L2 amplifies contrast information and discards luminance information

We next wanted to determine where and how these distinct photoreceptor-to-lamina neuron transformations occur. We first checked where within the neuron L2 enhances its contrast response and discards luminance information. To do so, we recorded calcium responses from different neuronal regions: its dendrites, along the axons and in the axon terminals (Figure 30A). The response amplitude measured in dendrites was very small and this data set was very difficult to obtain. Since the dendrites of L2 are located in the lamina region, recording from them was more difficult due to the cornea and medulla covering the lamina. Thus, we only observed visible responses in dendrites in less than 50% of the flies tested, compared to almost 100% when imaging from L2 and L3 axon terminals. Responses from the L2 dendrites were small, reaching maximum 10-20% dF/F (Figure 30A). The response amplitude increased along the neuron and was much more pronounced in the axon terminals where it reached 100% dF/F.

Furthermore, L2 responses to full-field flashes were more sustained in the dendrites than in the axon terminals (Figure 30A). However, during the 10 second presentation of the stimulus, L2 responses returned to the (almost) same baseline in all three regions (Figure 30B). Taken together, the amplification of a weak postsynaptic transient component and the elimination of the sustained component appear to occur after the photoreceptor-to-L2 synapse along the L2 neuron.

Figure 30. L2 response amplitude increased along the L2 neuron.

(A) Average calcium signals (dF/F) recorded from different regions of L2 neurons: dendrites (dark blue), axons (blue) and axon terminals (bright blue). N = 9 (34) for L2 dendrites, N = 7 (23) for L2 axons and N = 23 (130) for L2 axon terminals. (B) Bar plots showing the plateau calcium response of L2 neurons recorded from different regions: dendrites, axons and axon terminals. N = 8 (25) for L2 dendrites, N = 7 (19) for L2 axons and N = 16 (436) for L2 axon terminals. All traces and bars show mean ± SEM.

A

2 s ON OFF

L2 >> GCaMP6f 0.5

dF/F 0.5 dF/F 0.5 dF/F

0 Plateau response (dF/F) 0.50 0.5

0 0.5

B

RESULTS This signal transformation within the L2 neuron could either be shaped by cell-autonomous mechanisms or by circuit interaction. To test the hypothesis that the L2 responses are circuit dependent, we genetically isolated L2 from all circuit interactions by ort rescue. As described above (Figure 25), the ort mutant was thought to abolish all photoreceptor to lamina neuron communication (Gengs et al., 2002; Rister et al., 2007).

To create ort mutants, we used a defined null allele containing a 569-bp deletion of the ort gene, which leads to a premature Stop codon in trans to a deficiency deleting the entire ort locus (ort¹/ort^Df) (Figure 31A-B).

Figure 31. Df(3R)BSC809 deletes the entire ort locus and ort¹ is an ort null allele.

(A) A snapshot of GBrowse (www.flybase.org) in the region 3R: 19 357 748 .. 3R: 19 865 672. ort gene region is shown in the ‘Gene Span’ track at the top as blue arrows and is marked by an arrow. The ort gene sequence location is 3R:19 659 748 .. 3R: 19 663 672. Yellow colored Df(3R)BSC809 in the ‘Deleted segment’ track is the deficiency used in the ort experiment. Df(3R)BSC809 deletes 3R: 19 403 413 .. 3R: 198 470 010. Note that the deficiency deletes the entire ort locus. (B) A snapshot of GBrowse in the region 3R:

19 658 793 .. 19 663 990. ort gene region is shown in the ‘Gene Spa’ track at the top as blue arrows. In the

‘Insertions, Deletions’ track ort1 deletion is the deletion affecting the ort gene. It deletes 3R: 19 662 047 .. 19 662 616.

Using cell-type specific ort expression in L2, we specifically restored the photoreceptor-to-L2 connection (Figure 32A). When we presented the flies with 100% full-field flashes and recorded from L2 in an ort mutant background, L2 neurons showed a small but non-zero response to light flashes (Figure 32A). This was unexpected, because previous

ort

A

B

RESULTS reports have reported that ort mutants are motion blind (Joesch et al., 2010; Rister et al., 2007). Despite this controversial result, we are confident with our result, because we, for the first time, used a defined null allele of the ort gene (Figure 31A-B). Thus, our results argue in favor of ort-independent phototransmission, making the other histamine-gated Cl^- channel gene hisCl1 the strongest candidate (Gengs et al., 2002;

Schnaitmann et al., 2018). When we re-expressed ort in an ort mutant background (ort¹/ort^Df), L2 responses increased in amplitude (Figure 32A). The initial period of the flash response recorded from these L2 ort rescue neurons did not differ from heterozygous controls. Interestingly, responses in an L2 ort rescue fly appeared to display a more sustained plateau response than heterozygous controls (Figure 32A). To test whether this argues for a luminance-sensitive component, we next recorded calcium signals from heterozygous control and L2 ort rescue flies in response to flashes of different intensities (Figure 32B). As expected, heterozygous L2 control axon terminals showed a normal, transient response to all intensity steps that returned to one common baseline. This could already be seen in the responses of single axon terminal (Figure 32B). In contrast, L2 ort rescue neurons displayed a plateau response in addition to its normal peak response. This plateau response depended on the intensity of the stimulus (Figure 32B). Quantification in many cells and across many flies by pooling these plateau responses of L2 ort rescue neurons for all steps ending at a given luminance confirmed that the calcium signal did not return to baseline. In addition, the plateau response inversely correlated with the stimulus intensity and showed a nonlinear preference for dim stimuli (Figure 32C). This suggests that L2 ort rescue neurons are not purely contrast-sensitive, indicating that elimination of the luminance-sensitive baseline in L2 neurons is circuit dependent.

RESULTS

Figure 32. L2 discards luminance information from photoreceptors in a circuit-dependent manner.

(A-C) L2 ort rescue experiment. ort null (ort¹/ort^Df) mutants are shown in light gray. Heterozygous L2 control responses are shown in bright blue. In an L2 ort rescue (dark blue), L2 is genetically isolated from its circuit environment, and is the only lamina neuron receiving functional input from photoreceptors. (A) Average calcium signals of L2 terminals to 5 s full-field flashes. In an ort null mutant background, L2 responds minimally to light flashes. L2 responses in an L2 ort rescue are similar to heterozygous controls (light blue), with a more pronounced sustained component. N = 6 (26) for ort null mutant control flies, N = 14 (83) for heterozygous ort control and N = 9 (74) for L2 ort rescue. (B) Calcium signals recorded from single L2 axon terminals. L2 controls show transient responses to intensity changes that return to one baseline. In an L2 ort rescue, L2 responses are transient, but do not return to one common baseline, and contain a sustained plateau component. (C) Bar plots showing the plateau calcium response of genetically isolated (dark blue) and control (light blue) L2 neurons at different luminance. N = 16 (140) for heterozygous ort control and N = 13 (143) for L2 ort rescue.

To test whether the peak L2 response were still contrast-sensitive, we adapted flies to a bright background and then provided two different consecutive OFF steps, of which the second step was always at 25% contrast but varied in luminance (see Figure 17A). The two OFF steps, A and the B step, were both 10 s long. This allowed L2 control neurons to return to baseline. We first checked whether L2 peak responses are contrast-sensitive.

From average calcium signals of L2 control and L2 ort rescue neurons we observed that L2 control neurons returned to one baseline as expected, but L2 ort rescue neurons did

B

RESULTS not (Figure 33A). However, when plotting peak responses of L2 ort rescue neurons, their responses did not differ from control L2 neurons (Figure 33A-E). Responses to the different 25% contrast steps of both control and ort rescue neurons were indistinguishable from each other. Thus, genetically isolated L2 neurons still carried contrast information in their initial response. We then analyzed the plateau component of the response (Figure 33F-J). L2 control flies returned to the same baseline, regardless of the contrast and luminance (Figure 33F-H). In contrast, plateau responses of L2 ort rescue neurons to the A and the B steps were not exactly the same (Figure 33I-J).

However, ANOVA comparison revealed that plateau responses to the A steps were not significantly different from each other. The same was true for responses to the B step.

When plotting plateau responses as a function of luminance, responses to the A step did not end at the exact same amplitude as the responses to the B step at the close by luminance (Figure 33J). However, responses between the A and the B steps at the close by luminance were not significantly different. This result suggests that L2 plateau responses in L2 ort rescue neurons do carry some luminance information.

RESULTS

Figure 33. Baseline of genetically isolated L2 neurons is not purely contrast-sensitive.

(A) Schematic of the stimulus, consisting of a 30 s adapting bright period, followed by two sequential OFF steps, each 10 s long. The light intensity of the step is illustrated by the gray-scale of the trace. Average calcium signals of L2 heterozygous ort control (above) and L2 ort rescue (bellow). Arrows indicate the peak L2 responses, which were taken for the consequent analysis. (B-E) Peak calcium responses of L2 heterozygous ort control (B,C) and L2 ort rescue (D,E) plotted as a function of contrast (B,D) or luminance (C,E). A linear regression model (black dashed line) was fit to the responses to the A step (B,D) or to the A and B steps individually (C,E). The dashed lines indicate the 95% confidence interval. Responses to the A step are illustrated by circles, responses to the B step by squares. (F) Schematic of the stimulus, as in (A).

Average calcium signals of L2 heterozygous ort control (above) and L2 ort rescue (bellow). Arrows indicate the plateau L2 responses, which were taken for the consequent analysis. (G-J) Plateau calcium responses of L2 heterozygous ort control (G,H) and L2 ort rescue (I,J) plotted as a function of contrast (G,I) or luminance (H,J). A linear regression model (black dashed line) was fit to the responses to the A step (G,I) or to the A and B steps individually (H,J). The dashed lines indicate the 95% confidence interval. Responses to the A step are illustrated by circles, responses to the B step by squares. N = 8 (66) for L2 ort rescue and N = 9 (80) for L2 heterozygous control in (A-J).

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RESULTS Finally, we noted that when ort was overexpressed in L2, L2 responded to light flashes in a transient manner, even more transient than responses recorded from control L2 neurons (Figure 34).

Figure 34. ort overexpression is not responsible for the plateau component in genetically isolated L2 Calcium responses of L2 control (light blue) and ort overexpression in L2 (dark blue) to periodic 5 s full-field flashes. N = 5 (39) for L2 control flies, N = 10 (156) for L2 ort overexpression.