Nonlinear Spatiotemporal Integration by Electrical and Chemical Synapses in the Retina

Abstract

Electrical and chemical synapses coexist in circuits throughout the CNS. Yet, it is not well understood how electrical and chemical synaptic transmission interact to determine the functional output of networks endowed with both types of synapse. We found that release of glutamate from bipolar cells onto retinal ganglion cells (RGCs) was strongly shaped by gap-junction-mediated electrical coupling within the bipolar cell network of the mouse retina. Specifically, electrical synapses spread signals laterally between bipolar cells, and this lateral spread contributed to a nonlinear enhancement of bipolar cell output to visual stimuli presented closely in space and time. Our findings thus (1) highlight how electrical and chemical transmission can work in concert to influence network output and (2) reveal a previously unappreciated circuit mechanism that increases RGC sensitivity to spatiotemporally correlated input, such as that produced by motion.

Figure 1. Paired stimuli reveal nonlinear lateral interactions

(A) Simplified diagram of chemical and electrical synapses in the excitatory ON circuitry of the retina. (B) Dye filled ON-S ganglion cell (black; gray shading is patch-pipette) over a simulated mosaic of type 6 cone bipolar cells (yellow hexagons) to illustrate that RGC dendrites receive convergent input from numerous parallel feed-forward bipolar circuits. Shaded white rectanges show dimensions of the paired bar stimulus used in the following experiments. (C–D) Example responses to positive contrast (C) or positive and negative contrast bars (D). Top row, light stimulus. Middle rows, example single trial responses to single or paired bar stimuli. Bottom row, mean responses (8 trials each). Responses in (C) and (D) are from same example cell. Stimulus timing (33 ms flash) is indicated by light gray boxes. (E) Overlaid average responses from (C) (left) and (D) (right). Dashed black lines show linear sum of single bar responses (colored traces). Solid black lines show measured paired bar response. Summary of nonlinear indices for responses to paired positive contrast bars or paired positive/negative contrast bars shown in middle panel. Gray lines are data from individual cells and filled black circles show mean±SEM (n=6 cells). Gray bars above traces show stimulus timing. All bars were 18 μm-wide, inter-bar spacing 18–22 μm. See also Figure S1.

Figure 2. Nonlinear interaction depends on stimulus contrast

(A) Mean excitatory currents in response to single (gray) or paired (black) bar presentations at different stimulus contrasts in an example ON-S RGC. Dashed lines shows sum of single bar responses. (B) Contrast-response relationship for same cell shown in (A). Symbols and error bars show mean±SEM. Filled black circles that fall above (within) the grayed area indicate supralinear (sublinear) interactions. (C) Population summary of single bar contrast-response relationship. Gray symbols show data from individual cells. Filled circles with error bars are mean±SEM across cells (n=19 cells) for data collected into approximately equal sized bins by contrast. Responses were normalized to the response at 315–450% contrast. Solid black curve and dotted lines show mean and SEM, respectively of bipolar population model output (see Figure 5 and associated text; xhalf =71±9% contrast, h=2.18±0.16, σ_bipolar=14±2% contrast). (D) Population summary of nonlinearity index versus stimulus contrast for same cells shown in (C). Line styles and symbols same as in (C). See also Figures S2, S4 and S5.

Figure 3. Gap junctions contribute to supralinear paired bar integration

(A) Schematic illustration of experiment in (B–D) to isolate gap junction-mediated input to ON cone bipolar cells. (B) Example response time-courses for type 6 bipolar cell in wild-type (left) or cx36−/− (right) retina when GTP-γ-S was included in the pipette solution. Saturating flashes (10 ms; 520 μm-diameter circular spot) were presented every 2 sec after achieving whole-cell configuration. Insets show example single trial responses immediately after break-in (black trace) or once responses had reached a steady-state level (gray). (C) Summary of peak flash responses in ON cone bipolar cells at different holding potentials >2min after dialysis with GTP-γ-S (n=4 cells; two type 6 bipolar, one each type 5 and type 7 bipolar cells). Inset shows mean flash responses from example type 6 bipolar cell held at −64 (darkest trace) to +16 mV (lightest). (D) Comparison of initial versus steady-state ON cone bipolar responses with GTP-γ-S in recording pipette in wild-type (left) or cx36−/− mice (right). Diamonds for cx36−/− data represent a recording from an unknown ON bipolar type. Other symbols follow legend for wild-type recordings. Recordings shown in (B–D) acquired in a retinal slices. In these experiments, NBQX (10 μM) was included in bath solutions to eliminate rod bipolar pathway-mediated signals. (E) Example single (gray) and paired (black) bar responses at different stimulus contrasts from example ON-S RGC in cx36−/− retina (flat mount). Stimulus contrasts (left to right): 5820, 7790 and 19600%. (F) Contrast-response relationship for cell shown in (E). Symbols show mean±SEM. (G) Summary of single bar responses for ON-S RGCs recorded in cx36−/− mice. Gray symbols are data from single cells, filled circles are mean±SEM (n=7 cells). (H) Summary of nonlinearity index versus stimulus contrast. Same symbol and color scheme as (G). Note different contrast scale in (F–H) compared to Figure 2B–D. See also Figure S3.

Figure 4. Linear or sublinear responses in AII amacrine cells

(A) Mean voltage responses at different stimulus contrasts in an example AII amacrine cell (flat mount retina). Resting membrane potential = −47 mV. Stimulus contrasts (left to right): 62, 110 and 281%. (B) Contrast-response relationship for cell shown in (A). Symbols show mean±SEM. (C) Summary of AII amacrine cell responses across contrasts. Filled gray symbols (single cell) and filled black circles with error bars (mean±SEM) show data from current-clamp recordings (n=5 cells; resting membrane potential = −48±2 mV). Open gray symbols (single cell) and white circles with error bars (mean±SEM) are from voltage-clamp recordings (n=4 cells). (D) Summary of nonlinearity index versus stimulus contrast. Same symbol and color scheme as in (C).

Figure 5. Simplified model of synaptic output from bipolar cell population

(A) Dendritic input to ON cone bipolar cells was modeled by filtering stimuli by a circular Gaussian profile matched to the anatomical dimensions of type 6 bipolar dendrites (top; σ = SD of Gaussian). Gap junction-mediated signals were modeled by spreading a fraction of dendritic signals to neighboring bipolar locations according to an exponential function (bottom; λ=space constant). (B) Example modeled spatial response profile (triangles) of a bipolar cell to narrow bars of light using parameters shown in (A). Solid curve is a Gaussian function fitted to the model responses. For five independently generated model populations, 2 SD diameter of Gaussian profile for model responses = 43.4 ± 0.4 μm (experimentally measured = 44 ± 8 μm (Schwartz et al., 2012)). (C) Effect of nonlinear bipolar output. Gray lines of increasing darkness are modeled nonlinear interactions for paired bar stimuli for bipolar output nonlinearities with increasing steepness (h = 1, 1.5, 2, 3; see inset). All other model parameters were kept constant. These curves show output from model without bipolar noise. (D) Effect of adding noise. Gray lines of increasing darkness show nonlinear interactions for increasing amounts of additive noise (σ_bipolar=10, 20, 30 and 40% contrast; see inset) upstream of a fixed synaptic nonlinearity (x_half=30% contrast, h=3). (E) Comparison of bar responses (top) and associated nonlinearity indices across contrasts (bottom) from an example ON-S RGC and output of the model (solid lines) (c_half =37%; h=2.52; σ_bipolar=14%). (F) Representation of model output from individual bipolar cells (black hexagons) across the population for single (left, middle) and paired bars (right) at stimulus contrasts producing robust supralinear paired bar responses (70%, top row) or sublinear interactions (300%, bottom row). Note different color scales for top versus bottom panels. Each panel is a 400 μm by 400 μm region of the bipolar population and shows the average bipolar output across 10 stimulus presentations with bipolar noise values generated by different random seeds. White dotted lines show boundaries of bar stimuli.

Figure 6. Nonlinear interactions extend over space and time

(A) Mean responses from an example ON-S RGC to stimuli presented at different inter-bar spacings. (B) Summary of nonlinearity index versus inter-bar spacing. Gray symbols are data from individual cells. Black circles show mean±SEM (n=8 cells). Solid black line shows exponential fit (λ=21 μm) to points between 36 and 126 μm. Paired bars were presented simultaneously (33 ms flash). Stimulus contrasts were held constant across locations for each cell. (C) Mean responses from an example ON-S RGC to stimuli presented with different time lags between first and second bar presentations. (D) Summary of nonlinearity index versus inter-bar time differences. Gray symbols are data from individual cells. Black circles show mean±SEM (n=6 cells). Black line shows exponential fit to data (τ = 47 ms). Stimulus contrast and positions were held constant across time lags (inter-bar distance 22 µm). Line style in (A) and (C) follows that used in Figure 2.

Figure 7. ON-S RGCs exhibit enhanced sensitivity to spatio-temporally correlated stimuli

(A) Stimulus paradigm for comparing spatiotemporally correlated (‘sequential’, left) or uncorrelated (‘randomized’, right) stimuli. (B) Mean excitatory current (top row; whole-cell) and spike (bottom rows; cell-attached) responses from the same example ON-S RGC at different bar contrasts. Raster plots in middle rows show spike responses to twenty presentations of each stimulus type. Here, ‘sequential’ and ‘randomized’ trials are separated for visual clarity, but stimulus trials were interleaved in the experiment. Lines and light shaded regions in peristimulus time histograms in bottom row show mean ± SEM spike rate, bin width = 30 ms. (C)–(D) Summary responses across contrasts for excitatory input (C) and spikes (D) for same cell as shown in (B). Averaged responses in (B)–(D) are from 25 trials of each stimulus type. (E) Summary of sequential versus randomized stimulus response as function of stimulus contrast for excitatory currents (filled circles, n=5 cells) and spikes (open circles, n=6 cells). Symbols and error bars in (C)–(E) are mean±SEM (error bars mostly obscured by symbols in (C–D)). Bar speed = 0.86 mm/s (stimulus duration = 0.416 s). See also Figure S6.