T-Type Ca²⁺ Channels Boost Neurotransmission in Mammalian Cone Photoreceptors

Mouse cone photoreceptors exhibit I_LVA and I_HVA

First, we examined the voltage dependence of I_Ca in mouse cone photoreceptors. We prepared horizontal slices of the retina (Feigenspan and Babai, 2017) in which a horizontal cut was made through the outer plexiform layer to reveal cone photoreceptor terminals (Fig. 1A, white arrowhead). Cone photoreceptor terminals were discriminated from rod photoreceptor terminals (Fig. 1A, black arrowhead) by their size, anatomic localization, and I_Ca amplitude (see Materials and Methods). Cone photoreceptor terminals are approximately threefold bigger and localize closer to the inner retina than the relatively small rod photoreceptor terminals. Additionally, cone photoreceptors display I_Ca amplitudes that are six to eight times bigger than that of rod photoreceptors. To isolate I_Ca, we used a cesium and TEA-Cl-based intracellular solution with 2 mm CaCl₂ in the bath solution as the charge carrier. To exclude feedback from horizontal cells, the extracellular solution contained 8 µm CNQX, which is a potent AMPA and kainate receptor antagonist (Feigenspan and Babai, 2015; Babai et al., 2016). For the recording, we used pipettes with ∼10 MΩ resistance pulled from borosilicate glass (Sutter Instrument), and we achieved a final access resistance of 20–30 MΩ. Series resistance was compensated up to 66%. We rejected recordings with a larger than −30 pA leak current at −69 mV V_h. Cone photoreceptors were held at −69 mV, and I_Ca was measured by using a ramp voltage protocol from −89 mV (800 ms) to +49 mV with a speed of 0.1875 mV/ms (Fig. 1B1,B2). Surprisingly, the I_Ca trajectory showed two peaks, one at a lower (Fig. 1B2, LVA) and one at a higher (Fig. 1B2, HVA) membrane potential (V_m) range. The double peak seen in the current–voltage profile of cone photoreceptor I_Ca is comparable to I_Ca in certain types of retinal bipolar and horizontal cells where both HVA (L-type) and LVA (T-type) Ca²⁺ channels are present (Hu et al., 2009; Feigenspan et al., 2020). Therefore, we next measured I_Ca using the same voltage ramp protocol but starting from a more depolarized V_h value of −39 mV (800 ms). At this slightly depolarized V_m, LVA T-type Ca²⁺ channels typically show strong inactivation (Perez-Reyes, 2003), while HVA L-type Ca²⁺ channels only inactivate moderately (Taylor and Morgans, 1998). Consequently, only I_HVA should appear in the current trajectory. As a result, we found that the amplitude of I_LVA significantly decreased, but that of I_HVA did not change (Fig. 1B2,C1,C₂). The peak of I_LVA and I_HVA localized at approximately −40 mV and approximately −20 mV, respectively (Fig. 1C3), and the presence of the two current types yielded a wide range of responsive V_m. The first peak suggested the presence of T-type Ca²⁺ channels because the typical T-type I_Ca reaches its maximal amplitude between −40 and −10 mV (Ertel et al., 1997). However, unlike T-type I_Ca in other tissues, I_LVA was not fully inactivated at −39 mV, a potential that is close to the physiological V_m in darkness (−40 mV). In summary, patch-clamp recordings from the terminal of cone photoreceptors indicate the unexpected presence of an I_LVA, suggesting the expression of T-type Ca²⁺ channels in cone photoreceptors additionally to L-type Ca²⁺ channels.

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Figure 1.

The presence of I_LVA and I_HVA in cone photoreceptors. A, Example transmission microscope image of a patch-clamp recording from a cone photoreceptor terminal in a mouse horizontal retinal slice. White arrowhead, Cone photoreceptor terminal; black arrowhead, rod photoreceptor terminal. B₁, Example whole-cell patch-clamp recording from mouse cone photoreceptor terminals. Currents were monitored in response to a voltage ramp protocol from −89 to +49 mV, with a speed of 0.1875 mV/ms. Prepulse duration was 800 ms at different V_h values (black, −89 mV; gray, −39 mV). B₂, Example trace shows the current–voltage relationship of I_Ca. Arrowheads indicated I_LVA and I_HVA components. C₁–C₃, Peak amplitudes of I_LVA: V_h = −89 mV: −12.72 ± 2.15 pA, n = 12; V_h = −39 mV: −6.90 ± 1.53 pA, n = 12; p < 0.0001, paired t test. Peak amplitudes of I_HVA: V_h = −89 mV: −19.66 ± 3.33 pA, n = 12; V_h = −39 mV: −20.19 ± 3.45 pA, n = 12; p = 0.4511, paired t test. I_Ca peak position: LVA: −42.48 ± 0.46 mV, n = 12; HVA: −20.75 ± 0.71 mV, n = 12; p < 0.0001, Mann–Whitney test.

Pharmacological isolation of LVA and HVA I_ca

The pore-forming subunits of the T-type Ca²⁺ channel family consist of three members: Ca_v3.1 (α1G), Ca_v3.2 (α1H), and Ca_v3.3 (α1I), which show similar activation and inactivation kinetics (Klöckner et al., 1999; Perez-Reyes, 2003). Nickel blocks all three T-type Ca²⁺ channel members at higher concentrations, but it selectively blocks Ca_v3.2 channels at lower concentrations (Lee et al., 1999, 2002; Kang et al., 2006). We applied 100 µm nickel and stimulated cone photoreceptors with a ramp voltage protocol described in Figure 1. Example I_Ca responses are illustrated in Figure 2A1. We used a pressure-controlled puffing system (model ALA-VM8, ALA Scientific Instruments), which allowed the fast and local application of nickel and CNQX (8 µm) containing solution through a 75-µm-diameter tube directly onto cone photoreceptor terminals. Control I_Ca was measured during the puff application of the bath solution to exclude changes caused by mechanical effects. We found that the application of nickel caused a ∼70% and a ∼50% reduction of I_HVA and I_LVA components, respectively (Fig. 2A1,A2). Nickel is a commonly described ion that blocks LVA Ca²⁺ channels, but HVA Ca²⁺ channels, especially L-type Ca²⁺ channels, also show nickel sensitivity (Zamponi et al., 1996; Bradley et al., 2004; To et al., 2020). Therefore, the reduction of the I_HVA component is unsurprising. To further analyze the origin of the I_LVA component, we puffed 1 µm mibefradil, a T-type Ca²⁺ channel blocker (Martin et al., 2000), onto cone photoreceptor terminals. However, we only found a slight tendency for reduced amplitudes for both I_LVA and I_HVA (Fig. 2B1,B2). We did not use a higher concentration of mibefradil to isolate T-type Ca²⁺ channels because it has been shown that mibefradil is also an antagonist for L-type Ca²⁺ channels (Leuranguer et al., 2001; Lee et al., 2006; To et al., 2020), and Cl^–, K⁺, and Na⁺ channels (Heady et al., 2001). Next, we tested 5 µm Z944, a selective T-type Ca²⁺ channel antagonist (Casillas-Espinosa et al., 2015), which significantly decreased I_LVA amplitude but unexpectedly increased the I_HVA component (Fig. 2C1,C2), revealing Z944 as a possible agonist for L-type Ca²⁺ channels in cone photoreceptors. In general, the sensitivity of I_LVA to nickel and Z944 suggests the presence of T-type Ca²⁺ channels in cone photoreceptors, and the sensitivity of I_HVA to Z944 suggests that the L-type Ca²⁺ channel in cone photoreceptors may have unique molecular properties.

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Figure 2.

Pharmacological separation of I_LVA from I_HVA. A₁, Example current–voltage relationship of ramp-evoked I_Ca in mouse cone photoreceptors in the presence and absence of 100 μm NiCl₂. Arrowheads indicate I_LVA and I_HVA component (LVA, HVA). A₂, Peak I_LVA amplitude: control: −11.03 ± 1.80 pA, n = 5; nickel (Ni²⁺): −6.22 ± 1.13 pA, n = 5; p = 0.0030, paired t test. Peak I_HVA amplitude: control: −37.45 ± 5.25 pA, n = 5; nickel (Ni²⁺): −11.01 ± 1.52 pA, n = 5; p = 0.0022, paired t test. B₁, Example current–voltage relationship of evoked I_Ca in the presence and absence of 1 μm mibefradil (Mibe). B₂, Peak I_LVA amplitude: control: −11.15 ± 0.94 pA, n = 6; mibefradil: −9.81 ± 0.93 pA, n = 6; p = 0.0641, paired t test. Peak I_HVA amplitude: control: −37.04 ± 3.48 pA, n = 6; mibefradil: −35.06 ± 3.27 pA, n = 6; p = 0.1712, paired t test. C₁, Example current–voltage relationship of evoked I_Ca in the presence and absence of 5 μm Z944. C₂, Peak I_LVA amplitude: control: −12.51 ± 1.58 pA, n = 7; Z944: −6.05 ± 0.82 pA, n = 7; p = 0.0003, paired t test. Peak I_HVA amplitude: control: −36.61 ± 4.06 pA, n = 7; Z944: −49.25 ± 6.04 pA, n = 7; p = 0.0371, paired t test. D₁, Current response to a voltage ramp protocol from −89 to +61 mV, with a speed of 0.1875 mV/ms in the presence (gray) and absence (black) of 3 mm CsCl. Prepulse duration was 800 ms. D₂, Example trace shows current–voltage relationship of I_Ca during 3 mm CsCl application. D₃, Peak I_LVA amplitude: control: −15.72 ± 0.87 pA, n = 7; CsCl: −15.71 ± 1.25 pA, n = 7; p = 0.9786, paired t test. Peak I_HVA amplitude: control: −29.42 ± 3.71 pA, n = 7; CsCl: −32.09 ± 4.05 pA, n = 7; p = 0.0933, paired t test. E₁, Example current–voltage relationship of evoked I_Ca in the presence and absence of 10 μm nifedipine (Nife). E₂, Peak I_HVA amplitude: control: −37.45 ± 5.25 pA, n = 5; nifedipine: −19.73 ± 6.42 pA, n = 5; p = 0.0022, paired t test. Peak I_LVA amplitude: control: −10.83 ± 0.87 pA, n = 5; nifedipine: −10.86 ± 1.00 pA, n = 5; p = 0.9082, paired t test. F₁, Example current–voltage relationship of evoked I_Ca in the presence and absence of 1 μm TTX. F₂, Peak I_LVA amplitude: control: −12.80 ± 0.26 pA, n = 5; TTX: −12.92 ± 0.47 pA, n = 5; p = 0.7628, paired t test. Peak I_HVA amplitude: control: −34.51 ± 0.81 pA, n = 5; TTX: −33.61 ± 1.02 pA, n = 5; p = 0.2789, paired t test. G₁, Example current–voltage relationship of evoked I_Ca in the presence and absence of 10 μm ω-conotoxin GVIA (Cono). G₂, Peak I_LVA amplitude: control: −12.05 ± 1.77 pA, n = 5; Cono: −11.81 ± 2.06 pA, n = 5; p = 0.7978, paired t test. Peak I_HVA amplitude: control: −31.37 ± 2.92 pA, n = 5; Cono: −31.03 ± 3.60 pA, n = 5, p = 0.7205; paired t test. H₁, Example current–voltage relationship of evoked I_Ca in the presence and absence of (3 mm CsCl, 10 μm nifedipine, 5 μm Z944 and 100 μm nickel). H₂, Peak I_LVA amplitude (LVA), −10.72 ± 1.53 pA; peak I_HVA amplitude (HVA), −40.16 ± 5.78 pA; peak I_Ca amplitude in the presence of L-type and T-type Ca²⁺ channel blockers (Mix), −3.0 ± 0.33 pA. LVA versus mix, p = 0.005; HVA versus mix, p = 0.0028; n =5, unpaired t test.

Next, we confirmed whether other non-T-type Ca²⁺ channels contribute to I_LVA. We tested whether the hyperpolarization-activated cation current (I_h), which is responsible for shaping the photoreceptor light response (Akopian and Witkovsky, 1996), affects Ca²⁺ currents by puff application of 3 mm CsCl (Fig. 2D1,D2). We found no significant change in I_LVA and I_HVA amplitudes (Fig. 2D3), suggesting that the measured I_LVA was because of T-type Ca²⁺ channels and not hyperpolarization-activated cation channels. To inhibit the I_HVA component, we used 10 µm nifedipine, an L-type Ca²⁺ channel blocker. Nifedipine has a high affinity to L-type and a very low affinity to T-type Ca²⁺ channels (Stengel et al., 1998). Nifedipine significantly blocked the I_HVA component, but left the I_LVA component untouched (Fig. 2E1,E2). This indicates that the I_LVA component was not because of the activation of HVA L-type Ca²⁺ channels, further providing evidence for the presence of LVA T-type Ca²⁺ channels in mouse cone photoreceptors. Finally, we verified whether tetrodotoxin (TTX)-sensitive Na⁺ channels or ω-conotoxin GVIA-sensitive N-type Ca²⁺ channels influenced I_LVA or I_HVA. During puff application of 1 µm TTX (Fig. 2F1,F2) or 1 µm ω-conotoxin GVIA (Fig. 2G1,G2), we found no significant difference in I_HVA and I_LVA Ca²⁺ current amplitudes (Fig. 2F2,G2). Measuring I_Ca in the presence of a mixture of antagonistic compounds, including T-type and L-type Ca²⁺ channel blockers (10 µm nifedipine, 5 µm Z944, and 100 µm nickel additionally to 8 µm CNQX and 3 mm CsCl), resulted in a rather small residual current (Fig. 2H1,H2), which indicates a minimal nonspecific effect. In conclusion, the results from our pharmacological experiments strongly indicate the presence of T-type Ca²⁺ channels in addition to L-type Ca²⁺ channels in mouse cone photoreceptors.

Biophysiological characterization of I_LVA and I_HVA

In the next set of experiments, we measured the electrophysiological properties of I_LVA and I_HVA components. I_LVA was isolated by puff application of 8 µm CNQX, 3 mm CsCl, and 10 µm nifedipine containing extracellular solution, thereby removing any postsynaptic feedback, I_h, and I_HVA components. In this condition, I_LVA of cone photoreceptors was recorded in response to a series of voltage step stimulations (50 ms) from V_h = −69 mV (Fig. 3A). Furthermore, based on the results of our pharmacological experiments (Fig. 2), we chose 8 µm CNQX, 3 mm CsCl, and 5 µm Z944 to isolate I_HVA. In this condition, I_HVA produced typical high-voltage-activated current trajectories in response to a series of voltage steps (Fig. 3B). The average I_HVA and I_LVA amplitude relationships to V_m (I–V) in cone photoreceptors are illustrated in Figure 3C1. Relative conductance values of I_HVA and I_LVA were fitted with a Boltzmann function to estimate the voltage dependency of the activation (Fig. 3C2). As expected, the half-activation (V₅₀) of I_HVA shifted to the right by ∼10 mV compared with V₅₀ of I_LVA, but slope values were comparable to each other (Fig. 3C3). Additionally, the time course of activation and inactivation of I_LVA was measured on maximal current amplitude traces, which were chosen from the step series. Maximal I_LVA amplitudes were typically evoked by a voltage step from −69 to −41 mV (50 ms; Fig. 3D1). Fast inactivation was estimated by fitting the decay phase of the current with an exponential function (Fig. 3D1) and found to be <30 ms (Fig. 3D2). Time to I_LVA peak values stayed at ∼13 ms (Fig. 3D2). In general, the time courses of activation and inactivation kinetics of cone photoreceptor I_LVA were very similar to currents produced by other neuronal T-type Ca²⁺ channels (Klöckner et al., 1999). The time course of I_HVA inactivation was measured as the ratio of I_HVA amplitude at the beginning and the end of the current trace (Fig. 3E1). I_HVA showed an ∼15% reduction during a voltage step to −17 mV (50 ms) from V_h = −69 mV (Fig. 3E2). HVA L-type Ca²⁺ currents typically show little fast inactivation in photoreceptors (Barnes and Hille, 1989; Yagi and Macleish, 1994). To determine the steady-state inactivation properties of I_LVA and I_HVA in cone photoreceptors, currents were elicited by test pulses either to −39 mV (I_LVA) or −19 mV (I_HVA) preceded by a series of conditioning potential steps ranging from −100 to 0 mV (duration, 300 ms; Fig. 3F,G). The inactivation was calculated by dividing the test pulse-evoked current amplitudes with maximal current amplitudes (I/I_max; Fig. 3H1). Data points were fitted by a Boltzmann function. Results indicated that V₅₀ parameters of I_HVA inactivation were shifted to the right by ∼15 mV, while slope values stayed similar (Fig. 3H2). Interestingly, the activation and inactivation curves of both I_LVA and I_HVA revealed a noninactivating current window in the range of the physiological V_m (−60 to −30 mV) where channel inactivation is incomplete and thus regulates steady-state Ca²⁺ levels (Fig. 3H1).

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Figure 3.

Physiological properties of I_LVA and I_HVA. A, Representative examples of I_LVA in cone photoreceptors evoked by 50 ms voltage steps to different potentials (V_h = −62, −38, −32, −20, −8, and 52 mV) in the presence of 8 μm CNQX, 3 mm CsCl, and 10 μm nifedipine. B, Representative examples of I_HVA evoked by 50 ms voltage steps to different potentials (V_h = −62, −38, −32, −20, −8, and 52 mV) in the presence of 8 μm CNQX, 3 mm CsCl, and 5 μm Z944. C₁, Current–voltage relationship of I_LVA and I_HVA currents evoked by voltage steps from −77 to 43 mV by 6 mV steps. C₂, Voltage dependence of steady-state activation of I_LVA and I_HVA (g/g_max). Data points were fitted with the Boltzmann function (dashed lines). C₃, Mean V₅₀: I_LVA, −44.17 ± 1.98 mV; I_HVA, −34.38 ± 1.78 mV; n = 6; p = 0.0043, unpaired t test. Mean activation slope factor (slope): I_LVA, −4.77 ± 0.70; I_HVA, 4.99 ± 0.83; n = 6; p = 0.8417, unpaired t test. D₁, Typical maximal I_LVA in cone photoreceptors evoked by a voltage step from −69 to 41 mV (50 ms). The dashed red line indicates I_LVA decay fitted with an exponential function. D₂, Corresponding time to I_LVA peak, 13.18 ± 0.81 ms. Decay, tau of single exponential function: 28.82 ± 2.38 ms. E₁, Typical maximal I_HVA in cone photoreceptors evoked by a voltage step from −69 to −17 mV (50 ms). E₂, Corresponding fast inactivation ratio, 0.85 ± 0.03. F, Example traces showing the steady-state inactivation of I_LVA. A 300-ms-long conditioning pulse over a series of V_h, from −100 to −20 mV (20 mV increments), was applied before the test pulse (−39 mV, 50 ms). G, Example traces showing the steady-state inactivation of I_HVA. The 300-ms-long conditioning pulse over a series of V_h values from −100 to −20 mV (20 mV increments) was applied before the test pulse (−19 mV, 50 ms). H₁, Steady-state inactivation of I_LVA and I_HVA were calculated by normalizing the test pulse-evoked current amplitude to the maximum current amplitude and were plotted over the prepulse V_m. Data points were fitted with Boltzmann function and illustrated as solid black (I_HVA) and gray (I_LVA) lines. Dashed lines are the Boltzmann fits of the I_LVA and I_HVA activation kinetics from C₂. H₂, Mean V₅₀: I_LVA, −54.82 ± 0.81 mV; I_HVA, −40.11 ± 2.76 mV; n = 8 and n = 5; p < 0.0001, unpaired t test. Mean inactivation slope factor (slope): I_LVA, 5.06 ± 0.49; I_HVA, 5.45 ± 0.87; n = 8 and n = 5; p = 0.6773, unpaired t test.

Ca²⁺ spikes are present in cone photoreceptors

LVA T-type Ca²⁺ channels are suggested to play a crucial role in the activity of several types of neurons in the CNS by generating low-threshold spikes and activating higher-threshold ion channels, which induce a burst of firing and oscillatory behavior (Perez-Reyes et al., 1998; Perez-Reyes, 2003). To investigate the impact of T-type Ca²⁺ channels on cone photoreceptor V_m changes, we performed whole-cell current-clamp recordings. Because the Cs⁺-based and TEA-Cl-based pipette solution used in the previous experiments blocks K⁺ currents, thereby increasing the input resistance of the cells and the probability of spiking, we examined whether cone photoreceptors generate spikes in the presence of a K⁺-gluconate-based intracellular solution. In the current-clamp configuration, we first injected steady currents to keep V_m hyperpolarized (approximately −60 mV) and then depolarized cone photoreceptors stepwise by steady current injection to mimic different levels of light exposure. Figure 4A1 shows an example of the V_m changes of a cone photoreceptor in response to a current injection series from −24 to +6 pA (Δ5 pA, 1 s). When cone photoreceptor V_m was held at a hyperpolarized level of approximately −60, and approximately −50 mV by steady current injection, we did not observe any spike behavior. When V_m reached a threshold of approximately −40 mV, V_m showed a fast rise, which generated an immediate large spike (arrowhead) and additional smaller spikes with some delay (Fig. 4A1). The amplitude of a spike is classically dependent on how long the responsible ion channels are open at the rising phase. Therefore, for the higher spike peaks L-type Ca²⁺ channels could be responsible as they do not inactivate strongly (Fig. 3E). To determine the ion channel involvement in the observed spikes, we performed puff application of several pharmacological agents during the current injections described above. We could not evoke spikes during the puff application of 100 µm nickel onto the cone photoreceptor terminal (Fig. 4A2). In this condition, the V_m at the beginning of the stimuli stayed well below that seen in control conditions, and the injected current–V_m plot stayed rather linear in the range of physiological V_m (Fig. 4A3). We also tested the effect of Z944 on cone photoreceptor spike generation. The 5 µm Z944 blocked I_LVA but increased I_HVA in the previous experiment (Fig. 2C1,C2). Here, spikes could still be generated by current injections; however, peak V_m stayed slightly, but significantly, below control values (Fig. 4B1–B3). This suggests that an increased level of HVA L-type Ca²⁺ current could partially compensate for the reduced level of LVA T-type Ca²⁺ current considering spike generation. This assumption is also supported by the results of the next experiment, where the application of the L-type Ca²⁺ channel blocker nifedipine (10 µm) blunted the initial V_m rise (Fig. 4C1,C2). However, peak V_m values (Fig. 4C₃, gray trace) were less linear than what was observed in the presence of nickel (Fig. 4A3, gray trace). The slight depolarization remaining in the presence of nifedipine at more than −40 mV V_m suggests that the activation of T-type Ca²⁺ channels helps to boost the rising phase of the depolarization to then activate L-type Ca²⁺ channels. Next, we tested for the possible role of Na⁺ channels in spike generation. During puff application of 1 µm TTX, the depolarization evoked by a series of current injections was not altered (Fig. 4D1–D3). Additionally, by puffing 3 mm CsCl, we also found that I_h did not significantly influence cone photoreceptor spiking behavior (Fig. 4E1–E3). Altogether, the experiments indicate that the spiking behavior in cone photoreceptors at depolarized V_m is because of the cooperative activation of both T-type and L-type Ca²⁺ channels.

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Figure 4.

Ca²⁺ spikes present at cone photoreceptors. A₁, Example of cone photoreceptor V_m change to current injections (6, 1, −4, −9, −14, and −24 pA) in current-clamp mode using K⁺-gluconate-based intracellular pipette solution. Arrowhead labels the initial fast rise of the V_m (spike) at the beginning of the stimulus. A₂, Example trace of cone photoreceptor V_m change to the same level of current injections in the presence of 100 μm nickel (Ni²⁺). A₃, V_m amplitude versus stimulus intensity plot. Significance was tested with a paired t test among each stimulus intensity (−22 pA, p = 0.7346; −18 pA, p = 0.1548; −14 pA, p = 0.2918; −10 pA, p = 0.3324; −6 pA, p = 0.0389; −2 pA, p < 0.0001; 2 pA, p < 0.0001; 6 pA, p = 0.2151; n = 6). B₁, B₂, Example traces of cone photoreceptor V_m change to current pulses (0, −5, −10, −15, −20, and −25 pA), in the absence (control, B₁) and presence (gray, B₂) of 5 μm Z944. B₃, V_m amplitude versus stimulus intensity plot. Significance was tested with a paired t test in each stimulus intensity (−25 pA, p = 0.9846; −20 pA, p = 0.9583; −15 pA, p = 0.9467; −10 pA, p = 0.1408; −5 pA, p = 0.3731; 0 pA, p = 0.0485; 5 pA, p = 0.0498; 10 pA, p = 0.2916; n = 7). C₁, C₂, Example traces of cone photoreceptor V_m change to current pulses (2, −4, −10, −16, and −22 pA), in the absence (control; C₁) and presence (Nife; gray; C₂) of 10 μm nifedipine. C₃, V_m amplitude versus stimulus intensity plot. Significance was tested with a paired t test in each stimulus intensity (−35 pA, p = 0.1143; −30 pA, p = 0.1352; −25 pA, p = 0.1362; −20 pA, p = 0.9151; −15 pA, p = 0.5775; −10 pA, p = 0.0150; −5 pA, p = 0.0199; 0 pA, p = 0.050; n = 6). D₁, D₂, Example traces of cone photoreceptor V_m change to current pulses (0, −8, −12, −16, and −24 pA), in the absence (control; D₁) and presence (TTX; gray; D₂) of 1 μm tetrodotoxin. D₃, V_m amplitude versus stimulus intensity plot. Significance was tested with paired t test in each stimulus intensity (−25 pA, p = 0.296; −20 pA, p = 0.1322; −15 pA, p = 0.2485; −10 pA, p = 0.1641; −5 pA, p = 0.6813; 0 pA, p = 0.1703; 5 pA, p = 0.3681; n = 4). E₁, E₂, Example traces of cone photoreceptor V_m change to current pulses (2, −4, −10, −13, and −19 pA), in the absence (control; E₁) and presence (Cs⁺; gray; E₂) of 1 μm CsCl. E₃, V_m amplitude versus stimulus intensity plot. Significance was tested with paired t test in each stimulus intensity (−25 pA, p = 0.2575; −20 pA, p = 0.2498; −15 pA, p = 0.2172; −10 pA, p = 0.1939; −5 pA, p = 0.1366; 0 pA, p = 0.0712; 5 pA, p = 0.0512; n = 5).

Self-generating spike behavior at depolarized V_m

In the next set of experiments, we asked whether cone photoreceptors generate spikes spontaneously at dark V_m. Therefore, we injected a steady current for 10 s in the current-clamp configuration to keep the V_m of cone photoreceptors close to −40 mV, a typical dark V_m. As in the previous experiment, we used K⁺-gluconate-based intracellular pipette solution to keep input resistance more physiological. Remarkably, in this condition, cone photoreceptors repeatedly generated spikes. Figure 5A1 shows a representative example of cone photoreceptor spiking behavior when cone photoreceptor V_m was adjusted to approximately −40 mV by injecting −3 pA current. Puff application of 100 µm nickel onto the cone photoreceptor terminal blocked spikes completely in seven of seven cells (Fig. 5A2), and the effect could be washed out after a few minutes. The application of 10 µm nifedipine similarly blocked spike activity in six of six cells (Fig. 5B2), and the effect could be washed out after several minutes. Additionally, we tested the effect of 5 µm Z944 on spontaneous spike generation in cone photoreceptors. In this condition, spikes could still be observed with a frequency similar to control conditions (Fig. 5C1–C3), but the peak amplitude and the half-width of the spikes were significantly altered (Fig. 5C3). These results fit well with the examined effect of Z944 on I_LVA and I_HVA (Fig. 2). In summary, the effect of Z944 on spike kinetics and amplitude and the elimination of spontaneous spikes by nickel and nifedipine indicate that both L-type and T-type Ca²⁺ channels are responsible for the spontaneous spike generation in cone photoreceptors. Because spikes are typically associated with Na⁺ channels, we also examined whether 1 µm TTX inhibits spike generation. We found no significant difference in spike frequency (p = 0.4712, n = 4) and peak amplitude (p = 0.3820, n = 4) compared with control conditions. Examination of spontaneous spike behavior in the presence of 3 mm CsCl also showed no significant changes in spike frequency and peak amplitude (p = 0.2641, p = 0.2131, respectively, n = 4), suggesting no role of I_h in spike generation. In I = 0 recording mode, cone photoreceptors showed an average V_m of −37.01 ± −4.23 mV (n =11). At this potential, cone photoreceptors also consistently generated spontaneous spikes. In general, the analysis of control spikes indicated an average frequency of ∼1.5 Hz, a peak amplitude of approximately −13.5 mV, a baseline of approximately −40.5 mV, and a half-width of ∼32 ms (Fig. 5D). These parameters are different from Na⁺ action potential parameters measured in a typical mammalian central neuron (Bean, 2007). To further analyze the kinetics of the spontaneous spike events in cone photoreceptors, we picked representative, individual spikes from the recordings at depolarized V_m (approximately −40 mV), as illustrated in Figure 5E1. Initially, we created trajectory plots of spike rate of change (Fig. 5E2), and we measured the rise rate of V_m. We determined the spike threshold as 7.5% of the maximal rise rate at 0.22 mV/ms (Fig. 5E3, gray horizontal line). This value yielded an average of −36.2 ± 1.05 mV (Fig. 5E4) for the activation threshold of spontaneous spikes (gray horizontal line), which is very close to the typical dark V_m (Ingram et al., 2020). We found a maximal rise rate of ∼3.1 mV/ms (Fig. 5E4). This value is 50–150 times slower than was measured for Na⁺ action potentials (Hodgkin and Huxley, 1952), further strengthening the finding that spikes in cone photoreceptors are not generated by Na⁺ channels. Altogether, these experiments showed that mouse cone photoreceptors are able to develop self-generating spike activity around the dark V_m. A plausible explanation for this oscillatory behavior in spike generation is the periodic activation and inactivation of LVA T-type Ca²⁺ channels, which further activate L-type Ca²⁺ channels (Perez-Reyes, 2003).

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Figure 5.

Spontaneous spike activity at dark V_m. A₁–C₂, Example V_m changes in control condition and during 100 μm nickel (Ni²⁺), 10 μm nifedipine (Nife), and 5 μm Z944 application. C₃, Peak V_m of spontaneous spike events: control, −13.3 ± 2.50 mV; Z944, −19.45 ± 3.13 mV; n = 7; p = 0.0337, paired t test. Spontaneous spike frequency: control, 1.45 ± 0.38 Hz; Z944, 1.1 ± 0.32 Hz; n = 7; p = 0.2462, paired t test. Half-width: control, 38.0 ± 2.50 ms; Z944, 50.68 ± 3.13 ms; p = 0.0145, paired t test; n = 7. D, Summary of spike parameters generated in control condition. Frequency, 1.47 ± 0.23; peak V_m, −13.56 ± 1.72 mV; baseline V_m, −40. 57 ± 0.50 mV; half-width, 31.96 ± 1.72 ms. E₁, Example of a spontaneous spike in cone photoreceptors. E₂, Time derivative of the V_m of the trace in E₁ plotted against V_m. E₃, Zoomed in region of the rising phase from E₂. Dashed gray line, Exponential fit to estimate the average of the trace; straight gray lines, 7.5% of the maximal rise rate reveals the V_m threshold of the spike. E₄, V_m at 0.22 dV/dt: −36.17 ± 0.29 mV; n = 14; maximal V_m rise rate: 3.17 ± 0.24 mV/ms; n = 14. F₁, Example V_m changes at near body temperature (33°C). F₂, Frequency of spikes, 1.05 ± 0.17 Hz; peak V_m, −10.65 ± 2.05 mV; baseline, −35.52 ± 1.1 mV; half-width, 27.77 ± 4.3 ms. Room temperature versus 33°C: frequency, p = 0.2610; peak V_m, p = 0.3458; baseline, p = 0.0002; half-width, p = 0.4844, unpaired t test; n = 12 and n = 5, respectively. G₁, Example trace showing V_m changes evoked by full-field light flash (300 ms, ∼130 W/cm² irradiance) in dark-adapted cone photoreceptor. G₂, V_m before light stimulation (Dark V_m), −39.65 ± 0.99 mV; light response evoked V_m hyperpolarization (V_min), −53.96 ± 2.46 mV; offset of the light response (V_max), −3.61 ± 4.08 mV.

Another important question is whether these Ca²⁺ spikes have physiological relevance. Therefore, we investigated spontaneous spike behavior near body temperature (33°C; Fig. 5F1,F2). Spontaneous spikes were also evoked at higher temperature by current injection values that were used during the experiments in room temperature (room temperature, −1.41 ± 0.74 pA; 33°C, −2.8 ± 1.02 pA; p = 0.1349, n = 12 and n = 5, respectively, unpaired t test). We also tested whether spikes could be generated by natural stimuli. We performed current-clamp (I = 0) experiments in dark-adapted retinae using dim red light during slicing and recording. Full-field light flashes (irradiance, ∼130 W/cm²) evoked a typical light response in cone photoreceptors and an additional spike at light offset (Fig. 5G1). Cone photoreceptors initially stayed at V_m = approximately −39 mV (Fig. 5G1, Dark V_m), then the light flash induced a fast hyperpolarization to approximately −54 mV (Fig. 5G2, V_min). Spikes evoked by light offset reached a peak approximately V_m = 0 mV (V_max), as observed for spikes generated by current injections (Fig. 4, controls). These experiments illustrate that mouse cone photoreceptors generate Ca²⁺ spikes in dark and at light offset. Therefore, cone photoreceptors seem to process light information by both gradual V_m change and spike generation.

Spike activity increases intracellular Ca²⁺ levels in cone photoreceptor terminals

We showed that cone photoreceptors are able to generate spikes in response to a V_m jump and spontaneously at depolarized V_m and provided pharmacological evidence that spikes were produced by the activation of voltage-sensitive T-type and L-type Ca²⁺ channels. Here, we wanted to further investigate whether spikes stimulate the influx of Ca²⁺ into the cone photoreceptor terminal. Therefore, to detect spatially averaged intracellular Ca²⁺ changes, we used 100 µm Fluo-4 Ca²⁺ indicator (Thermo Fisher Scientific) in the pipette solution. Fluo-4 has been shown to be sufficiently sensitive (K_d = 345 nm) to detect low concentrations of free intracellular Ca²⁺ (Gee et al., 2000). In the experiments, we targeted cone photoreceptor terminals in the horizontal retina slice preparation with the patch electrodes and measured Ca²⁺ signals parallel to current-clamp measurements. First, we injected negative currents to keep the cells at hyperpolarized V_m, then we injected positive currents to depolarize the cells to a level that is close to the threshold of spike generation (approximately −40 mV). Figure 6A shows an example of a cone photoreceptor held at approximately −80 mV by injecting −13 pA current followed by a V_m step to more positive values for 5 s by injecting −4 pA current. In this example, the cone photoreceptor showed spikes at the start of the stimulation but shortly after (∼2 s), V_m remained steady at approximately −50 mV without spikes (Fig. 6A, black trace). The V_m of −50 mV is more negative than the activation threshold of the T-type Ca²⁺ channels (approximately −40 mV). Accordingly, intracellular Ca²⁺ concentration (ΔF/F₀) rose at the start of the stimulus and then declined slowly to baseline values. This indicates that the current injection activated voltage-sensitive Ca²⁺ channels at the beginning of the stimulus, which resulted in an intracellular Ca²⁺ rise in the terminal, but because of the absence of further spike generation at −50 mV, Ca²⁺ was slowly removed from the terminal. In the next example, V_m was changed to a more depolarized level by a current step to −2 pA (Fig. 6B). During the presence of this depolarizing current, V_m of the cone photoreceptor showed continuous spike activity at ∼1 Hz, and Ca²⁺ signals were elevated to a steady level. Additionally, because of the spike activity, a clear concurrent rise of the Ca²⁺ signal was observed (Fig. 6B, arrows). The relationship between spikes and intracellular Ca²⁺ concentration was analyzed by measuring the linear correlation between spike timing and Ca²⁺ signal peaks (Fig. 6C). We found a significant correlation (p < 0.0001, R² = 0.9992), indicating that spikes were generated by Ca²⁺. We found an average ΔF/F₀ of 1, and the Ca²⁺ signal peak could be detected ∼30 ms after spike peaks (Fig. 6D). Altogether, these experiments show that during self-generating spike activity at around dark V_m, the intracellular Ca²⁺ concentration rises from baseline level and stays at a steady high level, while individual spikes can further increase Ca²⁺ levels temporarily in cone photoreceptor terminals. Additionally, the local Ca²⁺ concentration rise suggests that both T-type and L-type Ca²⁺ channels localize at the cone photoreceptor terminal membrane.

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Figure 6.

Spikes cause intracellular Ca²⁺ rise in cone photoreceptor terminals. A, Example of simultaneous current-clamp recording (from −13 to −4 pA, 5 s) and Ca²⁺ imaging with 100 μm Fluo-4 in the cone photoreceptor terminal. Pictures in the top panel were taken from the patched cone photoreceptor terminal at the time points labeled with arrows (t₁–t₃). B, Example of simultaneous current-clamp recording and Ca²⁺ imaging during stronger stimulation (from −13 to −2 pA, 5 s). Arrows indicate Ca²⁺ rise because of the individual spike events. C, Linear relationships between the timing of spike and Ca²⁺ signal peak (p < 0.0001, R² = 0.9992). D, Relative Ca²⁺ change during spikes (ΔF/F₀), 1.08 ± 0.33; Δt between spike peak amplitude and Ca²⁺ signal peak (Δt), 30.58 ± 8.36 ms.

Both LVA and HVA Ca²⁺ channels contribute to synaptic vesicle release

To investigate the role of LVA T-type Ca²⁺ channels in synaptic vesicle release, first, we took advantage of the biophysical properties of the channels. At depolarized V_m (−41 mV), I_LVA showed partial (∼46%) inactivation, but I_HVA inactivation was only ∼15% (Fig. 3). We therefore, compared synaptic vesicle release evoked by a test stimulus (−19 mV, 25 ms) preceded by an ∼15-s-long, either hyperpolarized (V_h = −69 mV; Fig. 7A1, black) or depolarized (V_h = −39 mV, Fig. 7A1, gray) potential. Evoked synaptic vesicle release in cone photoreceptors was monitored by C_m measurements with the Sine + DC method (HEKA 10 Amplifier, HEKA Elektronik). We found that I_Ca charge (QI_Ca) was ∼1.5 pC in the presence of LVA and HVA Ca²⁺ channels (V_m = −69 mV). When V_m was held at −39 mV, QI_Ca decreased to ∼0.8 pC, significantly diminishing evoked synaptic vesicle release (Fig. 7A2). The number of released synaptic vesicles in cone photoreceptors was estimated by taking into account the single synaptic vesicle capacitance contribution, which has a typical value of 43.7 aF in mouse photoreceptors based on a 37.3 nm synaptic vesicle diameter (Fuchs et al., 2014). This yields ∼1730 synaptic vesicles at V_h = −69 mV and ∼1030 synaptic vesicles at V_h = −39 mV; thus, ∼41% fewer synaptic vesicles were released by the inactivation of LVA T-type Ca²⁺ channels. This result indicates that LVA T-type Ca²⁺ channels contribute to Ca²⁺-dependent synaptic vesicle release at cone photoreceptors. Next, we examined the contribution of HVA L-type Ca²⁺ channels to evoked synaptic vesicle release by the application of 10 µm nifedipine that reduced I_HVA by ∼68% and left I_LVA unaltered (Fig. 2E1,E2). Synaptic vesicle release was triggered by a test pulse (−19 mV, 25 ms) from V_h = −69 mV (Fig. 7B1), and nifedipine was applied with a pressure-controlled puffing system. QI_Ca showed a significant, ∼49%, reduction in the presence of 10 µm nifedipine, but evoked synaptic vesicle release was only reduced by ∼16% (Fig. 7B2). This result indicates that LVA T-type Ca²⁺ channels contribute more to synaptic vesicle release than HVA L-type Ca²⁺ channels (Fig. 7A1) and therefore have a boosting effect on synaptic vesicle release evoked by a strong stimulus (−19 mV, 25 ms) at mouse cone photoreceptors. As this is an unexpected finding, we further examined the effect of LVA T-type Ca²⁺ channels on synaptic vesicle release at cone photoreceptors. In the following experiments, we included 10 mm bis(2-aminophenoxy)ethane-N,N,N′,N′-tetra-acetic acid (BAPTA) in the pipette solution. BAPTA is a Ca²⁺ buffer with a faster on-rate than EGTA has, intercepting Ca²⁺ influx at the active zone via Ca²⁺ channels to reveal nanodomain or microdomain organization (Adler et al., 1991). The presence of BAPTA in the pipette did not change QI_Ca compared with EGTA (5 mm EGTA; Fig. 7B, black: QI_Ca = 1.16 ± 0.19 pC, n = 6; 10 mm BAPTA; Fig. 7C, black: QI_Ca = 1.28 ± 0.10 pC, n = 5, p = 0.6236, unpaired t test). However, 10 mm BAPTA caused a significant reduction in ΔC_m (5 mm EGTA, Fig. 7B, black: 90.00 ± 10.73 pC, n = 6; 10 mm BAPTA, Fig. 7C, black: 35.16 ± 5.18 pC, n = 5, p = 0.0018, unpaired t test). The ∼61% reduction of C_m indicates that most of the evoked synaptic vesicle release was triggered by Ca²⁺ influx through nanodomain-organized Ca²⁺ channels. Moreover, 39% of the evoked synaptic vesicle release in cone photoreceptors is either not sensitive to BAPTA or is Ca²⁺ independent. Nifedipine application, in the presence of BAPTA, caused a reduction of QI_Ca (Fig. 7C2) similar to what we observed with the EGTA-containing intracellular solution (Fig. 7B2). However, ΔC_m values decreased by ∼48% in the presence of the fast Ca²⁺ buffer (Fig. 7C2), which exceeded the degree of reduction that we measured in the presence of 5 mm EGTA (∼16%; Fig. 7B2). The stronger nifedipine sensitivity of the release in the presence of BAPTA indicates that most of the HVA L-type Ca²⁺ channels are organized into nanodomains at the active zone (Bartoletti et al., 2011). Moreover, the smaller nifedipine sensitivity in the presence of EGTA (Fig. 7B,C, gray) suggests that LVA T-type Ca²⁺ channels provide an additional Ca²⁺ source to trigger and boost synaptic vesicle release and that they locate close to the release sites. The mild stimuli evoked QI_Ca in the presence of 5 mm EGTA and 10 mm BAPTA reached similar values, but the synaptic vesicle release was significantly different (Fig. 7D). The reduced ΔC_m levels with BAPTA and the maintained ΔC_m levels with EGTA further strengthen the finding that LVA T-type channels are present close to the release sites and are able to boost synaptic vesicle release. To further examine the boosting effect of LVA T-type Ca²⁺ channels, we measured cone photoreceptor synaptic vesicle release in response to mild (−39 mV, 25 ms) and strong (−19 mV, 25 ms) depolarizing steps. Stimuli were applied pairwise to the same cone photoreceptor using a pipette solution containing either 5 mm EGTA or 10 mm BAPTA (Fig. 7E,F). At V_m = approximately −39 mV, the LVA T-type Ca²⁺ current has a maximal amplitude, while HVA L-type Ca²⁺ channels are activated only slightly (Figs. 1, 3). Consequently, mild stimulation should activate mainly LVA T-type Ca²⁺ channels, and strong stimulation should activate both Ca²⁺ channel types. We found that a mild stimulus significantly diminished QI_Ca compared with the strong stimulus in both Ca²⁺ buffering conditions (Fig. 7E,F). However, in the presence of 5 mm EGTA, the mild stimulus reduced synaptic vesicle release by only ∼12% (Fig. 7E), which is consistent with the result shown in Figure 7B, where mainly LVA T-type Ca²⁺ channels dominated evoked synaptic vesicle release. This shows that LVA T-type Ca²⁺ channels are capable of triggering synaptic vesicle release with high efficiency (∼88%) when photoreceptors are quickly depolarized from the light to the dark membrane potential (−39 mV). On the other hand, the comparison of mild and strong depolarization steps in the presence of 10 mm BAPTA revealed an ∼70% difference in evoked synaptic vesicle release (Fig. 7F). This demonstrates that LVA T-type Ca²⁺ channels are less effective in boosting synaptic vesicle release when the fast Ca²⁺ buffer is present; hence, they likely locate close to HVA L-type Ca²⁺ channels.

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Figure 7.

LVA Ca²⁺ channels boost synaptic vesicle release. A₁, Black, Example traces showing I_Ca and C_m induced by a voltage step (from −69 to −19 mV, 25 ms) at cone photoreceptors. Stim, Stimulus; G_m, membrane conductance; G_s, series conductance; gray, I_Ca and C_m induced by a voltage step (from −39 to −19 mV, 25 ms). Labeling similar to black traces. A₂, QI_Ca: V_h = −69 mV: 1.35 ± 0.19 pC; V_h = −39 mV: 0.83 ± 0.06 pC; n = 7; p = 0.0003, paired t test; C_m: V_h = −69 mV: 75.62 ± 4.69 fF; V_h = −39 mV: 45.02 ± 2.90 fC; n = 7; p = 0.0003, paired t test. B₁, Black, Example traces of I_Ca and C_m jump induced by voltage stimulus (from −69 to −19 mV, 25 ms), Patch pipette contained 5 mm EGTA. Gray, Same as black but in the presence of 10 μm nifedipine. B₂, QI_Ca: control, 1.16 ± 0.19 pC; Nife, 0.59 ± 0.09 pC; n = 6; p = 0.0047, paired t test; C_m: control, 90.99 ± 10.73 fF; Nife, 76.23 ± 9.52 fF; n = 6; p = 0.0007, paired t test. C₁, Black, Example traces of I_Ca and C_m change induced by a voltage step (from −69 to −19 mV, 25 ms). Pipette contained 10 mm BAPTA. Gray, Same as black but in the presence of 10 μm nifedipine. C₂, QI_Ca: control: 1.28 ± 0.10 pC, n = 5; Nife: 0.74 ± 0.04 pC, n = 5; p = 0.0021, paired t test; C_m: control: 35.16 ± 5.18 fF, n = 5; Nife: 18.45 ± 2.23 fF, n = 5; p = 0.0063, paired t test. D₁, Black, Example traces of I_Ca and C_m jump induced by a voltage step (from −69 to −39 mV, 25 ms). Pipette contained 5 mm EGTA. Gray, Same as black but with 10 mm BAPTA-containing intracellular solution. D₂, QI_Ca: 5 mm EGTA, 0.36 ± 0.04 pC; 10 mm BAPTA, 0.34 ± 0.03 pC; n = 5 and n = 4; p = 0.7302, unpaired t test; C_m: 5 mm EGTA, 83.68 ± 13.76 fF; 10 mm BAPTA, 16.61 ± 3.99 fF; n = 5 and n = 4; p = 0.0159, unpaired t test. E, Paired comparison of (−19 mV, 25 ms; strong) and mild (−39 mV, 25 ms; mild) stimulus evoked QI_Ca with 5 mm EGTA containing intracellular solution. Mild, QI_Ca = 0.36 ± 0.04 pC; strong, QI_Ca = 1.09 ± 0.1 pC; n = 5; p = 0.0005, paired t test. Mild, ΔC_m = 83.68 ± 13.76 fF; strong, ΔC_m = 95.24 + 12.42 fF; n = 5; p = 0.0021, paired t test. F, Paired comparison of strong (−19 mV, 25 ms) and mild (−39 mV, 25 ms) stimulus-evoked QI_Ca with 10 mm BAPTA containing intracellular solution. Mild: QI_Ca = 0.33 ± 0.03 pC; strong: QI_Ca = 1.12 ± 0.04 pC; n = 4; p = 0.0005, paired t test; mild: ΔC_m = 16.61 ± 3.99 fF; p = 0.0005; strong: ΔC_m = 55.97 ± 9.44 fF; n = 4; p = 0.01, paired t test.

Cone photoreceptors express Ca_v3.2 channel mRNA

To investigate which LVA T-type Ca²⁺ channels are present in cone photoreceptors, we performed RT-PCR experiments on sorted rod and cone photoreceptors from dissociated retinae of Rac3-eGFP mice, which express eGFP in cone photoreceptors (Gong et al., 2003; Fuchs et al., 2014). For fluorescence-activated cell sorting, eGFP fluorescence and FSC/SSC were used to sort cone and rod photoreceptors, respectively (Fig. 8A1,A2). The unlabeled sorting of rod photoreceptors by FSC/SSC is possible because of the high backscatter of heterochromatin in adult rod photoreceptor somata (Solovei et al., 2009; Feodorova et al., 2015). This method was optimized and validated by the use of CD73, a cell surface marker of cone/rod photoreceptor common precursors and mature rod photoreceptors (Koso et al., 2009). We performed nested RT-PCR with primers that have been validated and successfully deployed on mouse islet cDNA (Vignali et al., 2006). The results of the nested RT-PCR of sorted photoreceptors indicate that cone photoreceptors express Ca_v3.2, whereas rod photoreceptors do not express any LVA T-type Ca²⁺ channel (Fig. 8B1). To verify our sorting strategy, we also checked for the expression of β-actin (Actb), the cone photoreceptor marker short-wave-sensitive opsin 1 (Opn1sw), and the rod photoreceptor marker rhodopsin (Rho; Fig. 8B2). While Actb was present in all analyzed samples, Opn1sw was enriched in cone photoreceptors and Rho in rod photoreceptors, corroborating our sorting strategy (Fig. 8B2). On whole-retina cDNA, application of the primers led to specific bands for all three LVA T-type Ca²⁺ channels Ca_v3.1 (271 bp), Ca_v3.2 (305 bp), and Ca_v3.3 (258 bp; Fig. 8C1), as well as for the markers Actb (196 bp), Opn1sw (175 bp), and Rho (81 bp; Fig. 8C2). No bands were detectable in the negative control, in which reverse transcriptase was omitted during cDNA synthesis of whole-retina mRNA, to exclude amplification of genomic DNA (Fig. 8C1,C2). This confirms the nested RT-PCR results and demonstrates the mRNA expression of Ca_v3.2 in cone photoreceptors. Furthermore, data of previous work using “Drop-Seq” analysis of individual cells also suggests the presence of Ca_v3.2 mRNA in cone photoreceptors (Macosko et al., 2015).

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Figure 8.

Analysis of T-type Ca²⁺ channels in sorted photoreceptors. A₁, FACS strategy for isolating rod photoreceptors by FSC/SSC. A₂, FACS strategy for isolating cone photoreceptors by GFP fluorescence of Rac3-eGFP mice. B₁, Nested RT-PCR analysis of sorted cone and rod photoreceptor samples with LVA-specific primer pairs for Ca_v3.1, Ca_v3.2, and Ca_v3.3. A distinct band is present for the cone photoreceptor sample using Ca_v3.2 primers, whereas no bands can be observed for the sorted rod photoreceptor population. B₂, Conventional RT-PCR with primers for Rho, Opn1sw, and Actb on sorted rod and cone photoreceptors. Strong bands are visible for Opn1sw and Rho in cone and rod photoreceptor samples, respectively, as well as for Actb. C₁, C₂, RT-PCR analysis of whole-retina and negative control samples, in which reverse transcriptase was omitted during cDNA synthesis (retina, –RT). For whole retina, the following expected bands are present: Ca_v3.1 (271 bp), Ca_v3.2 (305 bp), Ca_v3.3 (258 bp), Rho (81 bp), Opn1sw (175 bp), and Actb (196 bp). No bands are visible for retina –RT samples.