Cone bipolar cell synapses generate transient versus sustained signals in parallel ON pathways of the mouse retina
- Department of Neurobiology and Biophysics, University of Washington, United States
- Department of Biology, University of Victoria, Canada
- Department of Neuroscience, University of Minnesota, United States
- Department of Neuroscience, Brown University, United States
Figures
Figure 1 with 1 supplement
Distinct temporal spiking response properties of ON-transient (ON-T) versus ON-sustained (ON-S) retinal ganglion cells (RGCs).
(A) Maximum intensity projection z-stack fluorescence images of ON-T (left, cyan) and ON-S (right, magenta) RGCs dialyzed with Alexa Fluor 594 during whole-cell patch-clamp recording. Scale bar 50 µm. (B) Characteristic spiking responses to a light increment for example ON-T and ON-S RGCs. (C) Example spiking responses to repeated trials of spatially uniform (520 µm spot diameter) Gaussian noise stimulus (top black trace) presented at a mean luminance of ~2000 P*/S-cone/s (~3300 R*/rod/s and ~1650 P*/M-cone/s). ON-T (cyan) and ON-S (magenta) RGCs were recorded sequentially in the same piece of retina. Average spike responses (bottom) calculated from 20 repetitions of the noise stimulus. (D) ON-T and ON-S spike-triggered averages (STAs) derived from responses to non-repeated noise stimuli. Colored lines and shaded areas show mean ± SEM STA waveforms from ON-T (n = 15) and ON-S (n = 13) RGCs. The STA time axis is reversed to facilitate comparison with linear filters in Figures 2 and 4. The mean line obscures the SEM shading for much of the filter in both cell types. (E) Quantification of STA kinetics. Open circles show measurements from individual cells and filled circles with error bars show mean ± SEM values. Significance values from Wilcoxon rank sum tests.
Figure 1—figure supplement 1
Noise-evoked responses are biased toward large amplitude events in ON-T compared to ON-S retinal ganglion cells (RGCs).
(A) Spike event peak spike rate cumulative distributions for ON-T (cyan; n = 6 cells; 131 spike events) and ON-S RGC (magenta; n = 7 cells; 300 spike events). Firing rates from average responses to a repeated noise stimulus (see Figure 1B, C) were normalized to the maximal firing rate in each cell before combining events across cells. (B) EPSC amplitude cumulative distributions for ON-T (n = 13 cells; 275 EPSCs) and ON-S (n = 10 cells; 365 EPSCs) RGCs. Same stimulus and normalization procedure as in (A).
Figure 2 with 2 supplements
Distinct excitatory synaptic input to ON-T and ON-S retinal ganglion cells (RGCs).
(A) Excitatory synaptic currents measured in example ON-T (cyan) and ON-S (pink) RGCs in response to the same Gaussian noise stimulus used in Figure 1. Traces show average currents from 10 repetitions of the same stimulus and are baseline subtracted to facilitate comparison. (B) Linear filters computed from responses to non-repeated noise stimulus presentation. Lines and shaded areas show mean ± SEM of responses from ON-T (n = 13) and ON-S (n = 15) RGCs. The mean line obscures the SEM shading for much of the filter in both cell types. (C) Quantification of filter kinetics. (D) Excitatory currents measured from example ON-T and ON-S RGCs in response to a 0.5-s step increase in light intensity (50% contrast; mean response to five repetitions of stimulus). (E) Quantification of step response kinetics (n = 19 ON-T RGCs; n = 13 ON-S RGCs). In (C) and (E), open circles show measurements from individual cells and filled circles with error bars show mean ± SEM values. Significance values from Wilcoxon rank sum tests.
Figure 2—figure supplement 1
Linear–nonlinear models of excitatory synaptic input to ON-T and ON-S retinal ganglion cells (RGCs).
(A) Linear filters computed from responses to non-repeated noise stimulus presentation. Lines show filters from individual ON-T (cyan; n = 13) and ON-S (magenta; n = 15) RGCs. Filters were normalized to the peak amplitude of the primary inward component to facilitate visual comparison of temporal responses. Same cells as shown in average filters in Figure 2B. (B) Nonlinearities derived by plotting the predicted response (convolution of linear filter and stimulus) against the measured excitatory currents for each RGC shown in (A). Linear predictions were calculated using non-normalized filters.
Figure 2—figure supplement 2
Inhibitory synaptic input to ON-T and ON-S retinal ganglion cells (RGCs).
(A) Inhibitory synaptic currents measured at Vhold = +10 mV (reversal potential for excitation) in example ON-T (cyan) and ON-S (pink) RGCs in response to same Gaussian noise stimulus as in s 1–2. Traces show average currents from 10 repetitions of the same stimulus and are baseline subtracted to facilitate comparison. (B) Linear filters computed from responses to non-repeated noise stimulus presentation. Lines and shaded areas show mean ± SEM of responses from ON-T (n = 8) and ON-S (n = 8) RGCs. (C) Quantification of filter kinetics in different RGCs. Zero-cross times were not significantly different (p = 0.57). Significance values from the Wilcoxon rank sum test. (D) Peak amplitudes of excitatory and inhibitory conductances (Gexc and Ginh, respectively) calculated from average current responses to repeated noise stimuli such as shown in Figure 2A and Figure 2—figure supplement 2A. Lines connect peak Gexc and Ginh values measured from the same RGCs. Circles with error bars show mean ± SEM across ON-T RGCs (n = 6) and ON-S RGCs (n = 7). (E) Summary of peak excitatory/inhibitory conductance ratios. Open circles show measurements from individual cells and filled circles with error bars show mean ± SEM values. No significant difference between ON-T and ON-S RGCs; p = 0.10, Wilcoxon rank sum test.
Figure 3 with 4 supplements
Identification of bipolar cell subtypes presynaptic to ON-T retinal ganglion cells (RGCs).
(A) Maximum-projection z-stacks of two dye-filled ON-T RGCs (green) in whole-mount retina from Gjd2-EGFP (red) transgenic mice. (B) Traced skeletons of dendritic arbors of the two ON-T RGCs from EM volume. (C) An EM micrograph showing a ribbon synapse (arrowhead) on a BC axonal terminal onto the dendrite of a ON-T RGC. (D) An example skeleton for each presynaptic BC type (types 5i, 5t, 5o, X, 6, and 7) of ON-T RGCs. Top: whole-mount view, bottom: side view. (E) Bar plot showing the proportion of input synapses to ON-T RGCs from each BC type. Mean ± SEM (n = 3 RGCs, 2 from this study and 1 from Ding et al., 2016).
Figure 3—figure supplement 1
Characterization of Gjd2-EGFP+ bipolar cells.
(A) Vertical section of the Gjd2-EGFP mouse line showing EGFP positive bipolar cells axons (green) stratifying in the ON sublamina of the inner plexiform layer (IPL), co-labeled with cholinergic amacrine cell (magenta, ChAT antibody). Transmitted light image of the retina in blue. (B) Vertical section of the Gjd2-EGFP mouse line showing EGFP positive bipolar cells (green) colocalized with the T5 marker CaBP5 (magenta). (C) En face image of a whole-mount retina from the Gjd2-EGFP mouse line showing EGFP positive bipolar cell bodies (green) co-labeled with the T5 bipolar cell marker CaBP5 (magenta). (D) Same field as in (C) with cell bodies of EGFP positive bipolar cells outlined (green) by application of a digital edge outlining filter in ImageJ. (E) Position of cell body centroids for EGFP positive bipolar cells in a larger field of view (20X objective) outlined by green circles. (F) Density recovery profile analysis showing the density profile as a function of distance from the reference neuron for EGFP positive bipolar cells (green histograms) and for an equal amount of neurons randomly positioned in the same space (gray histograms) showing the presence of a characteristic exclusion distance for EGFP positive bipolar cells, hallmark of a retinal mosaic.
Figure 3—figure supplement 2
ON-T and ON-S retinal ganglion cell (RGC) dendrites ramify in distinct inner plexiform layer (IPL) sublaminae.
(A) Side projections from 2P z-stack images of dye-filled RGCs (ON-T: top, cyan; ON-S: bottom, magenta) and EGFP signal (yellow) in retinas from Gjd2-EGFP mice. RGCs are the same cells as shown in en face view in Figure 1A. (B) Quantification of fluorescence signals from dye-filled RGCs in Gjd2-EGFP mouse retina as in (A). Colored lines and shaded areas show mean ± SEM fluorescence intensity with respect to distance from peak EGFP (yellow) fluorescence intensity (n = 6 ON-T, cyan and n = 4 ON-S, magenta, RGCs).
Figure 3—figure supplement 3
Gjd2-EGFP+ type 5 bipolar cells provide ~40% of total synapses onto ON-T retinal ganglion cells (RGCs).
(A) An example ON-T RGC biolistically labeled by Cerulean Fluorescent Protein (blue) and PSD95-mCherry (green) in retina from a Gjd2-EGFP (red) transgenic mouse. Top: en face view, bottom: side views. (B) Top: Dot map of PSD95-mCherry apposed (magenta) or not apposed (green) to GFP+ BC axon terminals. Bottom: Magnified view of the raw images and the dot map for a segment of dendritic arbor in the white box. (C) Quantification of total excitatory synapse density across the dendritic arbor of ON-T RGCs. (D) Quantification of excitatory synapses in ON-T dendrites that are adjacent to GFP+ axon terminals.
Figure 3—figure supplement 4
Classification of three subtypes of type 5 bipolar cells.
(A) Whole-mount views of the axon terminals of types 5i, 5t, and 5o bipolar cells in the serial block face scanning electron microscopy (SBFSEM) volume in Figure 3. The tiling of the space validates the categorization of the three subtypes of type 5 bipolar cells. (B) Identification of Gjd2-EGFP+ bipolar cells as type 5i bipolar cells, supported by correlated light (left) and electron (right) microscopy images. The reconstructed skeleton of the type 5i bipolar cell in (B) is delineated by dashed lines in (A).
Figure 4 with 1 supplement
Bipolar cell subtypes presynaptic to ON-T and ON-S retinal ganglion cells RGCs have indistinguishable temporal response properties.
(A) Maximum-projection z-stacks of fluorescent dye-filled bipolar cells targeted for electrophysiological recording in retinal slices from Gjd2-EGFP (left), Grm6-tdTomato (middle), and Gus8.4-EGFP (right) transgenic mice. (B) Responses to spatially uniform Gaussian noise stimulation (same as in Figures 1 and 2) in example type 5i (cyan), type 6 (magenta), and type 7 (green) bipolar cells. Voltage traces are average responses to 10 repetitions of the noise stimulus. Scale bar = 5 mV. (C) Linear filters computed from responses to non-repeated noise stimulus presentation. Lines and shaded areas show mean ± SEM of responses (type 5i, n = 11; type 6, n = 7; type 7, n = 6). (D) Quantification of response kinetics from linear filters. No significant differences across types (p = 0.19 zero-cross times; p = 0.89 biphasic index; Kruskal–Wallis test). (E) Responses to step light increment. Lines and shaded areas show peak-normalized mean ± SEM responses across type 5i bipolar cells (n = 8) and type 6 bipolar cells (n = 5). (F) Quantification of step response kinetics. Time to peak and steady-state response amplitude were not different between type 5i and 6 bipolar cells (p = 0.72 and p = 0.35, respectively; Wilcoxon rank sum test).
Figure 4—figure supplement 1
Linear–nonlinear models of bipolar cell voltage responses.
(A) Individual linear filters for all type 5i (cyan; n = 11), type 6 (magenta; n = 7), and type 7 (green; n = 6) bipolar cells computed from responses to non-repeated noise stimulus presentation. Filters were normalized to the peak amplitude of the primary inward component to facilitate visual comparison of temporal responses. Same cells as shown in average filters in Figure 4B. (B) Nonlinearities derived by plotting the predicted response (convolution of linear filter and stimulus) against the measured voltage response for each bipolar cell shown in (A). Linear predictions were calculated using non-normalized filters.
Figure 5 with 1 supplement
Presynaptic inhibition is not required for kinetic differences between bipolar cell inputs to ON-T versus ON-S retinal ganglion cells (RGCs).
(A) Example EM micrograph showing a ribbon dyad (red arrowhead) between a BC and an ON-T RGC and an amacrine cell, with an adjacent feedback inhibitory synapse (yellow asterisk). (B) Bar plots showing the percentage of RGC/AC ribbon dyads with feedback inhibition. Mean ± S.D. (n = 2 ON-S RGCs 85 dyads analyzed, 2 ON-T RGCs with 83 dyads analyzed). (C) Excitatory currents measured in response to light step increment in control conditions (cyan) and in the presence of GABA receptor antagonists (10 µM SR95531 and 50 µM TPMPA; dashed gray) in an example ON-T RGC. Traces are mean response from six repeats of stimulus presentation. (D) Summary of steady-state amplitudes measured from step responses in control or with GABA receptor antagonists. Step-evoked currents were significantly more transient when GABAA/C receptors were blocked (p = 0.031; Wilcoxon signed rank test; n = 6 RGCs). (E) Control excitatory currents (cyan) or with addition of strychnine (1 µM; dashed gray) to bath in an example ON-T RGC. (F) Summary of strychnine experiments. No significant difference between conditions (p = 0.44; Wilcoxon signed rank test; n = 5 RGCs). In (D) and (F), open circles with lines show measurements from individual ON-T RGCs. Filled circles with error bars show mean ± SEM.
Figure 5—figure supplement 1
Presynaptic inhibition does not affect response kinetics but does affect spatial integration for excitatory inputs to ON-T retinal ganglion cells (RGCs).
(A) Step-evoked (100% contrast, 520-µm spot diameter, 500-ms duration) excitatory synaptic currents in example ON-T (cyan) and ON-S RGCs measured in artificial cerebrospinal fluid (ACSF). Traces show mean responses to 10 trials and are baseline subtracted to facilitate comparison. (B) Summary of light step-evoked steady-state response amplitude for different stimulus sizes measured in ACSF. Note that excitatory inputs in ON-T are more transient than those in ON-S RGCs across all stimulus sizes, but responses in both RGC types are more sustained than when measurements were performed using Ames’ solution (compare to Figure 2D, E). (C) Summary of total response, measured as the integrated excitatory current response during the light step, versus stimulus spot size. Responses were normalized to the maximal response in each cell prior to averaging. Excitation in ON-T RGCs is tuned to smaller spot sizes than in ON-S RGCs, and this tuning relies in part on GABAergic inhibition. Symbols with error bars in (B) and (C) show mean ± SEM (ON-T control n = 12; ON-S control n = 6; ON-T GABA-R block n = 5; ON-T Gly-R block n = 5).
Figure 6
Distinct glutamate input kinetics at ON-S versus ON-T retinal ganglion cell (RGC) dendrites.
(A) ON-S RGC dendrites expressing iGluSnFR in a Kcng4-Cre mouse were filled with sulforhodamine 101 (left) after measuring their spiking response to a 200-µm spot extracellularly (right). Yellow bands indicate stimulus duration. (B) iGluSnFR expressing ON-S and ON-T dendrites at different depths of the inner-plexiform layer (Left) were imaged using two-photon microscopy. Example iGluSnFR signals extracted from small regions of interest (ROIs 1–8) from ON-S and ON-T RGC dendrites illustrate the kinetic differences in their respective inputs. Black, mean responses; gray, individual trials. (C) Distribution of steady-state response amplitudes (mean fluorescence during last 1 s of stimulus) of the individual (gray) and average (black) ROIs from ON-S and ON-T dendrites evoked by a 200-µm static spot (n = 7 FOVs, 6 retinas for ON-S and 5 FOVs, 5 retinas for ON-T RGC dendrites, *p < 0.001; t-test between ROIs).
Figure 7
The distinction between sustained and transient responses remains when amacrine cell activity is blocked.
(A) iGluSnFR measurements of glutamate release in control conditions and in the presence of TTX (which blocks spike-dependent amacrine cell output); or in the presence of NBQX, which blocks AMPA receptor-mediated input to all amacrine cells (as well as horizontal cells). (B) Ratio of steady state to transient response across ROIs before (top) and during (bottom) application of TTX/NBQX.
Figure 8
Stronger flash-evoked suppression of excitatory currents in ON-T versus ON-S retinal ganglion cells (RGCs).
(A) Paired flash-evoked excitatory currents measured at different inter-flash intervals (50, 100, 200, and 400 ms) in an example ON-T (top; cyan) and an ON-S (middle, magenta) RGC. Bottom black traces show voltage responses to the same stimulus measured in an example type 5i bipolar cell. Arrows at the top indicate timing of the first flash (black) and second flashes (gray). Flashes were 400% contrast and 10-ms duration. Traces show mean responses to five repeats of each paired-flash interval. (B) Filled circles with error bars show mean ± SEM paired-flash ratios at different inter-flash intervals across all ON-T RGCs (cyan; n = 6), ON-S RGCs (magenta; n = 5), and type 5i bipolar cells (black; n = 6).
Figure 9 with 1 supplement
Bipolar cell subtype-specific differences in ribbon size (volume).
(A) 3D reconstructions showing segmented axon stalk and axonal terminal (gray) and all the synaptic ribbons within the axonal terminal for a type 5i (cyan) and a type 6 (magenta) BC. (B) Violin plot showing distribution of ribbon volumes (each data point is a ribbon) for the type 5i and 6 BC in (A). The boxes indicate the interquartile range (thick lines, 25–75%), median value (white dot), and 1.5 times interquartile range (thin line). Shaded gray areas represent rotated kernel density plots. (C) 3D reconstructions showing segmented dendritic arbor (gray) and all the synaptic ribbons presynaptic to these segments of ON-T (cyan) or ON-S (magenta) retinal ganglion cell (RGC) dendrites. (D) Violin plot with scattered dots showing ribbon volumes for each ribbon presynaptic to a stretch of reconstructed ON-T or ON-S RGC dendrite. The color of each ribbon (dot) indicates the associated BC type. (E) An EM micrograph (left) and 3D reconstruction (right) of a ribbon and its adjacent vesicles. The ribbon is indicated by the red arrow in the EM image. In the 3D reconstruction, yellow dots indicate the adjacent vesicles surrounding the presynaptic ribbon (red) in the BC terminals (gray). Postsynaptic ON-T RGC is in blue. (F) Scatter plot showing ribbon size in volume in relationship with the number of adjacent vesicles. The gray line represents the linear regression model fitted to the data. Linear regression: β = 6.98e−06, p < 0.001, r2 = 0.66.
Figure 9—figure supplement 1
Bipolar cell subtype-specific differences in ribbon surface areas.
(A) Violin plot showing distribution of ribbon surface areas (each data point is a ribbon) for the type 5i and 6 BC in Figure 9A. (B) Violin plot with scattered dots showing ribbon surface areas for each ribbon presynaptic to a stretch of reconstructed ON-T or ON-S retinal ganglion cell (RGC) dendrite. (C) Scatter plot showing ribbon size in surface area in relationship with the number of adjacent vesicles. The gray line represents the linear regression model fitted to the data. Linear regression: β = 0.0003, p < 0.001, r2 = 0.69.
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Cone bipolar cell synapses generate transient versus sustained signals in parallel ON pathways of the mouse retina
eLife 13:RP98817.
https://doi.org/10.7554/eLife.98817.3
