Two-photon imaging of nonlinear glutamate release dynamics at bipolar cell synapses in the mouse retina

Abstract

Alpha/Y-type retinal ganglion cells encode visual information with a receptive field composed of nonlinear subunits. This nonlinear subunit structure enhances sensitivity to patterns composed of high spatial frequencies. The Y-cell's subunits are the presynaptic bipolar cells, but the mechanism for the nonlinearity remains incompletely understood. We investigated the synaptic basis of the subunit nonlinearity by combining whole-cell recording of mouse Y-type ganglion cells with two-photon fluorescence imaging of a glutamate sensor (iGluSnFR) expressed on their dendrites and throughout the inner plexiform layer. A control experiment designed to assess iGluSnFR's dynamic range showed that fluorescence responses from Y-cell dendrites increased proportionally with simultaneously recorded excitatory current. Spatial resolution was sufficient to readily resolve independent release at intermingled ON and OFF bipolar terminals. iGluSnFR responses at Y-cell dendrites showed strong surround inhibition, reflecting receptive field properties of presynaptic release sites. Responses to spatial patterns located the origin of the Y-cell nonlinearity to the bipolar cell output, after the stage of spatial integration. The underlying mechanism differed between OFF and ON pathways: OFF synapses showed transient release and strong rectification, whereas ON synapses showed relatively sustained release and weak rectification. At ON synapses, the combination of fast release onset with slower release offset explained the nonlinear response of the postsynaptic ganglion cell. Imaging throughout the inner plexiform layer, we found transient, rectified release at the central-most levels, with increasingly sustained release near the borders. By visualizing glutamate release in real time, iGluSnFR provides a powerful tool for characterizing glutamate synapses in intact neural circuits.

Figure 1.

Nonlinear release from bipolar cells explains frequency-doubled responses. A, Model for the Y-cell nonlinearity in ganglion cells. The subunits (OFF bipolar cells A-D) view a contrast-reversing grating (alternating between frames 1 and 2) and respond to the preferred contrast. If the nonlinear transformation occurred after linear integration of contrast, the bipolar cell response would be canceled when equal amounts of dark and light cover the receptive field (null phase, solid line, subunits B and D). If, instead, the nonlinearity occurred before contrast integration, the bipolar cell response would be frequency-doubled at the null phase (dashed line). In both cases, the postsynaptic ganglion cell sums the subunits and generates a frequency-doubled response. B, For a given subunit, the nonlinear response shown in A changes depending on the spatial phase of the grating.

Figure 2.

Imaging light-evoked glutamate release in the whole-mount retina. A, Two-photon fluorescence images of whole-mount mouse retina, in vitro. Transduction of adult retinas with AAV2/1-hSynapsin-iGluSnFR caused iGluSnFR expression in ganglion and amacrine cell membranes. z = 0 μm was the image plane that hemisected the ganglion cell somas; z increases toward the INL (z = 40 μm). B, Retinas were recorded on an upright microscope. Patterned light stimuli (peak wavelength 395 nm, 10⁴ R*/s for both rods and cones) were projected onto the photoreceptors (c) through the condenser lens. Stimulus-evoked release of glutamate from bipolar cells (b) was measured with two-photon fluorescence imaging of iGluSnFR-expressing amacrine cell (a) and ganglion cell (g; ON-type shown here) dendrites in the IPL. Red dashed line indicates the focal plane shown in D. C, Our conceptual model assumes diffuse expression of iGluSnFR throughout the cell membrane. iGluSnFR is expressed sufficiently near to synapses to report glutamate release. D, Two-photon fluorescence image of iGluSnFR-expressing dendrites of ganglion cells and amacrine cells in the IPL, 21 d after injection (whole-mount retina). E, A patterned light stimulus (1 Hz contrast-reversing “Y”) evoked a matching fluorescence response (bottom: heat map, average of 12 reversals, same area as shown in D). F, ON α cell excitatory current (V_hold = E_Cl) measured in a retina lacking cone function (Gnat2^cpfl3). The cell responded strongly to a 1 Hz contrast-reversing spot (100% contrast; 400 μm diameter, solid line at top) at a dim (∼10² R*/rod/s) mean level (“pre” recording). The cell responded to the scan laser (dashed line at top) but was saturated during the subsequent spot modulation. At a brighter mean (10⁴ R*/rod/s), the response to the scan laser was absent and the stimulus-evoked response modulation was minimal. Upon return to the dim mean, the response could again be measured (“post” recording), demonstrating that the laser did not irreversibly bleach the rods. G, PSF measurements of the microscope. A bead (0.5 μm diameter) embedded in 1% agarose was imaged in the radial (x-y) and axial (z) planes. The bead profile (blue line) was convolved with a Gaussian (red line) to fit (green line) the measured responses (black points). For the axial dimension (bottom plot), the green and red lines overlap. H, Two-photon fluorescence images (left) and color-coded fluorescence responses (right) obtained at the ON-OFF border in the central IPL. Color of each pixel represents the sum of the response amplitude to light ON (blue) and the response to light OFF (magenta) relative to baseline. Separation of pixels into intermingled blue and magenta regions (arrowhead) demonstrates that local responses were predominantly ON- or OFF-type; dendritic regions with substantial ON-OFF release (purple) were lacking. iGluSnFR expressed in ganglion cells and amacrine cells (top; hSynapsin promoter), and iGluSnFR expressed in Müller glia (bottom; GFAP promoter) gave similar results.

Figure 3.

iGluSnFR fluorescence parallels excitatory current across contrast levels. A, Two-photon fluorescence images of the dendritic arbor of an iGluSnFR-expressing OFF Y-type ganglion cell, targeted for whole-cell recording. The cell was filled with a red fluorescent dye (Alexa-568) through the patch pipette. iGluSnFR fluorescence intensity was averaged across the ROI indicated by the dashed outline. B, Simultaneously recorded excitatory currents (top) and fluorescence responses (bottom) evoked with contrast stimulation (150-μm-diameter spot) at two contrast levels (same cell as shown in A). The fluorescence response is reported as fractional change in fluorescence intensity compared with the baseline intensity (ΔF/F). Solid line and shaded region represent mean ± SEM of four repeats at each contrast level. C, Average excitatory current (left, shown inverted for comparison) and fluorescence response (right) to spots at five contrasts from 10% to 100%. Each curve shows the average response during the dark phase of the stimulus (mean of 12 periods). D, Contrast response functions based on the excitatory current (black) and fluorescence response (red) integrated over the duration of the response (C, pink area). Error bars indicate SEM across trials. E, Scatter plot of excitatory current and fluorescence response amplitude from 10% to 100% contrast (n = 4 cells, indicated by different colors). Responses were normalized by fitting a line to the data for each cell and scaling currents and fluorescence responses to the current amplitude of the fit at 100% contrast. Error bars indicate SEM across trials.

Figure 4.

Retinal bipolar cells under light-adapted conditions have strong inhibitory surrounds. A, Fluorescence responses to contrast-reversing spots of increasing diameter (top). Responses were recorded from iGluSnFR-expressing dendrites in the OFF and ON layers of the IPL (30 and 16 μm, respectively, distal to the ganglion cell layer). Traces represent the average fluorescence change in a 25 μm × 25 μm area under the center of the spot. Responses in the OFF layer often changed from OFF to ON responses for the largest spot size (response timing indicated with vertical dashed lines). Gray trace represents fluorescence signal during blank trials, when no spot was presented. B, Average size tuning functions for ON and OFF bipolar cells (ON: z = 16 μm, n = 14; OFF: z = 30 μm, n = 13). Data points indicate fluorescence change in the maximally responding 8.3 × 8.3 μm subregion in each of the imaged areas, including those shown in A; error bars represent SEM across subregions. Curves represent peak-to-trough modulation amplitude of the recorded response (see Materials and Methods). A negative amplitude in response to large spots (OFF layer) indicates a fluorescence increase during the light phase of the stimulus (ON response; as in A). The fitted lines indicate difference-of-Gaussians center-surround models (see Materials and Methods). The parameters for ON responses were as follows: k_center, 0.84 ΔF/F; k_surround, 0.75 ΔF/F; σ_center, 40.6 μm; σ_surround, 73.2 μm. The parameters for OFF responses were as follows: k_center, 0.69 ΔF/F; k_surround, 0.75 ΔF/F; σ_center, 32.9 μm; σ_surround, 82.4 μm. C, Simultaneously recorded size-tuning function of bipolar cells (red) and a postsynaptic ganglion cell (black). Bipolar cell tuning function reflects size tuning at the level of glutamate release; ganglion cell function shows tuning at the level of postsynaptic excitatory current. Curves represent peak-to-trough modulation amplitude of the response. Error bars indicate SEM across repeated trials. Inset, Two-photon fluorescence image of iGluSnFR expressing dendrites (green) and the dendrite of the recorded ganglion cell (red). D, Responses of ON (top) and OFF (bottom) bipolar cells to a small and a large spot stimulus presented at different distances from the bipolar cells' receptive field center. The ON bipolar cell response was more transient for a centered large spot compared with a centered small spot (solid and open arrowheads, respectively). Stimulus time course shown below centered stimulus traces. E, Response amplitudes of the OFF layer data shown in D. F, Quantification of the response to centered and peripheral, small and large spots (ON n = 4; OFF n = 5): a, response amplitude for a small, centered spot; b, response amplitude for a large, centered spot; c, response amplitude for a small, peripheral spot; d, response amplitude for a large, peripheral spot. See E for reference. Error bars indicate SEM across ROIs (25 μm × 25 μm area).

Figure 5.

Bipolar cell glutamate release in response to contrast-reversing gratings demonstrates linear integration by bipolar cells. A, Excitatory currents recorded in whole-cell patch-clamp from an iGluSnFR-expressing neuron. Responses show a transient inward current at each contrast reversal (blue lines; F2 response). The response is independent of spatial phase (spatial frequency, 10 cycles/mm). Shaded region represents SEM across trials. B, Two-photon fluorescence image of iGluSnFR-expressing dendrites (green) and an ON-type Y-cell filled with a red dye (Alexa-568; same cell as A). Dashed lines indicate ROIs used in the analysis. C, Fluorescence responses recorded simultaneously with the current recording shown in A (ROIs indicated in B). Each local region responds at the stimulus frequency (F1 response). Each region shows a null phase (arrowhead), as predicted by the model with linear integration by the bipolar cells before the stage of glutamate release (Fig. 1). D, Normalized response amplitude for the data shown in B,C. Response sign was either positive for initially rising responses or negative for initially falling responses. Each response is plotted twice (e.g., the response at 180 degrees is the inverse of the response at 0 degrees).

Figure 6.

A TPMPA-sensitive surround suppresses ON synapses during stimulation with high spatial frequencies. A, Fluorescence response during the null phase for glutamate release onto an ON Y-cell (red) and an OFF Y-cell (black); SEM shown in pink and gray, respectively. The contrast-reversing grating suppressed glutamate release in the ON layer, but not the OFF layer. B, Quantification of the suppression of glutamate release in the ON- and OFF layer during the null phase (ON, n = 16; OFF, n = 12). Each point represents one ROI. Points indicate the average fluorescence change during contrast stimulation relative to baseline (A, gray and blue regions, respectively). Summary points and error bars represent mean ± SEM. C, Fluorescence recordings from bipolar cell synapses in the ON Y-cell layer stimulated with contrast-reversing gratings in a small (150 μm; black) and large (1000 μm; red) aperture (top). The large stimulus caused TPMPA-sensitive suppression of release, both in the preferred and null phase. The null phase was approximate; there was a small residual response in some traces. D, The response offset and modulation amplitude were computed with a sinusoidal fit (green line) to the data. Response offset and modulation amplitude for all recorded responses to small (black) and large (red) stimuli in control conditions (n = 89 ROIs from 22 measurements). Data points next to each population indicate the mean difference ± SEM for each condition; asterisks indicate significant changes. E, Response offset (left) and modulation amplitude (right) for all recorded responses to small (black) and large (red) stimuli in control (solid) compared with TPMPA conditions (open symbols; n = 29 ROIs from 7 measurements). Summary points and error bars represent mean ± SEM. Asterisks indicate significant changes.

Figure 7.

Glutamate release in the ON Y-cell layer is less rectified and more sustained compared with the OFF Y-cell layer. A, Two-photon fluorescence image of the scan location (left) and light-evoked responses at this location recorded with line scans (2 kHz sampling; right). The line scan (dashed red line) intersects two dendrites of the simultaneously recorded OFF Y-cell (magenta). B, Same as A, for an ON Y-cell. C, Top row, Excitatory currents recorded from the OFF Y-cell shown in A during stimulation with contrast-reversing gratings (10 cycles/mm, 2 Hz). The difference in the response amplitude in alternate contrast-reversals suggests a slight imbalance in the number of bars stimulating the receptive field during alternate reversals. Middle rows, Fluorescence changes at two locations (1, 2 shown in A) on the dendrites recorded simultaneously with the currents. Here and in subsequent panels, SEM of fluorescence measurements is shown in pink. Bottom row (black), Sum of the normalized responses of two bipolar cell populations with opposite phase (red, blue = ROIs 1 and 2 shown above). D, Same as C, for an ON Y-cell. E, Average fluorescence response recorded with line scans across the dendrites of ON Y-cells after a light increment (left; n = 108) and OFF Y-cells after a light decrement (right; n = 54). Stimulus time course shown below each trace. Response waveforms were quantified by averaging the fluorescence signal in each colored region. F, Distribution and average ± SEM of the four measurements shown in E. G, Fluorescence change from the response just after stimulus onset (a, t_peak) to the response just before stimulus offset (b, t_peak + 160 ms) shows that responses in the synaptic layers where ON Y-cells stratify are relatively sustained. The difference between a and b is shown to the right of b. Error bars indicate SEM across ROIs. H, Simulation demonstrates how alternating sustained responses with a non-zero decay time-constant in combination generate transient responses at twice the frequency of the underlying responses.

Figure 8.

Temporal dynamics of bipolar cell glutamate release throughout the IPL. A, Above-threshold ON- and OFF responses (left and center, respectively; see Materials and Methods) to a small spot (150 μm, 1 Hz square-wave; bottom) recorded at 23 levels of the IPL (2 μm spacing) and aligned to the ON/OFF boundary (50%). Data show average of 14 stacks (see Materials and Methods for definition of ROIs). Shaded areas represent SEM across stacks. Stimulus time course shown below traces. Glutamate release in response to a contrast-reversing grating, sensed by a postsynaptic cell at each level of the IPL was simulated (right) by summing the recorded responses (left and center) with their 180-degree phase-shifted copies. B, Quantification of the temporal dynamics of glutamate release. Examples show two levels of the IPL: top, stratification level of OFF Y-cells; bottom, stratification level of ON Y-cells; a–c indicate measured sections of the response. C, Mean values of a–c (defined in B) at 23 levels of the IPL. White markers represent ON responses; black markers represent OFF responses. Shaded area represents SEM; n = 14 z-stacks. Asterisk indicates suppression of tonic glutamate release during the nonpreferred stimulus phase, which is present in ON- but not OFF layers. D, Comparison of the measured response amplitude at the stimulus frequency (F1, 1 Hz; dashed line, data shown in A, left and center), the simulated response amplitude at twice the stimulus frequency (F2, 2 Hz; solid line, data shown in A, right), and their ratio (F2: F1).