A mammalian retinal bipolar cell uses both graded changes in membrane voltage and all-or-nothing Na+ spikes to encode light

Abstract

Barlow (1953) studied summation in ganglion cell receptive fields and observed a fine discrimination of spatial information from which he inferred that retinal interneurons use analog signals to process images. Subsequent intracellular recordings confirmed that the interneurons of the outer retina, including photoreceptors, horizontal cells, and bipolar cells, respond to light with slow, graded changes in membrane potential. Analog processing may enable interneurons to discriminate fine gradations in light intensity and spatiotemporal pattern, but at the expense of the speed, temporal precision, and threshold discrimination that are characteristic of all-or-nothing Na(+) spikes. We show that one type of mammalian On bipolar cell, the ground squirrel cb5b, has a large tetrodotoxin (TTX)-sensitive Na(+) current. When recorded from in the perforated patch configuration, cb5b cells can signal the onset of a light step with 1-3 all-or-nothing action potentials that attain a peak amplitude of -10 to -20 mV (peak width at half-height equals 2-3 ms). When exposed to a continuous, temporally fluctuating stimulus, cb5b cells generate both graded and spiking responses. Cb5b cells spike with millisecond precision, selecting for stimulus sequences in which transitions to light are preceded by a period of darkness. The axon terminals of cb5b bipolar cells costratify with the dendrites of amacrine and ganglion cells that encode light onset with a short latency burst of spikes. The results support the idea that a spiking On bipolar cell is part of a dedicated retinal pathway for rapidly and reliably signaling dark to light transitions.

Figure 1.

cb5 bipolar cells have a large, transient Na⁺ current. A, Peak amplitude of the TTX-sensitive current in a series of bipolar cells (circles; n = 101) recorded in retinal slices. Cells were held at −70 mV and stepped to −30 mV. Horizontal lines indicate mean peak current. Cells were identified from fluorescence images obtained immediately after whole-cell recording. Cell types are ordered according to axon ramification depth in the IPL. B, Top, The membrane voltage of a cb5 bipolar cell was held at −70 mV and stepped to a series of potentials between −60 and 20 mV in 10 mV increments. The TTX-sensitive current is shown. The voltage command is plotted below. Bottom, Membrane voltage was maintained in 10 mV increments between −90 and −10 mV and stepped to a test potential of 0 mV. The TTX-sensitive current during steps from −90 to −40 mV is shown. Upper and lower sets of traces are from separate cells. C, Plots of channel activation and inactivation versus membrane voltage for cb5 cells during a step from −70 to −30 mV. Points show mean conductance values obtained by pooling measurements from 10 cells in the group with larger peak Na⁺ currents. The fits were obtained from a Boltzmann equation. For comparison, individual symbols plot the mean resting potentials of cb5 cells with large Na⁺ currents measured during subsequent experiments under three recording conditions: whole-cell recording (upward triangle), amphotericin perforated patch recording (diamond), and gramicidin perforated patch recording (downward triangle). Both vertical and horizontal bars show SD. Bars overlap with the symbol when not visible.

Figure 2.

Peak Na⁺ current amplitude and immunolabeling for calb and PKC in cb5 cells. The recording pipette contained Neurobiotin. A, A transient inward current in a bipolar cell during a step from −70 to −30 mV. B–D, Images of the retinal slice showing labeling for Neurobiotin, calb, and PKC. E, The superimposed images from B–D. The recorded cell is positive for calb and PKC. F, A smaller transient inward current in a different bipolar cell. G–J, The recorded cell is calb⁺/PKC⁻. Scale bar, 5 μm. Arrows mark the locations of tracer-labeled somas and axons. Outer plexiform layer (OPL); Inner nuclear layer (INL).

Figure 3.

Calb and PKC labeling is associated with a cell having a large Na⁺ current. A, The intensity of PKC labeling is plotted against the intensity of calb labeling for the somas of recorded (filled circles) and neighboring, unrecorded (black symbols) cells. Unrecorded cells with nearly equal PKC and calb label intensities were classified as cb5b (squares), whereas those with a high ratio of calb to PKC label intensity were classified as cb5a (triangles). Na⁺ current amplitude for the recorded cells is indicated by circle color (scale at right). B, Plot of Na⁺ current amplitude verses PKC to calb intensity ratio, normalized by comparison with nearby cb5a and cb5b cells. The excessive number of recorded cb5b cells in the sample is likely due to bias insofar as the ratio of cb5b:cb5a cells in the intact retina is only 1.4 (A. Light, Y. Zhu, J. Shi, S. Saszik, S. Lindstrom, L. Davidson, V. Chiodo, W. Hauswirth, W. and Li, S. DeVries, unpublished observations). The points corresponding to the results from the cells shown in Figure 2 are circled. Intensity is expressed in arbitrary units (a.u.).

Figure 4.

Current injections elicited action potentials in cb5 bipolar cells. A, Membrane voltage response of a cb5b bipolar cell during step current injections of 0–70 pA (listed at right, corresponding to responses of increasing amplitude). The timing of the current pulse is shown below. B, Membrane voltage response of a cb5a bipolar cell (peak transient response equaled −120 pA during a voltage-clamp step from −70 to −30 mV) to a series of current injections. C, Response of an On sublamina stratifying bipolar cell with a negligible Na⁺ current to a series of current injections. D, Plot of peak responses versus injected current for cb5b (top), cb5a (middle), and representative non-cb5 On bipolar (bottom) cells. Gray circles plot the responses of the cells in A–C. E, Response of a cb5b cell to a step current injection (90 pA) before and during TTX exposure. F, Response of a non-cb5 cell to a 70 pA current injection before and during TTX exposure. G, Superimposed cb5b bipolar and ganglion cell action potentials obtained during step current injections in a slice. Ganglion cell trace displaced upward by 2 mV from its resting potential. Cells were grouped into categories, cb5a, cb5b, and non-cb5, solely based on Na⁺ current amplitude during a step from −70 to −30 mV.

Figure 5.

Light flashes elicit all-or-nothing action potentials in a cb5b bipolar cell. A, Spontaneous activity during 4 consecutive traces. A flash (40 ms; horizontal bar) of light delivering 2750 photons-μm⁻² produced a spike doublet at the start of each trace. The voltage threshold for spike generation was obtained from an inflection point at start of each spike. B, Voltage responses during a series of flashes of increasing intensity (shown at right in units of photons-μm⁻²). A resting voltage of −55.3 mV was subtracted from the baseline. C, Vertical lines show the time at which a spike occurred. Sum of 9 traces at each intensity. Combined results from two separate repeats of the intensity series occurring between 3.75–20 and 22–42 min after gigaseal formation. Flash intensity in units of photons-μm⁻². D, E, At the end of the recording, the cell was filled with Neurobiotin by rupturing the membrane patch that occluded the tip of the whole-cell pipette. The recorded cell was positive for both calb and PKC. At right is a calb⁺ cb2 bipolar cell. The solid lines show the borders of the IPL, while the dashed line shows the approximate division between sublaminas a and b. F, Voltage response of a cb6a bipolar cell to light flashes of increasing intensity (at the right in units of photons-μm⁻² × 10⁻³). A flash delivering 46,730 photons-μm⁻² produced a half-maximal response as determined by fitting a Michaelis-Menten curve to the intensity–response relation. A resting voltage of −48.5 mV was subtracted from the baseline. A--E from one cell. Scale bar, 5 μm.

Figure 6.

Responses of On bipolar cells to random temporal flicker. A, Voltage response (black trace) of a cb5b cell during random binary flicker (blue trace, below, upward deflection corresponds to light-on; mean intensity equals 3.42 × 10⁶ photons-μm⁻² s⁻¹) under control conditions and in the presence of TTX (2 μm, red trace). B, Subsequent current response to a step in voltage clamp from −70 to −30 mV illustrating poor voltage control due to high pipette series resistance (∼340 MΩ). C, Extended period of flicker response obtained 20 min after the start of the recording. D, Current response during a step from −70 to −30 mV before, during, and after TTX application. E, Rapid depolarizing transients from C presented on a faster time base. Symbols show corresponding regions. The fourth sample was obtained at a later time during this continuous 15 s run. F, Spike timing during three consecutive traces stimulated with identical flicker sequences. G, Linear filters obtained using all of the response points (black trace) or just the points corresponding to the occurrence of spikes (STA, red trace). H, Flicker response from a different cb5b cell which showed discrete spikes that were suppressed by TTX. Gramicidin perforated patch recording. I, Purely graded flicker responses in a cb6a bipolar cell. J, Linear filter for the cb6a cell. Time in minutes (A–D) corresponds to the time after the start of data acquisition, which commenced soon after formation of the gigaseal.

Figure 7.

The axon terminals of cb5a/b cells costratify with the dendrites of On transient amacrine and ganglion cells. A, Left, Peristimulus histogram showing the spike rate in Hz of a single ganglion cell during a 2 s flash of light (lower horizontal bar in B, left). Bin width = 20 ms. Right, Raster plot showing the time of spike occurrence during 10 consecutive stimulus presentations on a faster time base. B, Left, Light-evoked EPSC recorded in the whole-cell configuration at a membrane potential of −70 mV. (right). EPSC response on a faster time base. C, Confocal image of a flat-mounted retina showing the tracer-labeled ganglion cell. D, Left, A 2-μm-thick optical section at the level of the dendrites of the Neurobiotin-labeled ganglion cell (green). cb5b cell terminals were labeled by antibodies to PKC (blue) and calb (red), whereas cb5a cell terminals were labeled only by calb. Right, Intensity of Neurobiotin (green), PKC (blue), and calb (red) labeling plotted as a function of IPL depth from top, 0%, to bottom, 100%. The white bar indicates the level of the image in D. E, Peristimulus spike histogram from another cell. F, Light-evoked EPSC on a slow (left) and fast (right) time base. G, The wide-field amacrine cell had neurites (H) that narrowly ramified within the layer formed by cb5a/b cell terminals. I, J, Peristimulus histogram and EPSC light responses for a ganglion cell with a relatively sustained response. K, L, Ganglion cell dendrites ramified between the band formed by cb5a/b cell terminals and the bottom of the IPL.