Cholinergic feedback to bipolar cells contributes to motion detection in the mouse retina

Abstract

Retinal bipolar cells are second-order neurons that transmit basic features of the visual scene to postsynaptic partners. However, their contribution to motion detection has not been fully appreciated. Here, we demonstrate that cholinergic feedback from starburst amacrine cells (SACs) to certain presynaptic bipolar cells via alpha-7 nicotinic acetylcholine receptors (α7-nAChRs) promotes direction-selective signaling. Patch clamp recordings reveal that distinct bipolar cell types making synapses at proximal SAC dendrites also express α7-nAChRs, producing directionally skewed excitatory inputs. Asymmetric SAC excitation contributes to motion detection in On-Off direction-selective ganglion cells (On-Off DSGCs), predicted by computational modeling of SAC dendrites and supported by patch clamp recordings from On-Off DSGCs when bipolar cell α7-nAChRs is eliminated pharmacologically or by conditional knockout. Altogether, these results show that cholinergic feedback to bipolar cells enhances direction-selective signaling in postsynaptic SACs and DSGCs, illustrating how bipolar cells provide a scaffold for postsynaptic microcircuits to cooperatively enhance retinal motion detection.

Keywords: AAV; Cre-DOG; acetylcholine; bipolar cell; direction-selective ganglion cell; intravitreal injection; patch clamp recording; starburst amacrine cell; wholemount retinal preparation; α7-nicotinic receptor.

Figure 1.. ChR2-evoked EPSCs in some bipolar cells were MLA sensitive

(A) In the presence of AMPA, kainite, and mGluR6 glutamate receptor blockers, ChR2-evoked EPSCs were recorded from bipolar cells. Averaged traces are shown for Off (top) and On (bottom) bipolar cells, in which EPSCs were blocked by application of MLA (100 nM, magenta). (B) In other bipolar cells, we first applied HEX (100 μM, green) that did not block ChR2-EPSCs, which were subsequently blocked by application of 100 nM of MLA. (C) A summary graph of ChR2-evoked EPSC amplitude in MLA-sensitive bipolar cells, which were insensitive to HEX but were blocked by MLA (p = 0.0001, mixed-model ANOVA). ChR2-evoked EPSCs returned after washout in some bipolar cells (gray). Data are represented as means ± SEM. See also Table S1.

Figure 2.. ChR2-evoked EPSCs in some bipolar cells were HEX sensitive

(A) In On and Off bipolar cells, ChR2-evoked EPSCs were blocked by application of HEX. (B) In other recordings, ChR2-EPSCs were unaffected by MLA but were HEX sensitive. (C) A summary graph of ChR2-evoked EPSC amplitude in HEX-sensitive bipolar cells. ChR2-evoked EPSCs were insensitive to MLA but were blocked by HEX (p = 0.0091, mixed-model ANOVA). ChR2-evoked EPSCs returned after washout in some bipolar cells (gray). Data are represented as means ± SEM. See also Table S1.

Figure 3.. A summary of ChR2-evoked EPSCs in bipolar cells

(A) 13 types of mouse bipolar cells project to different IPL depths shown in the retinal slice preparation. The dashed blue lines represent the Off-ChAT (top) or On-ChAT (bottom) bands. Types-1/2 and −3 Off bipolar cells stratify just above or below the Off-ChAT band, respectively. Types 5 or 7 On bipolar cells stratify just above or below the ON-ChAT band, respectively. (B) A representative HEX-sensitive type-5 bipolar cell filled with sulphorhod-amine-B (red) was identified by its axon terminal that stratified at the level of the On-ChAT band (YFP, yellow). (C) A summary graph showing the fraction of each bipolar cell type that showed no ChR2-evoked EPSCs (black), EPSCs sensitive to MLA (magenta), or EPSCs sensitive to HEX (green). See also Figure S2.

Figure 4.. A 3-SAC model reveals that cholinergic feedback to bipolar cells enhances the SAC centrifugal direction selectivity

(A) A 3-SAC model based on the digitized morphology of a tracer-labeled SAC. A simulated moving bar (20 μm wide by 400 μm tall) moved left to right, then right to left at a speed of 600 μm/s. (B) The simulated moving bar depolarized presynaptic bipolar cells (colored squares), which evoked EPSPs and calcium responses in the central SAC. Direction selectivity of motion responses was measured in the right-facing dendrites (point 1). Different dendritic compartments are shown by each color. (C) Without nicotinic feedback, a bar moving left to right evoked slightly larger EPSPs and calcium responses than for right to left motion, exhibiting weak direction selectivity. (D) With nicotinic feedback, the EPSPs and calcium response for motion from left to right were significantly enhanced, whereas motion from right to left evoked small EPSPs from signal backpropagation. See also Table S2.

Figure 5.. MLA, but not HEX, reduced the direction selectivity and charge transfer of IPSCs in On-Off DSGCs

(A) IPSCs were recorded in response to moving stimuli of 100% contrast. Hexamethonium (HEX, 100 μM) was applied to block non-α7 nicotinic receptors. In the presence of HEX, IPSCs were still direction selective. (B) A radar plot to show direction selectivity in the presence (red) and absence (black) of HEX. (C) DSI did not change by the HEX application, suggesting that HEX-sensitive receptors in bipolar cells did not have a role in direction selectivity in DSGCs. (D) IPSCs were recorded from an On-Off DSGC. An α7-nAChR antagonist, MLA (100 nM), was applied in the bath, which reduced the IPSCs. The effect of MLA was washed out. (E) A radar plot showing direction selectivity before (black) and after (red) the MLA application. MLA reduced the direction selectivity, shown by the vector length difference. (F) DSI of IPSCs (100% contrast) was significantly reduced, which was washed out (p < 0.05, repeated-measures ANOVA). (G) Charge transfer for the null-IPSCs was also significantly reduced by MLA application, which was washed out in some cells (p < 0.05, repeated-measures ANOVA).

Figure 6.. Direction selectivity in On-Off DSGCs was reduced after α7-nAChRs were eliminated from type-7 bipolar cells

(A) In wild-type mice (WT), On IPSCs were larger in response to null than preferred directional stimuli. IPSCs from individual DSGCs are shown in gray (n = 6 cells), and the average of these traces shown in red. In mutant-type (MT) mice both null and preferred stimuli-evoked IPSCs were reduced (n = 6 cells). The average of traces is shown in blue. (B) Summary graphs show the charge transfer of IPSCs in WT and MT. Both null- and preferred-evoked IPSCs were significantly reduced in mutant mice compared with WT. Data are represented as mean ± SEM. (C) Spike recording was conducted from an On-Off DSGC in wild-type retina. Direction selectivity was clearly present in response to 8%- and 80%-contrast moving stimuli. (D) The same recording was conducted from an On-Off DSGC in the type-7 α7-knockout (KO) mutant-type (MT) mouse. Spike activity was increased, and direction selectivity was reduced. (E) Summary graphs show the vector sum and DSI in wild-type mice (WT). Both parameters were high for all the stimuli, from 8 to 80% contrast, and ON and OFF responses. Data are represented as mean ± SEM. (F) Both the vector sum and DSIs in mutant mice were reduced. Compared with the WT DSGCs, both parameters for On responses recorded in response to 8%-, 30%-, and 80%-contrast stimuli were significantly reduced (*). However, Off responses did not exhibit significant differences between wild-type and mutant mouse DSGCs. See also Figures S3, S4, and S5. Data are represented as mean ± SEM.

Figure 7.. α7-nAChRs in bipolar cells contribute to SAC and DSGC direction selectivity

For an object moving from left to right, the neighbor SAC simultaneously provides lateral inhibition to the primary SAC as well as cholinergic excitation to α7-nAChRs in type-7 bipolar cells. This augments the proximal glutamate inputs to the primary SAC, enhancing the SAC distal dendrite’s response to centrifugal motion (from left to right here) and GABAergic output for the DSGC-null direction. The lateral inhibition from the neighbor SAC, as well as SAC dendrite compartmentalization, prevents backpropagation of the enhanced glutamate inputs for centripetal stimulation of the left-facing SAC dendrite.