Calcium spike-mediated digital signaling increases glutamate output at the visual threshold of retinal bipolar cells

Abstract

Most retinal bipolar cells (BCs) transmit visual input from photoreceptors to ganglion cells using graded potentials, but some also generate calcium or sodium spikes. Sodium spikes are thought to increase temporal precision of light-evoked BC signaling; however, the role of calcium spikes in BCs is not fully understood. Here we studied how calcium spikes and graded responses mediate neurotransmitter release from Mb-type BCs, known to produce both. In dark-adapted goldfish retinal slices, light induced spikes in 40% of the axon terminals of intact Mbs; in the rest, light generated graded responses. These light-evoked membrane potentials were used to depolarize axotomized Mb terminals where depolarization-evoked calcium current (ICa) and consequent exocytosis-associated membrane capacitance increases (ΔCm) could be precisely measured. When evoked by identical dim light intensities, spiking responses transferred more calcium (Q(Ca)) and triggered larger exocytosis with higher efficiency (ΔCm/Q(Ca)) than graded potentials. Q(Ca) was translated into exocytosis linearly when transferred with spikes and supralinearly when transferred with graded responses. At the Mb output (ΔCm), spiking responses coded light intensity with numbers and amplitude whereas graded responses coded with amplitude, duration, and steepness. Importantly, spiking responses saturated exocytosis within scotopic range but graded potentials did not. We propose that calcium spikes in Mbs increase signal input-output ratio by boosting Mb glutamate release at threshold intensities. Therefore, spiking Mb responses are suitable to transfer low-light-intensity signals to ganglion cells with higher gain, whereas graded potentials signal for light over a wider range of intensities at the Mb output.

Keywords: bipolar cell; calcium spike; graded potential; light response; threshold.

Fig. 1.

Experimental procedure. A: a light-evoked membrane potential (V_m) was recorded from an axon terminal of an intact Mb in current-clamp mode (left) and injected as a command potential into an axotomized terminal of another Mb in voltage-clamp mode (right). Alexa Fluor 488-filled Mbs (yellow) were imaged at the end of experiments. IPL, inner plexiform layer; OPL, outer plexiform layer; GCL, ganglion cell layer. Scale bars, 20 μm. B, bottom: injected light-evoked V_m (black, center) was flanked by sequences of sine and square waves. Top: the calculated Ca²⁺ current (I_Ca, red) in response to injected V_m (black) is shown on a fine timescale. Note that the I_Ca was noticeable when induced with light-evoked V_m but not with sine or square waves. C: trends of the membrane capacitance (C_m) before (black) and after (gray) injection of a light-evoked V_m were linearly extrapolated, and the difference in ΔC_m at the peak magnitude of the I_Ca represented exocytosis. Trends of the series conductance (G_s, middle) and membrane conductance (G_m, bottom) were analyzed similarly.

Fig. 2.

Light responses of Mb-type bipolar cells in dark-adapted retinas varied from spiking to graded. V_m in response to 500-ms-long light stimuli (shaded) recorded from axon terminals are shown for 10 representative Mbs. Light intensities (0.07, 0.20, 1.0, and 1,200 photons·μm⁻²·s⁻¹) are shown at top. The Mbs' ID numbers (1–10) are shown on left. Light responses with voltage transients >9 mV were considered as spiking (Mb1–Mb7) and those with transients ≤9 mV as graded (Mb8–Mb10). Arrows point to small voltage transients during plateau phase of the light response (Mb1 and Mb10, 1,200 photons·μm⁻²·s⁻¹).

Fig. 3.

Determining the functionally relevant spike magnitude. Ai, top: a light-evoked response with spike downscaled to 75%, 50%, 25%, and 0% of its original magnitude (dashed box, shown in detail in Aii), flanked with sine waves (shown schematically) was injected as a command potential (CP) into axotomized Mb terminals. Bottom: the exocytosis triggered in axotomized Mb terminals by the command potentials shown at top. Capacitance traces were averaged over 10 samples for better visibility. Noticeable exocytosis was triggered by the spike downscaled to 50% of its magnitude (green) but not with spikes downscaled to 25% (purple) or 0% (blue). Aii, top: expanded command potentials from dashed box shown in Ai. Bottom: I_Ca induced in axotomized Mb terminals by the command potentials shown at top. Note that the noticeable I_Ca is induced with spike downscaled to 50% (green) but not to 25% (purple) or 0% (blue). B: exocytosis triggered with the downscaled spikes (shown in Ai) in axotomized Mb terminals (n = 7). Note that noticeable exocytosis is triggered with spike with magnitude downscaled to 50% (green) but not 25% (purple) or 0% (blue).

Fig. 4.

Mb-type bipolar cells with spiking and graded responses. A: superimposed fluorescent and infrared images of Mb-generated spiking (Mb1 and Mb4) and graded (Mb10) responses shown in Fig. 2. Scale bar, 10 μm. Note the similarity in morphology of Mb-generated spiking and graded responses. Bi and Bii: responses of Mb with spiking (Bi) and graded (Bii) membrane voltage (V_m) to light with intensities of 0.07 photons·μm⁻²·s⁻¹ (top) and 0.2 photons·μm⁻²·s⁻¹ (bottom) in control Ames medium (Ctrl, black) and in the presence of 100 μM picrotoxin (PTX, red). Note that although PTX potentiated light-evoked responses, it did not turn spiking into graded (Bi) or graded into spiking (Bii) V_m. C: Mb light responses with cesium-based internal solution in current-clamp mode. Under the block of potassium channels with Cs⁺ and TEA, Mbs responded to light with nonspiking sustained depolarization up to −9.7 ± 9.9 mV (n = 7) with duration exceeding that of the light stimulus applied between 0 and 0.5 s. D: maximum magnitude of light-evoked V_m transient weakly correlated with Mb membrane resistance at command voltage −60 mV (Pearson's correlation coefficient 0.34 ± 0.13). Horizontal dashed line shows 9 mV threshold for spiking potentials. The input resistance of the dark-adapted Mbs was 365 ± 140 MΩ (n = 55), similar to ∼444 MΩ measured at the soma by Protti et al. (2000). Red and green circles represent Mbs selected for further analysis shown in E. E: mean response (n = 20) to a current step (42 pA) of 2 Mbs with the same input resistance but generating different maximum light-evoked V_m transients: 22.5 mV (red) and 1.54 mV (green). Note that the V_m depolarization was consistently larger for the Mb generating spiking responses (red), reaching the maximal difference of 2 mV at 1.6 ms after current onset. Black traces show double-exponential fits. Based on the fit parameters, capacitances of the axon terminals of Mbs with spiking and graded responses were similar, 3.3 ± 0.1 pF vs. 3.1 ± 0.2 pF, whereas the axon resistance was larger for the Mb with spiking responses, 87.6 ± 0.5 MΩ vs. 36.0 ± 1.9 MΩ.

Fig. 5.

Variability in I_Ca and exocytosis in axotomized Mb terminals. A: mean I_Ca (gray) in response to injection of a light-evoked V_m (black) into axotomized Mb terminals (n = 40). The shaded region around the mean shows ±SD. The polarity of the inward I_Ca was reversed for easier side-by-side comparison with the V_m. The V_m was recorded from axon terminal of Mb10 in response to light with intensity of 5.9 photons·μm⁻²·s⁻¹. The ratio of the mean I_Ca to its SD for the duration of the response was 2.1 ± 0.2. B: the transferred Ca²⁺ charge (Q^Ca) did not correlate with the series resistance: the Pearson's correlation coefficient was −0.06 ± 0.16. C: the exocytosis (ΔC_m) strongly correlated with Q^Ca: the slope of the linear fit (black) was 1.20 ± 0.17 fF/pC; the Pearson's correlation coefficient was 0.75 ± 0.11. D: the exocytosis (ΔC_m) strongly correlated with the initial capacitance of axotomized terminals: the slope of the linear fit (black) was 5.5 ± 1.3 fF/pF; the Pearson's correlation coefficient was 0.56 ± 0.14.

Fig. 6.

I_Ca of representative axotomized Mb terminals triggered by injection of light-evoked V_m changes. Light intensity corresponding to the shaded areas (in photons·μm⁻²·s⁻¹) and Mbs' ID numbers are shown at top right of each V_m trace (black). The polarity of the inward I_Ca (red traces) was reversed for easier side-by-side comparison with the corresponding V_m. Terminal 1 (left) was tested with spiking responses of Mb1 (see Fig. 2) to light with intensities of 0.07–1,200 photons·μm⁻²·s⁻¹ and with graded response of Mb10 to light with intensity of 5.9 photons·μm⁻²·s⁻¹. Terminal 2 (center) was tested with graded responses of Mb9 to light with intensities of 0.07–5.9 photons·μm⁻²·s⁻¹, 2 spiking responses of Mb1 to light with intensities of 1.0 and 5.9 photons·μm⁻²·s⁻¹, and graded response of Mb10 to light with intensity of 5.9 photons·μm⁻²·s⁻¹. Terminal 3 (right) was tested with graded responses of Mb10 to light with intensities of 0.07–78,000 photons·μm⁻²·s⁻¹. Horizontal black lines show zero baseline for I_Ca and resting potential −63 mV for V_m.

Fig. 7.

Ca²⁺ channels act as a high-pass filter of light-evoked V_m changes. Ai and Bi: Ca²⁺ transients (I_Ca, red) induced by injection of spiking (Ai) and graded (Bi) light-evoked V_m (black) into axotomized Mb terminals. Traces were reproduced from Fig. 6: Mb1 at 0.20 photons·μm⁻²·s⁻¹ (Ai) and Mb9 at 0.49 photons·μm⁻²·s⁻¹ (Bi). Note that I_Ca barely follows the sustained potentials. Aii and Bii: power spectrum of the V_m (black) and I_Ca (red) shown in Ai and Bi fell into 2 distinctly separated frequency bands: 0–16 Hz and 16–170 Hz. The 0–16 Hz band contained low-frequency components, whereas the 16–170 Hz band contained frequencies of the spike train (Aii) and V_m oscillations on top of graded potential (Bii). The normalized power of I_Ca was greater than that of V_m: 41.3 ± 0.5% vs. 6.4% (P = 0.001, n = 19) for the spiking response and 7.5 ± 0.7% vs. 0.2% (P = 0.02, n = 18) for the graded response.

Fig. 8.

Exocytosis and Ca²⁺ influx triggered by light-evoked potentials. A: ΔC_m (black) and Q^Ca (gray) triggered by light-evoked potentials in axotomized Mb terminals 1 (left), 2 (center), and 3 (right). The initial capacitance of axotomized Mb terminals 1, 2, and 3 was 3.29, 4.38, and 2.71 pF, respectively. Fit was done with the Hill equation. For ΔC_m, the intensity at half-maximal response (I_1/2, in photons·μm⁻²·s⁻¹) was 0.15 ± 0.01 for spiking potentials from Mb1, 0.41 ± 0.02 for graded potentials from Mb9, and 82 ± 25 for graded potentials from Mb10, whereas the Hill coefficients were 2.79 ± 0.50, 11.7 ± 2.9, and 1.9 ± 0.7, respectively. For Q^Ca, I_1/2 (in photons·μm⁻²·s⁻¹) was 0.18 ± 0.02 for spiking potentials from Mb1, 0.52 ± 0.06 for graded potentials from Mb9, and 462 ± 70 for graded potentials from Mb10, whereas the Hill coefficients were 2.89 ± 0.83, 2.25 ± 0.50, and 0.70 ± 0.06, respectively. Note that spiking light-evoked potentials from Mb1 and the most light-sensitive graded potentials from Mb9 almost saturated exocytosis at similar level (∼90 fF) at similar light intensity (0.49 photons·μm⁻²·s⁻¹). B, left: I_1/2 (in photons·μm⁻²·s⁻¹) for ΔC_m vs. that of for Q^Ca. Right: expanded area inside the dashed box shown on left. Note that for spiking potentials mean values of I_1/2 for ΔC_m and Q^Ca were almost equal (P = 0.1). For graded potentials mean value of I_1/2 for ΔC_m was less than that of for Q^Ca. C: Hill coefficient for ΔC_m vs. that for Q^Ca. Note that for spiking potentials mean values of the Hill coefficient for ΔC_m and Q^Ca were not significantly different (P = 0.1). For graded potentials, the mean value of the Hill coefficient for the ΔC_m was greater than that for the Q^Ca (P = 10⁻⁷ for Mb10 and P = 0.05 for Mb10). Gray error bars in B and C show SD about the mean.

Fig. 9.

Exocytosis triggered by Ca²⁺ influx in response command potentials generated from light responses. A: ΔC_m triggered in axotomized Mb terminals by Q^Ca transferred with light-evoked potentials recorded at axon terminals of Mb1 (black), Mb9 (red), and Mb10 (blue). Fits were done with the Hill equation. Hill coefficients were 0.95 ± 0.19, 4.14 ± 0.52, and 2.02 ± 0.17 for Mb1, Mb9, and Mb10, respectively. I_1/2s were 6.2 ± 1.8, 10.8 ± 0.4, and 65.9 ± 5.4 pC for Mb1, Mb9, and Mb10, respectively. B: expanded region shown in dashed box in A. Vertical dashed line marks the threshold for Q^Ca transferred with graded potentials. Note that below this threshold the Q^Ca transferred with spikes triggered exocytosis, whereas that transferred with graded potentials did not. C: efficiency of the exocytosis for Mb1 (black), Mb9 (red), and Mb10 (blue) as a ratio of ΔC_m to Q^Ca using the Hill fits from A. For spiking potentials from Mb1, exocytosis was the most efficient when triggered with Q^Ca of 0.34 ± 0.08 pC transferred with a single spike. For graded potentials from Mb9 and Mb10, exocytosis was the most efficient when triggered with Q^Ca of 14.3 ± 1.9 and 66.7 ± 7.9 pC, respectively.

Fig. 10.

Features of light-evoked spiking and graded Mb responses that affect exocytosis. A: the ΔC_m triggered with light-evoked spike trains in axotomized terminals increased with the rise in the number of spikes in the trains: 10.8 ± 4.3 fF (1 spike) < 44.9 ± 17.3 fF (8 spikes) < 80.0 ± 18.4 fF (14 spikes) < 90.2 ± 26.1 fF (28 spikes); P ≤0.05 for each pair of means (Newman-Keuls test, n = 19). Horizontal dashed line shows the approximate size of the rapidly releasable pool (RRP). B: the 1st spike in a train induced by current injection in axotomized terminals (n = 10, black) and evoked by light in terminals of intact Mbs (n = 10, red). Shaded areas are within SD about means. C, bottom: the cuts of a light-evoked spike train recorded from an intact Mb. The cut of the 1st spike (red) overlies the cut of the 1st and 2nd spikes (green), which overlies the cut of the 1st, 2nd, and 3rd spikes (blue), which in turn overlies the cut of all 4 spikes in the train (black). Top: I_Ca in a representative axotomized Mb terminal in response to the cuts of the spike train shown below. D: the exocytosis per spike (black) triggered in axotomized Mb terminals (n = 16) vs. the amplitude of a spike in a train shown in C. The spikes' ordinal numbers are shown at the data points. The slope of the linear fit (gray) is 4.50 ± 0.48 fF/mV. E: the exocytosis (black) triggered in axotomized Mb terminals (n = 18) by the graded light-evoked potentials from Mb9 (see Fig. 6). Peak amplitude of the light-evoked potentials and its scale bar are shown in red. F: the rising phase of the graded potentials from Mb10 evoked by light with intensities of 0.49 (blue), 1.0 (green), 35 (red), and 78,000 (magenta) photons·μm⁻²·s⁻¹. The fits (black) were done with the Hill equation. G: the exocytosis (black) triggered in axotomized Mb terminals (n = 10) by the graded light-evoked potentials from Mb10 (see Fig. 6). Peak amplitude of the light-evoked potentials and its scale bar are shown in red. The maximum slope of the potentials (from the Hill's fit) and its scale bar are shown in green. The duration of the light responses (above the threshold for I_Ca of −55 mV) and its scale bar are shown in blue.