM), L+M+S (LMS) and LM-S, the negative sign indicating a 16% contrast decrement. For a cell with balanced inputs the S ON and LM OFF responses should be equal and for a linear cell the LM-S response should equal the sum of individual S ON and LM OFF responses while an LMS stimulus would elicit no net modulation. For a typical response, shown in , the calculated Fundamental Fourier (F1) response amplitude to modulation of the S cone = 20 spikes/s, LM cones = 21 spikes/s, (LM-S)-cones = 41 spikes/s, and no significant response to inphase modulation of all three cones (LMS). This result was consistent across cells and showed that at low contrasts blue-ON cells receive relatively equally weighted (S:LM = 1.16 ± 0.24, mean ± sd, median = 1.13; n = 67; ) and linear (LM-S: S+LM = 0.86 ± 0.14, mean ± sd, median = 0.87; n = 67; ) S and LM inputs.
Summary of effects of GABAa/c and glycine receptor antagonists on the chromatic responses properties of the blue-ON cell.
A second stimulus protocol was designed to quantify the relative S vs
LM cone input weights more systematically. Complete datasets were achieved in a subset of 51 cells in the total sample of 89 in which cone weights were estimated. The stimulus varied S and LM cone contrast and phase stepwise from equal cone contrasts in phase to equal contrasts in anti-phase (). The data was fit by a simple linear model of cone summation (Diller et al., 2004
) and is described in detail in the Methods (see also caption). As expected, blue-ON cells peak response typically occurred when the S and LM cones were modulated at equal depth (equal cone contrasts) and in anti-phase and the response minimum was reached when the cones were modulated at equal depth and in phase (). Assuming linear summation, the fit quantifies the S:LM weight. Thus for the cell illustrated in the calculated S:LM cone input weight equaled 0.97. S and LM normalized and averaged response amplitudes and phases were averaged to give an input weight ratio (S:LM) of 1.09 ± 0.21 (mean ± sd; median = 1.56; n = 51)(, ).
S and LM cone contrast and temporal sensitivity
The relative balance of S and LM inputs was also tested at a range of lower and higher contrasts and temporal frequencies. Contrast sensitivity was compared using S and LM cone-isolating stimuli between 2–38% which were the maximal cone contrasts that we could achieve on a background of equal mean quantal catch for all three cone types. Both LM and S responses increased linearly with contrast (). The slope of a straight line fit to the data was used to quantify contrast gain (). The average contrast gain for the S was 1.26 ± 0.49 (mean ± sd; n = 8) and for the LM input 1.25 ± 0.25 (mean ± sd; n = 8). These overlap with contrast gain values calculated from extracellular recordings of L vs M and S vs LM opponent cells in the intact animal (Yeh et al., 1995
; Solomon et al., 2005
; Tailby et al., 2008
) and were somewhat lower than that for the well studied magnocellular-projecting ganglion cells in response to an achromatic stimulus (Crook, et al., 2008
). To illustrate this distinct difference we recorded from neighboring magnocellular projecting parasol cells using the same LM stimulus (parasol cells did not respond to S cone isolating stimuli) at an optimal spatial and temporal frequency (10.5 Hz). Parasol cells show extreme sensitivity to low contrasts with response amplitudes more than doubling to two-fold increases in contrast (, squares). The parasol cell contrast response functions were well fit with Naka-Rushton saturation functions (see Methods) and contrast gain values were calculated from the initial linear portion of the curve. On average the contrast gain was 4.85 ± 0.37 (mean ± sd; n = 3).
Figure 5 Blue-ON cells S and LM receptive fields have similar contrast and temporal sensitivities. A, Average contrast response functions measured with low spatial frequency (0.047 cpd) gratings with S (solid circles, n = 4) and LM (open circles, n = 4) cone isolating (more ...)
We compared LM and S temporal sensitivities by measuring temporal modulation transfer functions with cone isolating stimuli. A 2 mm diameter field was sinusoidally modulated at 25–48% contrast at temporal frequencies ranging from 0.61–78 Hz. S-ON and LM-OFF inputs were both sensitive to temporal frequencies up to 60 Hz with similar peak responses (S cone = 11 ± 7 Hz and LM cone = 7 ± 2 Hz, mean ± sd; n = 20)(). The inset plots the corresponding phase data for one cell. The phases of the S and LM inputs were initially approximately 180 degrees offset and converged with increasing temporal frequency. For each cell the linear portion of the phase data was fit with a straight line to calculate the latency. For the cell shown in the inset to , the S input latency was 42 ms and the LM 48 ms. Across cells the response to S cone stimuli showed slightly shorter latencies than that for LM stimuli (S mean latency ± sd = 40 ± 3 ms; LM mean latency ± sd = 46 ± 3 ms; n = 20)(). Our data are roughly consistent with those reported from measurements using comparable stimuli made in the intact eye in vivo (e.g. Yeh et al., 1995
). In Yeh et al, latencies of ~35 ms were found for both the S and LM cone driven responses at comparable light levels to those used here. The reason for this discrepancy is unclear though it could reflect effects of the in vitro maintenance on the temporal sensitivity of the retina. We have observed anecdotally a profound effect of retinal temperature on the response dynamics; while we attempt to keep our in vitro retinas as close to physiological temperature as possible (typically ~35–36 deg C) it is only possible to monitor temperature and a location in the superfusion chamber relatively distant from the recording site so precise temperatures and temperature fluctuations are not known. In another recent study recording from blue-ON cells in the in vitro retina much longer latencies of about 50 and 70 ms for the S and LM cone inputs respectively were reported (Field, et al., 2007
). However retinal illuminance was lower in this study and latencies were calculated from time to peak of the spike-triggered average derived from a white noise stimulus so it is possible that these methodological differences also contributed to the additional deviation from that found in vivo. Overall our data are consistent with previous work from recordings in the intact animal suggesting similar temporal and contrast sensitivities for S ON and LM OFF inputs to the blue-ON ganglion cell.
Coextensive S and LM cone receptive fields
To test whether blue-ON cells had center-surround or coextensive receptive field structure the S-ON and LM-OFF receptive fields of 55 blue-ON cells were mapped with either drifting sinusoidally modulated gratings as a function of spatial frequency, or sinusoidally modulated spots and annuli as a function of increasing diameter. Regardless of stimulus both receptive fields showed similar or identical spatial tuning (). For drifting grating stimuli, both the S and LM spatial frequency responses were low pass () with comparable peak responses at the lowest spatial frequencies tested and similar high spatial frequency cut-offs. To a growing spot centered on the cells receptive field (), LM and S response magnitudes together increased and peaked when the spot diameter expanded to fill an area that approximated the expected diameter of the dendritic tree of a small bistratified cell at the eccentricity of the recorded cell (Dacey, 1993
). The response plateaued at this diameter (usually between 75–200 μm) remaining elevated as the spot continued to grow () suggesting little or no surround antagonism. If a surround was present, increasing spot size to encroach on the surround should attenuate the response. To an annulus centered on the cells receptive field () the maximum response for both the LM and S inputs occurred when the inner diameter was smallest (9 μm). As the annulus center increased both ON and OFF inputs rolled off with no sign of a reversal in the response phase (inset) as would be indicative of a surround. Low pass S and LM spatial frequency responses were thus well fit with a single Gaussian. Examples in show the Gaussian fits (solid lines) and the inset in each plot shows the model 2D receptive field profile and calculated Gaussian radii (μm). S and LM cone driven receptive field sizes were nearly identical (S to LM diameter ratio from the Gaussian fits = 0.92 ± 0.13, mean ± sd; median = 0.94; n = 55; ) and the slope of the fit to LM vs S Gaussian radii in was equal to 0.97 (R2
= 0.90). The peak response amplitudes (~30 spikes/s) were also typically matched for the S and LM responses (S:LM response ratio = 1.19 ± 0.38, mean ± sd; median = 1.19; n = 55). Thus the S and LM fields are well matched in both size and sensitivity.
Figure 6 Blue-ON S and LM receptive fields are low pass and nearly co-extensive. Receptive fields were mapped with LM and S cone isolating drifting gratings as a function of spatial frequency, or spots and annuli as a function of center diameter. All spatial stimuli (more ...)
To summarize, the S-ON and LM-OFF inputs show similar contrast and temporal sensitivities; the two fields are also nearly equal in area and sensitivity, consistent with a spatially coextensive pattern (). To explore the retinal circuitry that generates the S-ON and LM-OFF responses () we used pharmacological manipulation to assess the role of excitatory ON pathway and inner retinal inhibitory pathway input to the small bistratified cell.
Opponent responses are not generated by inner retinal inhibitory pathways
A large percentage of inputs to the small bistratified cell are from amacrine cells (Calkins et al, 1998
; Ghosh and Grunert, 1999
). We therefore assessed whether inhibitory amacrine input had a major role in generating the balance of S vs LM inputs or shaping their coextensive receptive fields. We recorded responses from blue-ON cells in the presence of picrotoxin, a general GABAa/c receptor antagonist, and strychnine, a glycine receptor antagonist, both in combination and alone, using the same stimuli used to measure S vs LM input strength () and receptive field spatial structure (). Qualitatively the intracellular and spike histogram responses to cone isolating stimuli presented in clearly illustrate that the blue-ON cell’s S and LM opponency was not greatly enhanced or attenuated in the presence of picrotoxin (), strychnine (), or picrotoxin and strychnine together (). Further, the S and LM response amplitudes and LM-S vs LMS ratios calculated from the histograms were indistinguishable from the controls for all three conditions (). The sensitivity and balance of the S and LM cone input weights to incremental changes in contrast, measured with the protocol described in , was also maintained. For picrotoxin and strychnine alone (, open circles), and picrotoxin and strychnine together ( open circles), the responses, fits and relative S vs LM cone input weights () overlapped the controls ( solid circles). Blocking the inhibitory pathways also had no effect on the spatial extent of the S and LM receptive fields. Using cone isolating drifting gratings the S and LM receptive fields were measured in the presence of picrotoxin (), strychnine (), and picrotoxin and strychnine together (). For each condition the S and LM spatial frequency responses remained low pass, coextensive and well fit with a Gaussian receptive field model (solid lines). These results are summarized in , showing the S:LM radii and integrated volumes ratios. For all 3 conditions there was no change compared to the controls. Overall blue-ON cells remained opponent () with equally weighted S and LM inputs () and with intact, approximately co-extensive, low pass LM and S receptive fields (). However, for the intracellular recordings to high contrast S vs LM cone modulation (, upper traces) we noted subtle changes to S and LM amplitudes and kinetics after blocking GABAergic inhibition. In the presence of picrotoxin both the S and LM responses increased in amplitude, quantified as the amplitude Fourier component of the voltage response at the stimulus frequency (; picrotoxin/control S = 1.63 ± 0.14 and LM = 1.44 ± 0.22, mean ± sd; n = 3). The LM responses also hyperpolarized more slowly at the onset of the light stimulus (). The width of the LM response, measured at 1/3 the maximum response amplitude, increased from 264 ± 10 ms to 316 ± 13 ms (mean ± sd; n = 3) while the width of the S response was unchanged, from 246 ± 4 ms to 245 ± 8 ms (mean ± sd; n = 3). In the presence of strychnine the light responses were not significantly changed in amplitude (; strychnine/control S = 0.92 ± 0.12 and LM = 1.04 ± 0.07, mean ± sd; n = 3) or width (S control = 241 ± 6 ms and S strychnine = 243 ± 8 ms; LM control = 257 ± 14 ms and LM strychnine = 252 ± 19 ms, mean ± sd; n = 3). We conclude that inner retinal inhibitory pathways may play a role in shaping the amplitude and dynamics of the S and LM response but that these pathways are not essential for generating the LM-OFF component of the blue-ON cell response.
ON pathway block isolates an LM OFF field with center-surround organization
To directly test the hypothesis that input from the ON pathway generates the small bistratified cell’s S-ON response and that the LM-OFF response arises from a parallel OFF bipolar pathway synaptic input (Calkins et al., 1998
; Ghosh and Grunert, 1999
) we recorded responses to cone-isolating stimuli in the presence of the mGluR6 receptor agonist L-AP-4 to block ON pathway transmission (Slaughter and Miller, 1981
). The LAP-4 was also applied in combination with strychnine and picrotoxin to isolate excitatory OFF pathway input to the small bistratified cell. The addition of L-AP-4 abolished the SON response while sparing the LM-OFF response. Robust LM-OFF responses of similar amplitude were recorded to LM, LM-S and LMS cone modulation (). With S-ON input blocked the LM-S and LMS stimuli now elicited responses equivalent to pure LM stimuli. In 22 out of 22 blue-ON cells we recorded only LM-OFF responses in the presence of L-AP-4 or L-AP-4, picrotoxin and strychnine.
Figure 8 The receptive field of the LM OFF input isolated with L-AP-4 shows center-surround organization. A, Voltage responses and spike histograms to full field cone isolating pulses as described in (2 Hz, 16% contrast) before (above) and after (below) (more ...)
After elimination of the S-ON response by L-AP-4 we mapped the spatial receptive field of the isolated LM-OFF input for 13 cells and found responses at low spatial frequencies were strongly attenuated, indicating the presence of surround antagonism not observed when the S-ON component was present in the control situation. The presence of a surround in the isolated LM response was confirmed using spot and annular stimuli. Thus a 300 μm diameter spot elicited the expected LM-OFF response, but a 300 μm inner diameter annulus elicited an LM-ON response (). The surround was clearly observed by centering a spot on the cell’s receptive field and increasing its inner diameter. Initially, like the control, responses increased and peaked as the size of the spot covered the center but with increasing spot size the response amplitude decreased as the spot grew and stimulated the antagonistic surround (). Similarly the control LM OFF response to an annulus of increasing inner diameter peaks and then declines (, open circles) with no change in response phase ( inset, open circles). By contrast, in the presence of L-AP-4, picrotoxin and strychnine the LM-OFF response initially peaks but then declines until the annulus inner diameter reaches ~150 μm and isolates an LM-ON surround response as indicated by an approximately 180° shift in the response phase. ( inset, solid circles). The LM-ON response then peaks and declines as the inner diameter increases further (, solid circles). Finally, in the presence of either L-AP-4 alone (, left column) or L-AP-4 and the GABAa/c and glycine receptor antagonists (, right column), low spatial frequency drifting gratings stimulated the broad antagonistic surround changing the spatial tuning from low pass to band pass. L-AP-4 thus shifted the average peak response from a low spatial frequency (control: 0.047 ± 0 cpd; mean ± sd; n = 13) to a much higher spatial frequency (0.461 ± 0.217 cpd; mean ± sd; n = 13). The resulting spatial frequency responses were best fit with a Difference-of-Gaussian receptive field model rather than the single Gaussian model used for the controls (). From the fits the surround was estimated to be on average ~4 times larger than the center (control center radius/center radius after L-AP-4 alone, 1.35 ± 0.16 (mean ± sd; n = 7) and after L-AP-4, picrotoxin and strychnine, 1.34 ± 0.42 (mean ± sd; n = 6). The average sensitivity, measured as the amplitude of the peak response, was unchanged but more variable with the blockers present (control: 37 ± 9 spikes/s vs drug: 34 ± 17 spikes/s; mean ± sd; n = 13). Thus, responses in the presence of either L-AP-4 alone or L-AP-4, picrotoxin and strychnine reveal that blue-ON cells receive S-ON input via the ON pathway and are consistent with LM input from OFF cone bipolar cells that have center-surround organization. A hypothesis for the appearance of the surround in the LM OFF pathway when the ON pathway is blocked will be considered in the Discussion.
LM-ON surround may originate from a non-GABAergic feedback pathway
The surround response was robust in the presence of either L-AP4 alone or L-AP-4 with strychnine and picrotoxin (). The lack of effect of inner retinal inhibitory block on the center-surround structure of the isolated LM-OFF response suggested that a non-GABAergic and non-glycinergic pathway generated the surround. In previous studies we showed that the cone-driven surrounds of parasol ganglion cells were also maintained after block of inner retinal inhibition (McMahon et al., 2004
) but could be significantly attenuated by increasing the buffering capacity of the retina (Davenport et al., 2008
) consistent with the interpretation that the surround was mediated by a novel non-GABAergic outer retinal feedback mechanism (Kamermans and Spekreijse, 1999
; Hirasawa and Kaneko, 2003
; Kamermans and Fahrenfort, 2004
). We therefore used a similar protocol here, by adding HEPES buffer (20 mM) to the Ames medium to measure its effect on the center-surround organization for three of the cells in which the LM-OFF response had been isolated by L-AP-4, picrotoxin and strychnine. The addition of HEPES attenuated the LM-ON response leaving only the center LM-OFF response intact (), and with wash out of HEPES the LM-ON responses were partially recovered (). With HEPES buffering, the peak response to drifting gratings also now occurred at the lowest spatial frequency again (0.047 cpd), like the original LM field, and the low pass spatial frequency responses were fit with a Gaussian model (). The results for the cell in are summarized in the 2D Gaussian receptive field profiles () showing the LM receptive field before drug application (control, far left), the center and surround LM receptive field in the presence of L-AP-4, strychnine and picrotoxin (middle) and after the addition of HEPES (attenuating the LM-ON surround; right). Similar measurements for 2 other cells were also made and showed the same pattern (see caption). Thus, much like that shown previously for the LM surround of parasol ganglion cells (Davenport et al., 2008
) the ‘unmasked’ ON-surround of the LM-OFF pathway input to the blue-ON cells may be mediated by non-GABAergic outer retinal feedback (Kamermans and Spekreijse, 1999
; Kamermans and Fahrenfort, 2004
; Cadetti and Thoreson, 2006
Figure 9 In the presence of L-AP-4 the isolated LM surround was sensitive to HEPES. A, In the presence of L-AP-4, picrotoxin and strychnine the isolated LM input responds to the offset of a 300 μm spot and to the onset of an annulus with a 300 μm (more ...)