Nonlinearities measured with spots

The spots used to measure the receptive field center-surround structure, were shown once with black contrast (-100% contrast) and once with white contrast (+100% contrast). This stimulus design allowed us to investigate for each spot diameter size, the response to the

preferred (e.g. black) and non-preferred (e.g. white) contrast. A linear bipolar cell is expected to show a similar amount of hyper- and depolarization to the preferred and non-preferred spot. A nonlinear bipolar cell is expected to show a different amount of hyper- and depolarization to the preferred and non-preferred spot. We indeed found bipolar cells that showed such an equal amount of depolarization and hyperpolarization (see example cell in Fig.5.1A). However, almost 90% of our recorded bipolar cells showed unequal amounts of hyper- and depolarization i.e.

nonlinear responses. Thereby, they varied in their imbalance between hyper- and depolarization.

For example, the bipolar cell in Figure 5.1B showed a depolarization to the preferred stimulus of

~5mV, while the hyperpolarization was ~2mV or in other words ~40% of the depolarization.

The response of the bipolar cell in Figure 5.1C was even more nonlinear; the cell strongly depolarized to the preferred stimulus (~20mV) but did not show hyperpolarization to the non-preferred stimulus (rectification). We quantified the degree of hyper- and depolarization

imbalance by computing a hyper-depolarization index (HDi, see methods 3.6.1). Values close to 1 stand for a strong imbalance between the hyper- and depolarization, while values close to 0 stand for almost equal hyper- and depolarization (see the corresponding values for the example cells in Fig.5.1A-C). The population distribution of the hyper-depolarization index is shown in Figure 5.1D and ranged between 0-1 (n=23). Almost 70% of our cells showed values higher than 0.4, meaning that they showed much smaller amount of hyperpolarization to the non-preferred stimulus compared to the depolarization to the preferred contrast. Note, for almost all cells the observed imbalance came from a higher depolarization (depolarization > hyperpolarization). For two cells, the imbalance came from a higher hyperpolarization (hyperpolarization >

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depolarization). One of the two cells was the ON cell described in in Figure 4.2, here the

hyperpolarization to the non-preferred stimulus was much stronger than the depolarization to the preferred stimulus.

Figure 5.1. Output nonlinearity measured with spots. Comparison of the amount of depolarization (dep., red) and hyperpolarization (hyp., blue) to a black and white spot of 1 mm diameter. A. Example cell where the amount of depolarization and hyperpolarization to the black and white spot is very similar. B. Example cell that hyperpolarize, yet, the amount of depolarization is larger. C. Example cell with strong depolarization and zero hyperpolarization. D. Population distribution of the hyper-depolarization index (HDi, range 0-1, n=23), small values stand for an equal amount of hyper- and depolarization (like example cell in A), large values stand for an unequal amount (example cells B and C).

In Figure 5.1, we studied the imbalance between the hyper- and depolarization with the largest spot stimulus of 1 mm. Thus, we activated the center and surround together. We have seen in subchapter 4.4 that the activation of the surround can change the temporal properties.

Thus, we were wondering, whether the observed imbalance is influenced by the stimulation of the surround and investigated the imbalance during a stimulation limited to the center.

Figure 5.2A shows for an example cell the response traces to the large and small spot for both contrasts. For both spot sizes, the cell showed a similar ratio between the hyper- and depolarization. Over all recorded bipolar cells, we observed a clear correlation between the

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hyper-depolarization index (HDi) during center and large spot stimulation (r=0.87, p<0.001, n=17, Fig.5.2B). Further, we compared the imbalance in the hyper- and depolarization observed under a spot stimulus, with the imbalance during the full-field light steps. The ratio between the hyper- and depolarization stayed similar during a center and full-field stimulation (p= 0.33, n=17, Fig.5.2C). Therefore, we concluded that the degree of imbalance between hyper- and

depolarization, is similar under center and full-field stimulation and not changed by the surround.

Figure 5.2. Output nonlinearity under center and surround stimulation. A. Example cell where the amount of depolarization and hyperpolarization was similar under a large spot (1000 µm) and center spot (160 µm). B.

Population comparison of the hyper-depolarization index (HDi) under center and large spot stimulation, a clear correlation was observed (r=0.87, p<0.001, n=17). C. Population comparison of the HDi under a center spot and full-field light steps, the indices were similar, no significant difference was observed (p=

0.33, n=17).

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97 Excurse: Gray or no gray?

As for the receptive field center-surround characterization, 9 out of the 23 cells in Figure 5.1D were recorded with spots that switched between black and white. For 7 cells we recorded both stimuli and again tested, whether the different stimulus designs had an effect on the

imbalance between the hyper- and depolarization. We found that the hyper-depolarization index (HDi) was strongly correlated between the two stimulus designs (Fig.5.3A, r= 0.83, p=0.02, n=7).

As for the receptive field center-surround characterization, we excluded the possibility that the intermediate gray screen can change the imbalance between the hyper-depolarization. Note however, we found some cells, like the example in Figure 5.3B-C, where the hyper-depolarization ratio was more balanced during the black-white switch (Fig.5.3C, +4 mV depolarization, -4 mV hyperpolarization) than during the stimulus with gray intermediate screen (Fig. 5.3B,+5 mV depolarization, -2 mV hyperpolarization).

Figure 5.3. Output nonlinearity with different spot designs. A. Positive correlation among the hyper- depolarization indices (HDi) for spots with gray frames and spots without gray frames (r=0.83, p=0.02, n=7). B. Example cell under a center spot with gray preframes, the cell showed an unequal amount of hyper- and depolarization. C. Same cell as in B but under a center spot with a black-white switch (no gray-frames), here the cell showed a more equal amount of hyper- and depolarization.