I_h Channels Control Feedback Regulation from Amacrine Cells to Photoreceptors

<div><p>In both vertebrates and invertebrates, photoreceptors’ output is regulated by feedback signals from interneurons that contribute to several important visual functions. Although synaptic feedback regulation of photoreceptors is known to occur in <i>Drosophila</i>, many questions about the underlying molecular mechanisms and physiological implementation remain unclear. Here, we systematically investigated these questions using a broad range of experimental methods. We isolated two <i>I_h</i> mutant fly lines that exhibit rhythmic photoreceptor depolarization without light stimulation. We discovered that I_h channels regulate glutamate release from amacrine cells by modulating calcium channel activity. Moreover, we showed that the eye-enriched kainate receptor (EKAR) is expressed in photoreceptors and receives the glutamate signal released from amacrine cells. Finally, we presented evidence that amacrine cell feedback regulation helps maintain light sensitivity in ambient light. Our findings suggest plausible molecular underpinnings and physiological effects of feedback regulation from amacrine cells to photoreceptors. These results provide new mechanistic insight into how synaptic feedback regulation can participate in network processing by modulating neural information transfer and circuit excitability.</p></div

Blocking synaptic glutamate release from ACs suppresses the rhythmic depolarization in <i>I</i>_h mutant photoreceptors.

<p>(<b>A</b>) Ultrastructure of lamina cross-sections in wild-type and <i>I</i>_<i>h</i> mutant flies. The left panel shows the organization of the columnar neurons with synaptic connections in the lamina. Photoreceptor cells are shown in gray, L1–L2 neurons in black, and ACs in red. These neurons are present in all lamina columns, and single example profiles are shown arrayed across the lamina. The middle and the right panels show EM images of lamina cross-sections in wild-type and <i>I</i>_<i>h</i> mutant flies, respectively. Photoreceptor axons are colored in yellow and AC processes in blue. (<b>B</b>) Intracellular recording traces of <i>I</i>_<i>h</i> mutant flies with expression of TeTxLC using <i>L1L2-GAL4</i> and <i>Lai-GAL4</i> drivers. The fractions of photoreceptors that exhibit rhythmic depolarization are presented in the middle panel, and the time (t_3/4) required for a 3/4 recovery from the responses upon stimulation cessation is shown in the right panel. The numbers of recorded flies are listed. (<b>C</b>) Inactivation of ACs via ectopic expression of dORK^ΔC suppresses rhythmical depolarization in <i>I</i>_<i>h</i> mutant flies. The fractions of flies that exhibit ERG oscillation phenotype and the numbers of recorded flies are presented in the right panel. An ERG trace of flies expressing dORK^ΔC in wild-type ACs is also presented. (<b>D</b>) Intracellular recording traces of <i>I</i>_<i>h</i> mutant flies expressing <i>UAS-vGluT-RNAi</i> using different drivers. The fractions of photoreceptors that exhibit rhythmical depolarization are presented in the middle panel, and the time (t_3/4) required for a 3/4 recovery from the responses upon stimulation cessation are shown in the right panel. The number of recorded flies for each genotype is listed.</p

Depletion of I_h channels in ACs results in rhythmic depolarization in photoreceptors.

<p>(<b>A</b>) I_h channel expression levels in flies with I_h channel depletion using <i>UAS-I</i>_<i>h</i>-<i>RNAi</i> driven by anatomically restricted GAL4 drivers. A single copy of the GAL4 driver was used for each GAL4 line. Each lane was loaded with two fly heads. The I_h channel bands are indicated with arrows. (<b>B</b>) ERG traces of flies with I_h channels depletion using <i>UAS-I</i>_<i>h</i>-<i>RNAi</i> driven by anatomically restricted GAL4 drivers. A single copy of the GAL4 driver was used for each GAL4 line. (<b>C</b>) The fraction of flies that exhibit ERG oscillation phenotype in each genotype. The numbers of recorded flies for each genotype are listed. (<b>D</b>) Expression of I_h channels in <i>UAS-mCD8-GFP</i>,<i>UAS-I</i>_<i>h</i>-<i>RNAi/Lai-Gal4</i> (bottom) and control (top) flies. Dissected whole brains were stained with anti-I_h (red) and anti-GFP (green) antibodies. Note that I_h channel distribution in <i>UAS-mCD8-GFP</i>,<i>UAS-I</i>_<i>h</i>-<i>RNAi/Lai-Gal4</i> flies is comparable to control flies except in ACs.</p

Identification of glutamate receptor that mediates retrograde glutamate signaling from ACs to photoreceptors.

<p>(<b>A</b>) ERG traces of flies with iGluR depletion in photoreceptors. Photoreceptor-specific Rh1-GAL4 was used for iGluR screening. (<b>B</b>) Fractions of flies exhibit ERG oscillation phenotype and the numbers of recorded flies are presented.</p

RNAi lines used for iGluR screening.

<p>RNAi lines used for iGluR screening.</p

Expression of I_h channels in ACs restores a normal ERG response.

<p>(<b>A</b>) Expression of I_h channels in ACs suppresses ERG baseline oscillation. I_h channels were expressed using anatomically restricted GAL4 drivers. Flies possessed one copy of the indicated drivers. (<b>B</b>) The fraction of flies that exhibit the ERG oscillation phenotype in each genotype. The number of recorded flies for each genotype is listed. (<b>C</b>) Expression of I_h channels in <i>I</i>_<i>h</i> mutant (top) and <i>I</i>_<i>h</i>;<i>Lai-GAL4/UAS—I</i>_<i>h</i> (bottom) flies. Dissected whole brains were stained with anti-I_h (green) and anti-24B10 (red) antibodies. L, lamina; M, medulla. (<b>D</b>) Intracellular recordings of photoreceptors show that I_h channels expression in ACs suppresses rhythmical depolarization without light stimulation. The fractions of photoreceptors that exhibit rhythmic depolarization are presented in the right panel, and the numbers of recorded photoreceptors for each genotype are listed.</p

Expression patterns of endogenous I_h channels.

<p>(<b>A</b>) Localization of endogenous I_h channels in the adult fly head. Dissected whole heads were double stained with anti-I_h (green) and 24B10 (red, for photoreceptor membrane) antibodies. The images show a longitudinal view of the retina (R), lamina (L), and medulla (M). (<b>B</b>) Distribution of I_h channels in the lamina region. Images show a longitudinal view. (<b>C</b>) I_h channels expressed in L1 neurons. L1 neurons were labeled with mCD8-GFP under the control of the <i>L1-GAL4</i> driver. Two L1 somata are indicated by arrows. (<b>D</b>). I_h channels were highly expressed in L2 neurons. L2 neurons were labeled with mCD8-GFP under the control of the <i>L2-GAL4</i> driver. The upper panel shows a longitudinal view of the lamina, and the lower panel shows a cross view of the lamina. L2 somas are indicated by arrows. (<b>E</b>) I_h channels were expressed in ACs. ACs (arrows) are labeled with mCD8-GFP under the control of the <i>Lai-GAL4</i> driver. The upper panel shows a longitudinal view of the retina (R), lamina (L) and medulla (M), and the middle and lower panels show a longitudinal and cross-sectional view of AC processes.</p

<i>I</i>_h mutant lines exhibit ERG baseline oscillation.

<p>(<b>A</b>) Annotated transcriptions of the <i>I</i>_<i>h</i> gene. Two <i>piggyBac</i> insertion sites are marked with triangles. The RNAi recognized site and coding region used for antibody generation are labeled at the top. (<b>B</b>) <i>I</i>_<i>h</i> mutant lines exhibit ERG baseline oscillation. For ERG traces throughout all figures, event markers represent 5-s orange light pulses, and scale bars are 5 mV. (<b>C</b>) Fraction of flies that exhibit the ERG oscillation phenotype in each genotype. The numbers of recorded flies for each genotype are listed. (<b>D</b>) RT-PCR shows <i>I</i>_<i>h</i> mRNAs are transcripted in wild-type flies but are absent in <i>I</i>_<i>h</i> mutant flies. Primer pair CACGCGACCAATCTCATCC/ TCATGGAGTGTTACCCTCG, which can amplify all transcriptional variants, was used in RT-PCR analysis. The tubulin gene was used as a loading control. (<b>E</b>) Western blotting revealed four major I_h channel variants (indicated by arrows) expressed in wild-type flies but absent in <i>I</i>_<i>h</i> mutant flies. Note that the low-intensity bands presented in <i>I</i>_<i>h</i> mutant flies are nonspecific.</p

<i>I</i>_h mutant photoreceptors undergo rhythmic depolarization without light stimulation.

<p>(<b>A</b>) Intracellular recording traces of wild-type and <i>I</i>_<i>h</i> mutant photoreceptors. For intracellular recording traces, event markers represent 5-s orange light pulses, and scale bars are 5 mV. Measurements of the amplitude (Am), frequency (Fr), rise time (R_t), and decay time (D_t) of rhythmic depolarization are provided at the top. (<b>B</b>) The fraction of photoreceptors (R-cells) that exhibit oscillation phenotype. The numbers of photoreceptors recorded for each genotype are listed. (<b>C</b>) Measurement of the amplitude of light-induced depolarization (middle) and the time (t_3/4) required for a 3/4 recovery from the responses upon stimulation cessation (right). <i>n</i> = 10.</p