Modulation of Ca2+-activated K+ currents and Ca2+-dependent action potentials by exocytosis in goldfish bipolar cell terminals

  1. Mary J. Palmer1
  1. 1Neuroscience Group, Institute for Science and Technology in Medicine, Keele University, Keele ST5 5BG, UK
  1. Corresponding author M. J. Palmer: Huxley Building, School of Life Sciences, Keele University, Keele ST5 5BG, UK. Email: m.j.palmer{at}cns.keele.ac.uk
 
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Abstract

Retinal bipolar cells convey light-evoked potentials from photoreceptors to ganglion cells and mediate the initial stages of visual signal processing. They do not fire Na+-dependent action potentials (APs) but the Mb1 class of goldfish bipolar cell exhibits Ca2+-dependent APs and regenerative potentials that originate in the axon terminal. I have examined the properties of Ca2+-dependent APs in isolated bipolar-cell terminals in goldfish retinal slices. All recorded terminals fired spontaneous or evoked APs at frequencies of up to 15 Hz. When an AP waveform was used as a voltage stimulus, exocytosis was evoked by single APs, maintained throughout AP trains and modulated by AP frequency. Furthermore, feedback inhibition of the Ca2+ current (ICa) by released vesicular protons reduced depression of exocytosis during AP trains. In the absence of K+ current inhibition, step depolarizations and AP waveforms evoked a rapidly activated outward current that was dependent on Ca2+ influx (IK(Ca)). I therefore investigated whether proton-mediated feedback inhibition of ICa affected the activation of IK(Ca). A transient inhibition of IK(Ca) was observed that was dependent on exocytosis, blocked by high-pH extracellular buffer, of similar magnitude to inhibition of ICa but occurred with a delay of 2.7 ms. In addition, the amplitude of APs evoked under current clamp was inhibited by the action of vesicular protons released by the APs. Protons released via exocytosis may therefore be a significant modulator of Ca2+-dependent currents and regenerative potentials in bipolar-cell terminals.

Retinal bipolar cells are generally regarded as non-spiking interneurons that use graded changes in membrane potential to convey light responses from photoreceptors to amacrine cells and ganglion cells (Werblin & Dowling, 1969; Saito et al. 1979; Ashmore & Falk, 1980). However, it has been shown that a class of bipolar cell in goldfish retina (Mb1 cells) can respond to light with Ca2+-dependent action potentials (APs), which are generated in the large axon terminal (Protti et al. 2000). It is postulated that Ca2+-dependent APs are important for speeding up and amplifying bipolar-cell light responses (Zenisek & Matthews, 1998; Protti et al. 2000), but little is known about their properties or ability to evoke neurotransmitter release.

Ca2+-dependent regenerative potentials have been recorded directly from the axon terminals of dissociated bipolar cells (Burrone & Lagnado, 1997; Zenisek & Matthews, 1998) but they exhibit a much longer and more variable duration than Ca2+-dependent APs in bipolar cells in retinal slices (Protti et al. 2000). Ca2+-dependent APs also occur in rod photoreceptors (Xu et al. 2005) and immature mouse inner hair cells (Kros et al. 1998; Beutner & Moser, 2001), which are similar to bipolar cells in that exocytosis occurs at ribbon-type synapses (Prescott & Zenisek, 2005). Ribbon synapses support both a fast, phasic mode of release and slower tonic release during prolonged depolarizations (Mennerick & Matthews, 1996; Sakaba et al. 1997b; von Gersdorff et al. 1998; Rouze & Schwartz, 1998; Neves & Lagnado, 1999). In inner hair cells, exocytosis was observed as an increase in membrane capacitance in response to single APs (Beutner & Moser, 2001; Marcotti et al. 2003).

APs and regenerative potentials in bipolar-cell terminals, rod photoreceptors and inner hair cells are dependent on L-type Ca2+ channels and repolarization is likely to involve Ca2+-activated K+ channels (Burrone & Lagnado, 1997; Zenisek & Matthews, 1998; Protti et al. 2000; Marcotti et al. 2003, 2004; Xu et al. 2005). Large-conductance Ca2+-activated K+ (BK) channels have been localized to goldfish bipolar-cell terminals, where they occur in close association with Ca2+ channels and are activated rapidly by micromolar increases in [Ca2+]i (Sakaba et al. 1997a; Llobet et al. 2003). It has been shown recently that Ca2+ currents (ICa) at ribbon synapses are inhibited by vesicular protons released during exocytosis (DeVries, 2001; Palmer et al. 2003a). This is known to affect subsequent exocytosis from bipolar-cell terminals (Palmer et al. 2003a), but additional consequences have not been explored. It is possible that proton-mediated inhibition of ICa causes reduced activation of BK channels under physiological conditions. Released vesicular protons, therefore, have the potential to modulate bipolar-cell terminal APs via their effects on both ICa and Ca2+-activated K+ currents (IK(Ca)).

In order to further investigate Ca2+-dependent APs in bipolar-cell terminals, I have made recordings from isolated bipolar-cell terminals in goldfish retinal slices (Palmer et al. 2003b). This method avoids treatment of the retinal tissue with dissociating enzymes such as papain, which has been shown to affect the properties of IK(Ca) in hair cells (Armstrong & Roberts, 1998, 2001) and may explain the discrepancy in regenerative responses between dissociated and intact bipolar-cell preparations. I find that isolated terminals in retinal slices reliably fire spontaneous and evoked Ca2+-dependent APs with stereotyped waveforms at rates of up to 15 Hz. Exocytosis is evoked by single APs, is maintained throughout AP trains, and is sensitive to AP frequency. Depression of release during AP trains is reduced by proton-mediated feedback inhibition of ICa. In addition, I demonstrate that both IK(Ca) and AP amplitude are inhibited by released vesicular protons in bipolar-cell terminals.

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Methods

Retinal slice preparation

The experiments conformed with the guidelines laid down by the animal welfare committee of Keele University. Retinal slices were prepared from goldfish (Carassius auratus; 8–14 cm) after dark-adaptation for 1 h. Goldfish were killed by decapitation followed immediately by destruction of the brain and spinal cord. The eyeballs were removed and retinae dissected out and treated for 20 min with hyaluronidase to remove vitreous humor. Each retina was cut into four pieces, placed ganglion-cell layer down on filter paper and kept at 4°C, until needed, in medium containing (mm): NaCl 127, KCl 2.5, MgCl2 1.0, CaCl2 1.0, Hepes 5 and glucose 12 (∼260 mosmol l−1); pH was adjusted to 7.45 with NaOH. Vertical slices were cut at 250-μm intervals using a Narishige slicer (ST-20, Tokyo, Japan). Slices were transferred to the recording chamber and continuously perfused (1 ml min−1) with medium containing (mm): NaCl 108, KCl 2.5, MgCl2 1.0, CaCl2 2.5, NaHCO3 24 and glucose 12 (pH 7.4, ∼260 mosmol l−1); the solution was continuously gassed with 95% O2–5% CO2. For experiments requiring a higher concentration of extracellular pH buffer, 24 mm Hepes was added and the concentration of NaCl reduced to 84 mm to maintain osmolarity; pH was set to 7.4 with NaOH. Drugs were bath applied in the perfusing medium. Picrotoxin and nifedipine were obtained from Tocris (Bristol, UK). All other chemicals and salts were obtained from Sigma-Aldrich (Gillingham, UK). Slice preparation and recordings were performed at room temperature (20–23°C), in daylight conditions.

Identification of bipolar-cell terminals

Slices were viewed with visible light optics through a 40 × water-immersion objective and 1.6 × zoom (Zeiss Axioskop 2, Gottingen, Germany), and a CCD camera (Hitachi, Tokyo, Japan). Bipolar-cell terminals were identified by their size, shape and position in the slice, as well as depolarization-evoked ICa and membrane capacitance responses (Palmer et al. 2003b). The recorded terminals are likely to belong to the Mb1 class of goldfish bipolar cell, which are on-center cells with predominantly rod input and are characterized by a large bulbous axon terminal (Sherry & Yazulla, 1993). A subset of terminals were isolated as a result of severing of the bipolar-cell axon during the slicing procedure; this was determined from the capacitative current response to a −10-mV step from −60 mV (Palmer et al. 2003b). Only isolated terminals were used for this study.

Bipolar-cell terminal recordings

Whole-cell recordings were obtained using patch pipettes (5–8 MΩ) pulled from borosilicate glass (World Precision Instruments, Sarasota, FL, USA) using a Sutter puller (P- 2000, Novato, CA, USA). Pipettes were coated with dental wax to reduce their capacitance and filled with one of two intracellular solutions. The first contained (mm): potassium gluconate 106, Hepes 25, KCl 10, MgCl2 4.6, Mg-ATP 3, Na-GTP 0.5 and EGTA 0.5 (pH 7.2, ∼270 mosmol l−1). The second contained (mm): caesium methane sulphonate 115, Hepes 25, TEA-Cl 10, Mg-ATP 3, Na-GTP 0.5 and EGTA 0.5 (pH 7.2, ∼270 mosmol l−1). Series resistance was typically 10–15 MΩ and leak current was less than 20 pA at a holding potential (Vh) of −60 mV. Data acquisition was controlled by Patchmaster software (HEKA, Lambrecht, Germany) and signals were recorded via a double EPC-10 (HEKA) patch-clamp amplifier. Sampling rate was 10 or 20 kHz and signals were filtered at 3 kHz. Capacitance measurements were performed by the ‘sine + DC’ method (Gillis, 1995). In brief, a 1-kHz sinusoidal voltage command (30 mV peak to peak) was added to the Vh of −60 mV and the resulting current analysed at two orthogonal phase angles by the EPC-10 software emulation of a lock-in amplifier. These signals, together with the DC current, were used to generate values for membrane capacitance (Cm), membrane conductance (Gm) and series conductance (Gs; Gillis, 1995). Offline analysis was performed using IgorPro software (Wavemetrics, Lake Oswego, OR, USA).

Data analysis

Membrane potentials recorded under current clamp were corrected for a liquid-junction potential (LJP) of +11 mV after the experiments were performed. Exocytosis was measured as the increase in membrane capacitance (ΔCm), evoked by membrane depolarization: Formula where Cm(baseline) is the average Cm value during a 50- to 200-ms period prior to the depolarization, and Cm(response) is the average Cm value during a 50- to 200-ms period following the depolarization, after allowing time for evoked conductances to have decayed. ΔCm was not used if simultaneous changes in Gm or Gs were observed. For measurement of the peak inhibition of IK(Ca) during paired step depolarizations, the outward current at the peak of the inhibition of the first depolarization was compared with the outward current at the same time point during the second depolarization, if little or no inactivation of IK(Ca) occurred during the steps (e.g. Fig. 5C). If pronounced inactivation occurred such that the second depolarization evoked an outward current with a different profile from the first (e.g. Fig. 5B), the outward current at the peak of the inhibition was compared to the current amplitude at the same time point during a depolarization later in the recording when exocytosis had run down, or with the predicted amplitude at that time point extrapolated by eye from the uninhibited current later in the step. Pooled data are expressed as mean ±s.e.m. Statistical significance was assessed using paired and unpaired Students' t tests as appropriate, with P < 0.05 considered significant.

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Results

Properties of Ca2+-dependent APs in bipolar-cell terminals

In order to investigate the regenerative potentials exhibited by bipolar-cell terminals in retinal slices, whole-cell recordings were made from isolated (axon-severed) terminals under current clamp using potassium gluconate-containing intacellular solution. Approximately half of the terminals (7/13) fired spontaneous APs with no injected current, and sustained AP firing was evoked in the remaining terminals by injection of positive current (≤ 4 pA, n= 6; Fig. 1A). Spontaneous APs were inhibited by negative current injection, which caused strong hyperpolarization of the terminal (Fig. 1B). The properties of bipolar-cell terminal APs are presented in Table 1. AP frequency was dependent on the amount of injected current (Fig. 1C) and was most sensitive between −2 pA and +2 pA, with a linear slope of 1.5 Hz pA−1. AP amplitude and duration were stable when AP frequency was relatively low (Fig. 1D), but high-frequency APs exhibited a decreased amplitude. Further increases in positive current injection evoked small, fast voltage oscillations of variable amplitude, rather than regular APs (Fig. 1E and F). The maximum frequency at which a stereotyped AP waveform was maintained was approximately 15 Hz.

Recordings were also made in the presence of the ionotropic GABA receptor antagonist picrotoxin (50 μm) to determine whether endogenous activation of GABAA and/or GABAC receptors in bipolar-cell teminals affects regenerative potentials. No significant differences in AP properties were observed in the presence of picrotoxin (n= 8; Table 1 and Fig. 1C). In agreement with previous observations of the Ca2+ dependence of APs in bipolar-cell terminals (Protti et al. 2000), APs could not be elicited in the presence of the L-type Ca2+ channel antagonist nifedipine (100 μm, n= 9; Fig. 1G). Terminals in nifedipine exhibited a depolarized resting potential (−46 ± 5 mV, n= 9) and were more strongly depolarized by a + 25 pA current ramp than control terminals (n= 6; P < 0.01, Table 1 and Fig. 1G).

Ca2+ influx and exocytosis evoked by APs

To determine the Ca2+ influx and amount of exocytosis evoked by a bipolar-cell terminal AP, a typical AP waveform recorded under current clamp was used as a stimulus in voltage-clamp experiments. Ca2+ influx was measured as inward charge during the depolarization using Cs+-based intracellular solution to block K+ currents; exocytosis was measured as ΔCm following an AP waveform using Cs+- or K+-based intracellular solutions. On average, a single AP evoked Ca2+ influx of 1.24 ± 0.15 pC (n= 14) and ΔCm of 38 ± 3 fF (n= 29; Fig. 2A). In a subset of terminals, the response to an AP waveform was compared to the response to a 25-ms step depolarization to −10 mV. The AP waveform evoked 27 ± 2% of the Ca2+ influx and 44 ± 3% of the ΔCm evoked by the step depolarization (n= 8; Fig. 2A).

Bipolar-cell terminals show pronounced depression of exocytosis, which is probably due to depletion of vesicles close to Ca2+ channels and requires many seconds for full recovery (von Gersdorff & Matthews, 1997; Burrone & Lagnado, 2000; Palmer et al. 2003a). It was, therefore, unknown whether exocytosis could be sustained during a train of APs, or whether only the first AP would be effective in evoking release. A train of 10 or 20 APs was delivered at 5.6 Hz and ΔCm was measured after each AP. As can be seen from Fig. 2B, exocytosis was evoked by APs throughout the train. The total ΔCm in response to a 10-AP train was 134 ± 10 fF (n= 29). The first and second APs evoked 29 ± 2% and 14 ± 1% of the total ΔCm, respectively, (n= 29; Fig. 2C). Following the first two APs, release per AP was fairly constant during the rest of the train, with an average ΔCm/AP of 10 ± 1 fF (n= 29; Fig. 2C). In the subset of terminals in which a 20-AP train was delivered, ΔCm evoked by the twentieth AP (6.3 ± 1.1 pF) was not significantly different from ΔCm evoked by the 10th AP (7.1 ± 1.2 pF, n= 7). In the presence of the L-type Ca2+ channel inhibitor, nifedipine (100 μm), no exocytosis was observed in response to a 10-AP train (ΔCm= 0 ± 7 fF, n= 5).

The total Ca2+ influx during a 10-AP train was 20.5 ± 2.3 pC (n= 14). However, it was observed that the first AP evoked significantly less Ca2+ influx than subsequent APs (Fig. 2B). The first AP evoked only 56 ± 4% as much Ca2+ influx as the 10th AP, whereas the second AP evoked 91 ± 3% (n= 14; Fig. 2D). This is probably due to inhibition of ICa by vesicular protons released via exocytosis (DeVries, 2001; Palmer et al. 2003a), which will affect the first AP more strongly because it evokes more exocytosis than subsequent APs in the train. Inhibition of ICa by released protons has previously been shown to reduce synaptic depression to paired-pulse stimuli (Palmer et al. 2003a).

Effect of released vesicular protons on exocytosis evoked by APs

To determine the effect of released protons on ICa and exocytosis during a train of APs, recordings were made in a different set of terminals with the addition of 24 mm Hepes to the extracellular solution (NaCl replaced by Hepes to maintain osmolarity). This increase in extracellular pH buffering significantly reduced proton-mediated inhibition of ICa during step depolarizations (Fig. 3A;DeVries, 2001). In the presence of extracellular Hepes, the Ca2+ influx evoked by the first AP of a 10-AP train was increased to 94 ± 3% of that evoked by the 10th AP (n= 6); that is, the inhibition of ICa by vesicular protons was greatly reduced (Fig. 3B).

With 24 mm extracellular Hepes, the first AP accounted for significantly more of the total exocytosis evoked by a 10-AP train than in control conditions (40 ± 2%, n= 17; P < 0.01). This resulted from a significantly larger ΔCm evoked by the first AP (62 ± 4 fF, n= 17; P < 0.01; Fig. 3C) but no significant difference in the total release by 10 APs (165 ± 13 fF, n= 17; Fig. 3C). Therefore the inhibition of ICa by released vesicular protons under control conditions enables exocytosis to be more evenly distributed throughout an AP train.

Effect of AP frequency on exocytosis

The results of the current-clamp and voltage-clamp experiments indicate that depolarization of bipolar-cell terminals primarily modulates the frequency of AP firing (Fig. 1C), and that bipolar-cell terminals are able to maintain exocytosis in response to individual APs in a 5.6-Hz train (Fig. 2B). Therefore, I investigated whether the amount of exocytosis could be modulated by AP frequency. In one set of terminals, measurements were made of the ΔCm evoked by two trains of APs with identical durations (1.29 s) but different frequencies: three APs at 2.33 Hz and 10 APs at 7.78 Hz (Fig. 4A). The difference in these AP frequencies equates to approximately 3.8 pA of injected current (Fig. 1C). The 2.33-Hz train evoked ΔCm of 57 ± 6 fF, whereas the 7.78-Hz train evoked ΔCm of 104 ± 17 fF (n= 8; Fig. 4B). On average, the 7.78 Hz train evoked 76 ± 8% more exocytosis than the 2.33 Hz train (n= 8; P < 0.01; Fig. 4B). Bipolar-cell terminal exocytosis is therefore sensitive to changes in AP frequency.

Identification of IK(Ca) in bipolar-cell terminals

As discussed above, exocytosis has been shown to lead to inhibition of Ca2+ influx via the action of released vesicular protons. Ca2+ channels are colocalized with BK channels in bipolar-cell terminals and Ca2+ influx rapidly activates IK(Ca) under physiological conditions (Sakaba et al. 1997a). The activation of IK(Ca) may therefore be modulated by proton-mediated inhibition of ICa. Firstly, IK(Ca) was identified in bipolar-cell terminals by examining the membrane currents evoked by step depolarizations with K+-based intracellular solution. The experiments were performed in the presence of picrotoxin (50 μm). Steps to −10 mV evoked a transient inward ICa followed rapidly by net outward current, which peaked approximately 3 ms after the start of the depolarization (Fig. 5A). With maintained depolarization, the current exhibited a biphasic profile: during the first ∼25 ms, a variable degree of outward current inactivation occurred, whereas for the remainder of the depolarization (up to 1 s), the outward current slowly grew in amplitude (Fig. 5A). The mean current values for selected time points during a step depolarization are given in Table 2.

The outward current is likely to be mediated by a combination of IK(Ca) and voltage-gated K+ current (IK(V)) (Kaneko & Tachibana, 1985), which were dissected by inhibiting L-type Ca2+ channels with nifedipine (100 μm). In the presence of nifedipine, both the initial inward ICa and the outward current at 3 ms were greatly reduced (Fig. 5A and Table 2). The outward current developed slowly over the first 20 ms and was fairly constant for the remainder of the depolarization (Fig. 5A and Table 2). In addition, it was observed that step depolarizations to −30 mV in the presence of nifedipine evoked no outward current (−1 ± 1 pA at 200 ms, n= 8). Therefore, IK(Ca) comprises both a rapidly activating component that shows variable inactivation over tens of milliseconds, and a component that gradually increases in amplitude during prolonged depolarizations.

Inhibition of IK(Ca) by released vesicular protons

To determine whether the rapidly activated component of IK(Ca) was affected by proton-mediated inhibition of ICa, paired 25-ms step depolarizations to −10 mV (100-ms interval) were delivered to bipolar-cell terminals. The majority of recordings (24/28) were made in the presence of picrotoxin (50 μm) to eliminate feedback currents mediated by GABAA and GABAC receptors. A prominent transient inhibition of IK(Ca) was observed during the first depolarization in 23 of 28 terminals (Fig. 5B and C). The inhibition peaked 4.5 ± 0.1 ms after the start of the depolarization and reduced the outward current by approximately 62 ± 4% (reduced from 120 ± 11 to 51 ± 8 pA, n= 23). For comparison, in a different set of terminals recorded with Cs+-based intracellular solution in the presence of picrotoxin (50 μm), proton-mediated inhibition of ICa peaked at 1.8 ± 0.1 ms and reduced the inward current by 68 ± 10% (n= 5; Fig. 3A). The inhibition of IK(Ca) was absent during the second depolarization of a pair of step depolarizations, when exocytosis was depressed (Fig. 5B), and also during depolarizations late in the recording, when exocytosis had run down (Fig. 5B). In addition, inhibition of IK(Ca) was greatly reduced (n= 5) or not observed (n= 9) in terminals in the presence of 24 mm extracellular Hepes (Fig. 5D). These results are consistent with inhibition of IK(Ca) resulting from inhibition of ICa by released vesicular protons in bipolar-cell terminals.

Inhibition of IK(Ca) by released protons during AP trains

With K+-based intracellular solution, an AP waveform evoked a transient inward current followed by a more sustained outward current (Fig. 6A). In the presence of nifedipine (100 μm), both the inward charge and the outward charge during the AP were greatly reduced (control: inward, 0.14 ± 0.03 pC; outward, 0.52 ± 0.09 pC, n= 12; nifedipine: inward, 0.01 ± 0.01 pC; outward, 0.01 ± 0.00 pC, n= 5; Fig. 6B). Therefore, it is likely that the majority of the K+ current during an AP is mediated by IK(Ca). Following from the observation that ICa evoked by the first AP in an AP train is most inhibited by released protons, I investigated whether IK(Ca) evoked by the first AP is similarly inhibited. When a train of 10 APs was delivered at 5.6 Hz, it was observed that IK(Ca) was consistently smaller in response to the first AP than subsequent APs (Fig. 6C). The outward charge during the first AP was 78 ± 3% of that during the average of the second to 10th APs (n= 12; P < 0.01). Inhibition of IK(Ca) during the first AP was not observed in a different set of terminals in the presence of 24 mm extracellular Hepes: the outward charge during the first AP was 98 ± 5% of that during the average of the second to 10th APs (n= 10; Fig. 6D). Therefore, released vesicular protons inhibit both ICa and IK(Ca) during AP waveforms in bipolar-cell terminals.

Effect of released protons on AP amplitude

I investigated whether released vesicular protons modulate AP firing in bipolar-cell terminals by making current-clamp recordings in the presence of 24 mm extracellular Hepes. The most obvious effect of high-pH buffer was on the shape of spontaneous and evoked APs. In control recordings, occasional irregular APs were observed that had a longer duration as a result of a brief plateau at their peak and, sometimes, an unusually large amplitude (Fig. 7A). In 15% of control recordings (6/41), these events were more common than regular APs. However, in the presence of extracellular Hepes, the long-duration events were observed much more frequently and were predominant in 50% of recordings (8/16; Fig. 7A).

To determine whether an effect of released protons on AP amplitude could be observed, terminals were held at a membrane potential slightly negative to AP threshold for at least 10 s (to ensure recovery from vesicle depletion) before injection of a small amount of positive current to evoke AP firing (mean current increase from −2.9 ± 0.5 to 0.7 ± 0.5 pA, n= 18). The majority of the recordings (16/18) were made in the presence of picrotoxin (50 μm). Under normal pH buffering conditions, it was observed that the amplitude of the first evoked AP was, on average, smaller than that of subsequent APs: the first AP peak was 3.2 ± 0.6 mV more negative than the average peak of the third to 10th APs (n= 12, P < 0.01; Fig. 7B and D). The magnitude of the inhibition of the first AP was variable between terminals and was found to be positively correlated with the magnitude of the inhibition of IK(Ca) during paired 25-ms step depolarizations (Fig. 5B and C) in the same terminals (n= 12; r= 0.82, P < 0.01; Fig. 7C). In the presence of 24 mm extracellular Hepes, the amplitude difference between the first AP and average of the third to the 10th AP was reduced to 1.2 ± 0.5 mV (n= 6; P < 0.05 compared with control; Fig. 7B and D). These results suggest that vesicular protons released during Ca2+-dependent APs feed back to modulate AP amplitude in bipolar-cell terminals.

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Discussion

The results of this study demonstrate that bipolar-cell terminals in retinal slices reliably fire Ca2+-dependent APs which depolarize the terminal within the physiological range for bipolar-cell light responses (Ashmore & Falk, 1980). A single AP evokes ΔCm of ∼38 fF and exocytosis is maintained at a rate of ∼10 fF per AP during a 5.6-Hz AP train. Negative feedback of ICa via released vesicular protons limits the amount of exocytosis evoked by an AP and reduces synaptic depression during AP trains. Proton-mediated inhibition of ICa also inhibits IK(Ca) during step depolarizations and AP waveforms, and reduces the amplitude of APs evoked under current clamp.

AP generation required L-type Ca2+ channel activation, as shown by the absence of APs during application of nifedipine. The depolarizing effects of nifedipine on both the resting membrane potential and the depolarization evoked by current injection are likely to be due to a reduction in activation of IK(Ca), which normally limits the extent of depolarization of the bipolar-cell terminal (Burrone & Lagnado, 1997). The lack of outward current evoked by AP waveforms in the presence of nifedipine suggests that AP repolarization is predominantly mediated by IK(Ca). IK(V) activation in bipolar-cell terminals appears to require stronger and more prolonged depolarization than occurs during an AP. The major Ca2+-activated K+ channel subtype in bipolar-cell terminals is BK channels, which are colocalized with Ca2+ channels at active zones and rapidly activated (< 1 ms) by local micromolar increases in [Ca2+]i following Ca2+ channel opening (Sakaba et al. 1997a; Llobet et al. 2003). An Mb1 terminal has been estimated to contain approximately 300 BK channels with a single-channel conductance of 50 pS (Sakaba et al. 1997a). IK(Ca) observed in the present study was activated rapidly and caused a large increase in membrane noise (Fig. 5A), consistent with BK channel activation.

There was some variability between terminals in the extent of inactivation of IK(Ca) during the first 25 ms of depolarization. Two BK current components have been identified in frog saccular hair cells (Armstrong & Roberts, 2001) and adrenal chromaffin cells (Solaro et al. 1995), one non-inactivating and the other rapidly inactivating, which were mediated by two distinct channel populations. It is possible that the recorded terminals in this study comprised a heterogeneous population with distinct BK channel properties suited to their specific functions. It was noted that terminals with the most strongly inactivating currents were more likely to fire irregular, long-duration APs. IK(Ca) in bipolar-cell terminals showed a second component which grew in magnitude over hundreds of milliseconds. This current probably reflects activation of a population of BK channels that are located at a distance from Ca2+ channels (Sakaba et al. 1997a) and respond to a gradual increase in global [Ca2+]i during prolonged depolarizations (Kobayashi & Tachibana, 1995). The small outward current which remained in the presence of nifedipine and developed over tens of milliseconds is likely to be mediated by delayed rectifier K+ channels (Kaneko & Tachibana, 1985), although there may be a small contribution from voltage-dependent BK channel activation (Vergara et al. 1998). It has previously been found that the enzyme papain modifies the properties of both IK(Ca) and ICa in saccular hair cells (Armstrong & Roberts, 1998), for example by removing the inactivation of BK currents (Armstrong & Roberts, 2001). Effects such as these may explain the differences between regenerative potentials observed in this study and those reported in dissociated bipolar cells (Burrone & Lagnado, 1997; Zenisek & Matthews, 1998).

The amplitude and shape of APs in isolated terminals in retinal slices were similar to the properties of APs recorded in intact bipolar cells (Protti et al. 2000), although AP duration was shorter in the isolated terminals (16 ms compared with 41 ms in intact cells). In addition, AP frequency was much higher in isolated terminals, because APs in intact bipolar cells exhibited a prolonged refractory period (1–2 s) that arose from the retinal circuitry prior to the synaptic terminal (Protti et al. 2000). The APs exhibited by the synaptic terminal also differ significantly from light responses recorded from the same class of bipolar cell with sharp microelectrodes. Rod-dominant bipolar cells with bulbous axon terminals in carp retina exhibited light responses that comprised an initial transient depolarization, which peaked ∼150 ms after light onset, followed by a smaller sustained depolarization (Saito & Kujiraoka, 1982). Similar voltage responses were recorded from rod bipolar cells in the dogfish retina (Ashmore & Falk, 1980). Increasing light intensity caused both an increase in peak amplitude up to a maximum of ∼25 mV and a decrease in the rise time and latency of the responses.

The absence of fast APs from the light responses of intact bipolar cells is likely to arise from several factors. Firstly, intact bipolar cells have a significantly lower input resistance than isolated terminals (Palmer et al. 2003b), as a result of synaptic currents and other membrane conductances in the dendrites and cell body, which is expected to decrease the likelihood of AP generation. Electrical coupling to other bipolar cells (Kujiraoka & Saito, 1986) will have the same effect. Secondly, GABA input from amacrine cells to bipolar-cell terminals may be greater during light responses than under the conditions of the present study. AP firing would tend to be inhibited by both the decrease in input resistance resulting from ionotropic GABA receptor activation and inhibition of ICa via metabotropic GABA receptors (Zenisek & Matthews, 1998). In relation to this, spontaneous APs were rarely observed in low input resistance (light-adapted) intact bipolar cells in retinal slices, but the reliability of AP firing was increased in both light- and dark-adapted conditions by pharmacological activation of ICa (Protti et al. 2000). Under physiological conditions, the regenerative membrane properties of bipolar-cell terminals may contribute to shaping the time course of light responses and boosting transmitter release from the terminal, possibly in a manner regulated by GABA receptors. For example, it has been suggested that regenerative potentials in the terminal underlie the transient, large-amplitude component of the rod bipolar-cell light response (Burrone & Lagnado, 1997; Zenisek & Matthews, 1998; Protti et al. 2000).

The exocytotic response to a single AP waveform was 38 fF, equating to the release of approximately 1440 vesicles, or 26 vesicles per active zone (assuming a vesicle capacitance of 26.4 aF and 55 ribbons per terminal; von Gersdorff et al. 1996). This response is similar in size to exocytosis of the rapidly releasable pool of vesicles (33 fF; Mennerick & Matthews, 1996; Neves & Lagnado, 1999), which corresponds with the number of vesicles docked along the bottom of each ribbon (approximately 22 vesicles per ribbon, giving ΔCm of 32 fF; von Gersdorff et al. 1996). A single AP, therefore, appears to activate the rapid, phasic mode of bipolar-cell exocytosis. During a 5.6-Hz train of APs, steady-state release of 10 fF per AP was observed, equating to 56 fF, or 2120 vesicles, per second. Prolonged step depolarizations evoke a slow phase of exocytosis, which releases a pool of approximately 4400 vesicles over a period of about 1 s (Neves & Lagnado, 1999), similar to the number of vesicles tethered to the sides of the ribbons (∼4850; von Gersdorff et al. 1996). It is likely that AP trains stimulate release from this pool of vesicles. During a 5.6-Hz train, the pool is not depleted at its maximum rate and it is possible that replenishment with cytoplasmic vesicles prevents depletion (Gomis et al. 1999), thus enabling maintained release during a 20-AP train lasting 3.6 s.

Exocytosis from both bipolar-cell and photoreceptor terminals leads to inhibition of Ca2+ influx via acidification of the synaptic cleft (DeVries, 2001; Palmer et al. 2003a). This arises from the low pH of synaptic vesicles, which use a proton gradient to pump neurotransmitter into the vesicle (Liu & Edwards, 1997) and consequently release protons during exocytosis. L-type Ca2+ channels are sensitive to extracellular pH: lowering pH causes a decrease in conductance and a shift in activation to more positive potentials (Iijima et al. 1986; Prod'hom et al. 1987; Krafte & Kass, 1988; Barnes & Bui, 1991). The inhibition of Ca2+ influx by released vesicular protons in bipolar-cell terminals leads to inhibition of exocytosis and has the effect of reducing paired-pulse depression of release (Palmer et al. 2003a). In the present study, I found that vesicular protons also inhibit IK(Ca) in bipolar-cell terminals. The inhibition was dependent on exocytosis and was blocked by increasing the concentration of extracellular pH buffer. The 2.7-ms delay in peak inhibition of IK(Ca) compared with peak inhibition of ICa, and the similar magnitude of the two effects, are consistent with inhibition of IK(Ca) via a reduction in Ca2+ influx, rather than a direct effect of protons on BK channels. Similarly, Ca2+-activated Cl− currents (ICl(Ca)) in cone photoreceptors are inhibited by low extracellular pH and this can be fully accounted for by inhibition of Ca2+ influx (Barnes & Bui, 1991). Proton-mediated inhibition of ICa may therefore have knock-on effects on a variety of Ca2+-dependent cellular processes. However, unlike BK channels, Ca2+-activated Cl− channels have slow kinetics and are probably located at a distance from Ca2+ channels, requiring global [Ca2+]i increases within the terminal for activation (Okada et al. 1995). Thus, it is unclear whether ICl(Ca) will be modulated by synaptically released protons, which predominantly exert rapid and transient effects on ICa during phasic exocytosis.

The high sensitivity of BK channels to changes in Ca2+ influx is likely to be conferred by a combination of their low Ca2+ affinity, high degree of cooperativity, rapid kinetics and tight colocalization with Ca2+ channels at active zones (Roberts et al. 1990; Art et al. 1995; Sakaba et al. 1997a; Yazejian et al. 2000; Llobet et al. 2003; Sun et al. 2004). At physiologically relevant membrane potentials (e.g. −30 mV), [Ca2+]i in excess of 10 μm is required for significant BK channel activation (Magleby, 2003). Such high Ca2+ concentrations will only be achieved in microdomains around open Ca2+ channels, as a result of rapid buffering and diffusion away from entry sites (Heidelberger et al. 1994; Matthews, 1996; Beaumont et al. 2005; Schneggenburger & Neher, 2005). The activation of BK channels is therefore very tightly linked to activation of ICa, rather than to global [Ca2+]i (Yazejian et al. 2000), and is expected to strongly limit the amount of exocytosis from bipolar-cell terminals by rapidly hyperpolarizing the membrane following ICa activation. In view of this, the observed variable inactivation of BK channels may be important for enabling sustained exocytosis to occur.

Finally, it was observed that AP amplitude was inhibited by synaptically released protons. The amplitude of the first AP of a train was smaller than that of subsequent APs (which evoke less exocytosis), the magnitude of the inhibition was correlated with the amount of proton-mediated inhibition of IK(Ca) in the same terminals, and the inhibition was significantly reduced in the presence of increased extracellular pH buffer. Therefore, the amplitude of APs in bipolar-cell terminals is limited not only by the activation of BK channels but also by feedback inhibition of ICa. This decreases the amount of exocytosis evoked by the first AP and consequently reduces synaptic depression during an AP train, as shown by the voltage-clamp experiments measuring ΔCm. The high sensitivity of BK channels to a drop in Ca2+ influx during proton-mediated inhibition of APs enables the fine balance between ICa and IK(Ca) activity to be maintained. An interaction between these currents is also important for regulating cellular function in other types of neuron. For example, in the auditory hair cells of lower vertebrates, ICa and IK(Ca) underlie an electrical resonance that contributes to frequency selectivity (Art & Fettiplace, 1987; Art et al. 1995; Ramanathan et al. 1999). Frog hair cells are similar to goldfish bipolar cells in that Ca2+ channels and Ca2+-activated K+ channels are colocalized at ribbon-containing active zones (Roberts et al. 1990; Issa & Hudspeth, 1994), but it is currently unknown whether released vesicular protons modulate ICa and IK(Ca) in this system.

In summary, the results of the present study have demonstrated that bipolar-cell terminals in retinal slices readily fire Ca2+-dependent APs that evoke exocytosis. The amount of exocytosis is modulated by proton-mediated inhibition of ICa and is sensitive to AP frequency. Further work is required to determine the role of Ca2+-dependent APs in bipolar-cell light responses, their modulation by GABA input from amacrine cells, and whether their occurrence extends to bipolar cells of mammalian species. It has also been shown that inhibition of ICa by released vesicular protons has consequences in addition to modulation of exocytosis: both Ca2+-activated K+ currents and AP amplitude are inhibited as a result of this feedback process. Vesicular protons are likely to be a significant modulator of synaptic function in neurons with ribbon-type synapses.

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Figure 1. 
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Figure 1. 

Isolated bipolar-cell terminals in retinal slices fire spontaneous and evoked APs A, current-clamp recording from a bipolar-cell terminal with K+-based intracellular solution. This terminal did not fire APs spontaneously, but APs were evoked by injection of +4 pA of current. In this and subsequent Figures, membrane voltages have been corrected for liquid-junction potential. B, membrane voltage response from a terminal that fired spontaneous APs. Negative current injection (ramp from 0 to −10 pA) caused hyperpolarization and inhibited AP firing. C, modulation of AP frequency by current injection in control recordings (n= 13) and in the presence of picrotoxin (50 μm; n= 8). Data points show mean ±s.e.m.D, voltage responses from a terminal in which AP amplitude and duration were unaffected by changing AP frequency within the low range. This recording was made in the presence of picrotoxin (50 μm). E, voltage responses from a terminal firing high-frequency APs in which further current injection evoked small, variable voltage oscillations rather than regular APs. F, voltage response from a terminal to a current ramp from 0 to +25 pA. AP frequency increased and amplitude decreased with increasing current. G, voltage response to a 0- to +25-pA ramp from a terminal in the presence of the L-type Ca2+ channel antagonist nifedipine (100 μm) plus picrotoxin (50 μm). No APs were observed in the presence of nifedipine.

Figure 2. 
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Figure 2. 

Ca2+ influx and exocytosis in response to AP waveforms A, voltage-clamp recording from a bipolar-cell terminal with Cs+-based intracellular solution in the presence of picrotoxin (50 μm). A typical AP waveform previously recorded under current clamp was used as the stimulus, and membrane capacitance was measured before and after the depolarization using a voltage sinewave. The AP evoked an inward Ca2+ current (ICa) and an increase in membrane capacitance (ΔCm). For comparison, ICa and ΔCm evoked in the same terminal by a 25-ms step depolarization to −10 mV are shown on the right. B, ICa and ΔCm responses in the same terminal as A evoked by a 5.6-Hz train of 20 APs (stimulus shown at top). ICa was smaller in response to the first AP than subsequent APs in the train. ΔCm was greatest in response to the first AP, and exocytosis was evoked by APs throughout the train. C, mean ΔCm evoked by the first, second, and third to 10th APs in a 5.6-Hz train (n= 29). Data from recordings using K+-based intracellular solution were included. D, mean Ca2+ influx evoked by the first, second, and third to 10th APs in a 5.6-Hz train (n= 14).

Figure 3. 
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Figure 3. 

Released vesicular protons affect Ca2+ influx and exocytosis during AP trains A, ICa evoked by a pair of 25-ms step depolarizations to −10 mV (interval, 100 ms) in the presence of picrotoxin (50 μm), (first, black; second, grey). Under control extracellular pH buffering conditions (24 mm HCO3−), prominent inhibition of ICa by released vesicular protons occurred during the first depolarization, but not during the second when exocytosis was depressed. In a different terminal, the addition of 24 mm Hepes to the extracellular solution greatly reduced inhibition of ICa. B, ICa evoked by a 5.6-Hz train of 10 APs in the same terminals as A, (first AP, black; second to 10th APs, grey). The addition of 24 mm Hepes greatly reduced the inhibition of ICa during the first AP. C, in the presence of 24 mm Hepes, the first AP evoked a significantly larger ΔCm than in control conditions (left), but there was no significant difference in the total ΔCm evoked by 10 APs (middle: control, n= 29; Hepes, n= 17). There was significantly more Ca2+ influx during the 10-AP train in the presence of Hepes than in control conditions (right: control, n= 14; Hepes n= 6).

Figure 4. 
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Figure 4. 

Bipolar-cell terminal exocytosis is sensitive to AP frequency A, ΔCm evoked by two trains of APs (three APs at 2.33 Hz and 10 APs at 7.78 Hz) in one terminal (control conditions). Approximately twice as much exocytosis was evoked by the 7.78-Hz train as by the 2.33-Hz train. B, ΔCm evoked by the 2.33-Hz and 7.78-Hz AP trains in eight terminals (•) and mean ±s.e.m.ΔCm values (□).

Figure 5. 
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Figure 5. 

Inhibition of Ca2+-activated K+ current (IK(Ca)) by released vesicular protons A, membrane currents evoked by step depolarizations to −10 mV with K+-based intracellular solution in control conditions (black) and in the presence of nifedipine (100 μm; grey). To the right, the initial 20-ms period of depolarization is shown on an expanded time scale. Nifedipine inhibited ICa and both a rapidly activated component and a slowly developing component of IK(Ca). Recordings were made in the presence of picrotoxin (50 μm) to block GABA-mediated currents. B, membrane currents and ΔCm evoked by paired 25-ms step depolarizations to −10 mV (interval, 100 ms) in the presence of picrotoxin (50 μm). Black traces show responses early in the recording when the first depolarization evoked a large ΔCm; superimposed (grey traces) are responses later in the recording when exocytosis had run down. The first depolarization, associated with the largest ΔCm, showed a prominent transient inhibition of IK(Ca). C, membrane currents evoked by a pair of 25-ms depolarizations to −10 mV in the presence of picrotoxin (50 μm), (first, black; second, grey), in a terminal that showed little outward current inactivation during the depolarization. Inhibition of IK(Ca) was observed during the first depolarization. D, as for C, but with the addition of 24 mm Hepes to the extracellular solution. Inhibition of IK(Ca) during the first depolarization was greatly reduced.

Figure 6. 
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Figure 6. 

Inhibition of IK(Ca) during AP trains A, the current response to an AP waveform with K+-based intracellular solution in the presence of picrotoxin (50 μm). A brief inward ICa was followed by more sustained outward current. B, the current response to an AP waveform in a different terminal in the presence of nifedipine (100 μm). Both ICa and the outward current (IK(Ca)) were inhibited. C, the currents evoked by APs in a 5.6-Hz train (first AP, black; second to 10th APs, grey) in the presence of picrotoxin (50 μm). IK(Ca) was smaller during the first AP than during subsequent APs. D, mean outward charge of first AP as a percentage of the average outward charge during the second to tenth APs, in control conditions (n= 12) and in the presence of 24 mm extracellular Hepes (n= 10). The inhibition of IK(Ca) of the first AP is likely to result from inhibition of Ca2+ influx by released vesicular protons.

Figure 7. 
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Figure 7. 

Released protons modulate AP firing in bipolar-cell terminals A, example current-clamp recordings from a terminal in control conditions and a terminal in the presence of 24 mm extracellular Hepes. Long-duration, larger-amplitude spikes (*) were occasionally observed in control conditions, and were more prominent in the presence of Hepes. B, recordings from a control terminal and a terminal in the presence of 24 mm Hepes that mainly exhibited regular AP firing. The terminals were held below AP threshold for at least 10 s, followed by injection of a small amount of positive current (2–5 pA) to evoke AP firing. In control conditions, the first AP was noticeably smaller than subsequent APs in the train, whereas this difference was reduced in the presence of Hepes. Both recordings were made in the presence of picrotoxin (50 μm). C, a graph of peak inhibition of IK(Ca) during paired step depolarizations to −10 mV against the inhibition of the first evoked AP (difference between the first AP maximum amplitude and the average of the third to 10th AP maximum amplitudes) in the same terminals; the data were fitted with a straight line. D, mean peak AP amplitudes for the first evoked AP and average of the third to 10th APs in control conditions and in the presence of 24 mm extracellular Hepes. Hepes significantly reduced the inhibition of the first AP.

View this table:

Table 1. Properties of APs and Vm in bipolar-cell terminals

View this table:

Table 2. Properties of membrane currents evoked by step depolarizations to −10 mV with potassium gluconate-containing intracellular solution, in the presence of picrotoxin (50 μm)

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Acknowledgements

This work was supported by a Medical Research Council Career Development Award. The author wishes to thank Dr Mike Evans for advice and critical reading of the manuscript.

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Footnotes

    • Accepted February 23, 2006.
    • Received January 18, 2006.
    • Revision received February 20, 2006.
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  1. Published online before print February 23, 2006, doi: 10.1113/jphysiol.2006.105205 May 1, 2006 The Journal of Physiology, 572, 747-762.
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