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. Author manuscript; available in PMC: 2014 Feb 6.
Published in final edited form as: Neuron. 2013 Feb 6;77(3):516–527. doi: 10.1016/j.neuron.2012.11.024

Synaptic ribbons influence the size and frequency of miniature-like evoked postsynaptic currents

Bhupesh Mehta 1,5, Josefin Snellman 1,5, Shan Chen 4, Wei Li 4,*, David Zenisek 1,2,3,*
PMCID: PMC3569740  NIHMSID: NIHMS425565  PMID: 23395377

Abstract

Non-spiking cells of several sensory systems respond to stimuli with graded changes in neurotransmitter release and possess specialized synaptic ribbons. Here we show that manipulations to synaptic ribbons caused dramatic effects on mEPSC-like (mlEPSC) amplitude and frequency. Damage to rod-bipolar cell ribbons using fluorophore-assisted light inactivation resulted in the immediate reduction of mlEPSC amplitude and frequency, whereas the first evoked response after damage remained largely intact. The reduction in amplitude could not be recovered by increasing release frequency after ribbon damage. In parallel experiments, we looked at mlEPSCs from cones of hibernating ground squirrels, which exhibit dramatically smaller ribbons than awake animals. Fewer and smaller mlEPSCs were observed postsynaptic to cones from hibernating animals, depolarized cones were able to generate larger mlEPSCs. Our results indicate that ribbon size may influence mlEPSC frequency and support a role for ribbons in coordinating multi-vesicular release.

Keywords: ribbon synapse, retina, photoreceptor, bipolar cell, synaptic transmission, hibernation, fluorophore-assisted light inactivation

Introduction

In the visual, auditory and vestibular systems, photoreceptors, bipolar cells and hair cells have evolved to support tonic graded release of neurotransmitters in response to changes in sensory stimuli. These cells have specialized synaptic ribbons, proteinaceous structures to which dense arrays of vesicles are tethered (For review; (Matthews and Fuchs, 2010; Schmitz, 2009)). In rod bipolar cells of the retina, synaptic ribbons support both fast transient synaptic transmission, which signals temporal contrast, and tonic neurotransmitter release to signal luminance levels, which are detected as intermittent mEPSC-like events in postsynaptic amacrine cells(Jarsky et al., 2011; Oesch and Diamond, 2011). Similarly, photoreceptors signal changes in light level as tonic changes in neurotransmitter release (Jackman et al., 2009).

Asynchronous mEPSC-like events emanating from several ribbon-type synapses have been shown to increase in size with depolarization ((Li et al., 2009; Singer et al., 2004), however see also;(Glowatzki and Fuchs, 2002)) . These large events are thought to arise from the near simultaneous release of the contents of multiple vesicles. To distinguish these from the mEPSCs that arise from the release of a single vesicle (Fatt and Katz, 1952), we refer to these in this manuscript as miniature-like excitatory post-synaptic currents (mlEPSCs). The prevalence of multivesicular release from ribbon-type synapses suggests a possible role for the ribbon in facilitating these events.

Several studies have suggested a role for the ribbon in determining the size amount of neurotransmitter release in response to step depolarizations. Ribbon disruption has profound and specific effects on the kinetics of neurotransmitter release (Frank et al., 2010; Khimich et al., 2005; Snellman et al., 2011) and responses to sensory stimuli (Allwardt et al., 2001; Buran et al., 2010; Dick et al., 2003). Similarly, diurnal changes in ribbon number correlate with changes in the size of the readily releasable pool in goldfish bipolar cells (Hull et al., 2006). The effects on mlEPSCs have not previously been studied, however. Here, we examined the influence of the ribbon on mlEPSC amplitude and frequency using two approaches: 1) using fluorophore assisted light inactivation (FALI) to selectively and acutely damage the synaptic ribbon in mouse bipolar cells while recording effects on synaptic transmission postsynaptically; and 2) examining the effects of hibernation on ground squirrel cones, which causes the removal of most ribbon material from the membrane, leaving a short ribbon intact opposite postsynaptic cells ((Reme and Young, 1977) and unpublished observations). Our results here show that 1) unlike neurotransmitter release evoked by steps to 0 mV, mlEPSC frequency is dramatically reduced by FALI before a subsequent depolarization 2) FALI reduced the amplitude of mlEPSCs; 3) the shortened ribbons of hibernating animals exhibited a significant reduction in mlEPSC frequency with little effect on mlEPSC amplitude after accounting for the change in frequency. These results reveal differences in the ribbon-associated vesicle pools contributing to mlEPSCs at resting potentials and in response to voltage steps and support an important role for the ribbon in coordinating multivesicular release.

Results

Characterization of mlEPSCs at rod bipolar cell to AII Amacrine cell synapse

To study the properties of mlEPSCs released from a ribbon-type synapse, we first performed whole cell voltage clamp recordings of AII-amacrine cells (AIIs) under conditions where GABAergic and glycinergic synaptic transmission had been blocked (see Methods), without recording from a presynaptic rod bipolar cell. Under these conditions, recordings (e.g. figure 1A) from AII's exhibit frequent AMPA receptor mediated mlEPSCs (Morkve et al., 2002; Veruki et al., 2003). Application of the mGluR6 agonist L-AP4, which hyperpolarizes ON-type bipolar cells (Slaughter and Miller, 1981), reduced the frequency of these events by >80% (figure 1A-B), as previously reported (Singer et al., 2004). Upon voltage clamping a presynaptic rod bipolar cell (VH = -60mV) in the presence of 4uM L-AP4, we observed a significant increase in the frequency of spontaneous mlEPSC's (figure 1C-D), which was reversed upon killing the voltage-clamped RBC (fig 1C). These results suggest that although each rodent AII amacrine cell receives glutamatergic input from several rod bipolar cells (RBC) (Kolb and Famiglietti, 1974; Sterling et al., 1988; Tsukamoto et al., 2001), under conditions of paired recordings in the presence of L-AP4 the spontaneous activity that is observed is dominated by input from the single voltage clamped presynaptic RBC.

Fig. 1. Nature of mlEPSCs in rod bipolar cell – AII amacrine cell synapse.

Fig. 1

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(A) mlEPSCs recorded from AII amacrine cell, before, in presence of mGluR6 receptor agonist L-AP4 (4uM) and upon wash out of L-AP4. (B) bar graph showing effect of L-AP4 on mlEPSCs recorded from three AII-amacrine cells normalized to the number of mlEPSCs recorded before L-AP4 addition. Before L-AP4 addition, we recorded an average of 1219 mlEPSCs per minute, that reduced to 181 mlEPSCs per minute upon L-AP4 addition. Voltage clamping a presynaptic rod bipolar cell (VH -60mV), substantially increases the mlEPSCs recorded from the postsynaptic AII and the effect is reversed upon killing the RBC (C). (D) Bar graph showing normalized mlEPSC frequency from 3 experiments. Before voltage clamping the RBC, AII-amacrine cells displayed, on average, 248 events per minute in presence of L-AP4. (E) average of over 30 mlEPSCs taken for different ranges of amplitude (indicated in legend) of mlEPSCs from a single paired recording. Inset shows the same traces normalized to their peak amplitudes. These average mlEPSCs show remarkable similarity in kinetics and waveform. (F) effect of bipolar cell holding potential on frequency of AII mlEPSCs (n = 7). (G) Bar graph showing mlEPSC counts as small (mlEPSC amplitude <median+2SD) and big (mlEPSC amplitude >median+2SD) plotted for different holding potential of RBC (-80, -60 and -40 mV) from 7 experiments. Each value is normalized to the number of small mlEPSCs at -60 mV for that particular recording. On average, we recorded 573 mlEPSCs per minute with the bipolar cell held at -60 mV. There is a notable increase in the percentage of mlEPSCs with bigger amplitude at -40 mV of RBC's membrane potential (n=7, p<0.001; paired t-test). (AII VH = -60mV and RBC VH = -60 mV, unless until mentioned in figure). The median and SD used to define large and small events were determined by the responses measured with the RBC held at -60 mV.

The mlEPSC's recorded in the AII amacrine cells exhibit considerable heterogeneity in amplitude, consistent with coordinated multivesicular release (Singer et al., 2004). Random superposition of mlEPSCs is unlikely to account for the heterogeneity in event sizes we observe. The highest frequency of mlEPSCs we observed when a presynaptic bipolar cell was held at a resting potential of -50 or -60 mV was 37 events per second. Assuming a stochastic process at this rate, we would expect only 3.7% of mlEPSCs to be followed within 1 ms by another event, which is well within our ability to detect events. At the average rate observed at -60 mV, the coincidence rate of events within 1 ms of another mlEPSC is less than 1% of all events. Similarly, if large mlEPSCs arise from stochastic coincidental release of multiple vesicles, then one might expect that large mlEPSCs might exhibit slower kinetics than smaller mlEPSCs. To test this idea, we grouped the mlEPSCs into 6 subgroups based on their amplitude and averaged a random selection of each subgroup. Figure 1E shows the average mlEPSC for each subgroup and the inset shows the averages normalized to the peaks. The waveform and kinetics did not vary with amplitude (inset figure 1E), consistent with the larger mlEPSC's being a result of multivesicular release from the presynaptic bipolar cell, supporting the findings of Singer et al. (2004).

We next looked at the effect of presynaptic holding potential on the amplitude and frequency of mlEPSCs. To do so, RBCs were held at membrane potentials between -80 and -40 mV for 100 s and mlEPSCs were analyzed. Since RBCs exhibit a phasic component of increased neurotransmitter release upon depolarization(Jarsky et al., 2011; Oesch and Diamond, 2011; Singer and Diamond, 2006; Snellman et al., 2009), we analyzed mlEPSC properties beyond 60 s after the initiation of a voltage step to assay the properties of steady-state release. As expected for the calcium dependence of neurotransmitter release, the steady-state mlEPSC frequency is dependent on the holding potential of the presynaptic rod bipolar cell (figure 1F), as previously described (Jarsky et al., 2011; Oesch and Diamond, 2011). In addition, we found that the amplitude of mlEPSCs also exhibited a strong voltage dependence. To simplify the analysis across cell pairs, which exhibit considerable variability in amplitude from pair to pair, we divided mlEPSCs into small and large events. Small mlEPSCs were defined as any event that was less than the median amplitude + 2 standard deviations (S.D.) at -80 mV for any particular cell pair. Large events were defined as events that were greater than the median + 2 S.D. Figure 1G plots the number of small and large events for bipolar cells held at -80, -60 and -40 mV normalized to the number of small events at -80 mV. As shown in figure 1G, more positive membrane potentials favors bigger mlEPSCs.

Since mlEPSC frequency and amplitude both exhibited a strong voltage-dependence, we next tested the extent to which calcium entry through calcium channels was required for mlEPSCs or for the triggering of large mlEPSC. To do so, we used the voltage-gated calcium channel blocker Co2+. Figure 2A shows that 5mM CoCl2 was sufficient to block all evoked release in response to a voltage step to a presynaptic RBC, in our recordings, indicating effective block of voltage-gated calcium channels in the RBC. Of note, the opening of single calcium channels is sufficient to drive neurotransmitter release from RBCs (Jarsky et al., 2010), further supporting the idea that nearly all channels are blocked by CoCl2 in these experiments. By contrast, mlEPSC frequency was only reduced approximately 60% by the addition of 5mM CoCl2 in pairs where the RBC was held at a membrane potential of -60 mV. Both the evoked release and mlEPSC frequency recovered following washout of CoCl2 (figure 2A-C). We also performed experiments with the L-type calcium channel blocker nifedipine. With nifedipine ((100-400 μM), we were only able to block evoked release by 58 +/- 22 % (n = 3 pairs), perhaps due to poor penetration of the compound into the slice. In qualitative agreement with the results with Co2+, we found that the large reduction in evoked release was accompanied by a considerably small reduction in mlEPSC frequency (33 +/5%; n = 5 cells). These results suggest that true calcium-channel independent “spontaneous” release represents a smaller fraction of the release we observed when a presynaptic bipolar cell was held at -60 mV.

Fig. 2. Opening of voltage gated calcium channels is required for multivesicular release.

Fig. 2

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In double voltage clamped pair of a rod bipolar cell and an AII-amacrine cell, step depolarization of rod bipolar cell from -60 mV to 0mV triggers a postsynaptic EPSC recorded from AII amacrine cell. (A) Effect of 5mM cobalt chloride (CoCl2) on evoked release. Note that CoCl2 reversibly blocks all evoked release from the RBC. (B) Recording of mlEPSCs from AII amacrine cell with rod bipolar cell held at -60 mV, showing effect of CoCl2 on amplitude of mlEPSCs. (C) Bar graph showing the effect of CoCl2 (5mM) on mlEPSC frequency from 4 experiments. In all four AII-RBC pairs, before CoCl2 addition the AII amacrine cell displayed on average 454 mlEPSCs per minute. (D) Distribution of mlEPSC amplitudes before and after CoCl2 addition. Note that results show significant reduction in the number of larger events upon CoCl2 application (n=4 pairs). (E) displays reduction in the distribution peak by two units on plotting subtracted mlEPSCs count before cobalt minus after CoCl2, and mlEPSC counts with respect to their amplitude in presence of CoCl2 (n = 4 pairs). (AII VH = -60mV, RBC VH = -60 mV)

Also apparent in the results was that the addition of Co2+ reduced the amplitude of the mlEPSCs (e.g. figure 2B), consistent with a role for calcium channels in coordinating multivesicular events (Graydon et al., 2011; Li et al., 2009). To analyze this effect, we measured the size of mlEPSCs in the presence and absence of CoCl2 and found that blockade of calcium channels resulted in a dramatic reduction in the size of mlEPSCs (figure 2D), with the mean amplitude reduced from 9.19 +/- 0.07 pA to 6.69 +/- 0.09 pA (P = <0.001). If one assumes that the mEPSCs observed in CoCl2 represent the true ‘spontaneous’ release from all presynaptic bipolar cells, one can recover the component of release evoked from calcium entry through calcium channels by subtracting the CoCl2 distribution from the distribution in the absence of channel blockers. Figure 2E shows those results from this analysis. From figure 2E, one can see that calcium entry through calcium channels favors larger, presumptive multivesicular events. Similarly, partial blockade of calcium channels using nifedipine (100 - 400 μM) decreased the average mlEPSC size by 33.3 +/- 7% (n = 4502 events before nifedipine; 3026 after). Our results are consistent with a role for calcium channels in coordinating multivesicular release (Graydon et al., 2011) and are consistent with the idea that multivesicular release may provide a mechanism for distinguishing between true spontaneous mEPSCs from evoked mlEPSCs (Singer et al., 2004).

Effect of Synaptic Ribbon Damage on mlEPSCs

To test the role of the synaptic ribbon in mlEPSC release, we used fluorophore assisted light inactivation (FALI) (Hoffman-Kim et al., 2007) to selectively and acutely damage the ribbon (Snellman et al., 2011), while monitoring release post-synaptically in a post-synaptic AII . In brief, this method takes advantage of the generation of singlet oxygen molecules upon excitation of a fluorophore to generate local damage near the fluorophore. To selectively target the ribbon, we used the whole-cell patch electrode to load RBCs with fluorescein-conjugated short peptides, that selectively binds to RIBEYE, (Zenisek et al., 2004) the most abundant protein in synaptic ribbons (Schmitz et al., 2000). As reported earlier (Snellman et al., 2011), illumination of RBCs containing this peptide resulted in only a small reduction of the first evoked response following illumination, whereas the second response and consecutive responses were significantly reduced. By contrast, we observed a reduction in the frequency and amplitude of spontaneous mlEPSCs within seconds of the initiation of the FALI inducing illumination (figure 3B), when the RBC was held at a membrane potential of -60 mV. Unlike the evoked component (e.g. figure 3C), the effect on mlEPSCs occurred prior to a depolarizing step (figure 3B), suggesting that mlEPSCs arise from a separate ribbon-associated pool of vesicles than those involved in the transient response to depolarization. As a control, we performed similar experiments with cells loaded with a fluorescein-labeled scrambled peptide, and observed no change in the spontaneous or evoked release upon illumination (fig. 3F,G,H,I). Figure 3E summarizes the effects of illumination on the frequency of mlEPSCs at -50 or -60 mV across 12 RBC:AII pairs with the ribbon binding peptide, and 2 RBC:AII pairs with the scrambled peptide (figure 3J). For the 12 RBC:AII pairs (figure 3E), the average absolute frequency observed per minute before FALI was 1087 ±178 and 658 ±101 after FALI.

Fig. 3. Fluorophore Assisted Light Inactivation of ribeye affects vesicle release from active zone.

Fig. 3

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(A) EPSCs observed in an AII amacrine cell upon depolarization of the synaptically connected rod bipolar cell, filled with the ribbon binding fluorescein labeled peptide before FALI. (B) Recording of mlEPSCs during period when FALI is induced by illumination with blue light (black bar), while bipolar cell is held at a membrane potential of -60 mV. Note that the mlEPSCs are rapidly affected during the 15 second illumination. Small gray bars indicate the inserts plotted from the 10 seconds recording, before and after FALI. (C) The first evoked response after FALI from the same cell pair. Note that the response is similar to the evoked response before FALI (A), despite dramatic effect on mlEPSCs. On average, the EPSC in response to the first depolarization after FALI had a charge that was 81.1 +/- 5.5% of the EPSC prior to illumination. (D) The response to a second voltage step after FALI. Note that the amplitude is substantially reduced. (E) Bar graph showing significant reduction in frequency of mlEPSCs after FALI from 12 RBC-AII pairs, normalized to the frequency of events before FALI (*p<0.001; paired t-test). (F-J) Same as A-E, except with scrambled version of the peptide in the presynaptic pipette. No noticeable alteration in the frequency and amplitude of evoked EPSC or mlEPSCs was observed in the postsynaptic AII amacrine cell upon blue light stimulation (n = 2). (VH RBC = -50 mV or -60mV, VH AII= -60mV).

Figure 4 investigates the effect of ribbon damage on the amplitude of mlEPSCs. Figure 4A shows the size of each mlEPSC before (black ●) and after (gray ◇) illumination of the ribbon-binding peptide for one experiment. As is apparent in figure 4A, ribbon damage resulted in a dramatic reduction in the number of large mlEPSCs. Figure 4B shows a histogram of the mlEPSC sizes from the same experiment as in 4a, whereas figure 4C summarizes the results across 12 synaptic pairs. Figure 4C compares the effect of ribbon damage on small mlEPSCs (defined as less than median + 2S.D. before FALI) to the effect on large mlEPSCs. As shown in figure 4C, illumination of the ribbon-bound peptide had a substantially greater effect on bigger mlEPSCs (reduced by 75%) than on small mlEPSCs (reduced by 20%). These results suggest that a functional ribbon is required for the coordination of multivesicular release.

Fig. 4. FALI of ribbons affect multivesicular release.

Fig. 4

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(A) Distribution of individual minis from a single experiment plotted over time, before (black ●) and after (gray ◇) FALI. Note that each dot represents the amplitude of a single mlEPSC. (B) Overall mlEPSC histogram of event amplitude before (black) and after (gray) FALI for a single pair. Note the dramatic drop in the number of larger events observed. (C) Normalized mlEPSC counts plotted as small (<Median+2SD) and big (>Median+2SD) before and after FALI from 11 RBC-AII pairs. A preferential loss of bigger amplitude mlEPSCs or multivesicular events is observed (C; n = 12 pairs, *p<0.001; paired t-test). The median and SD were defined for each pair as response measured with the RBC held at -60 mV (n = 6) or -50 mV (n = 6) before FALI, and the overall data is normalized to the frequency of small mlEPSCs before FALI. Overall, the data presented in this panel comes from 6 rod bipolar cells held at -50 mV, which contributed 7663 mlEPSC before FALI and 4487 mlEPSCs after FALI, and 6 RBCs held at -60 mV, which contributed 4229 mlEPSCs before FALI and 2801 after FALI. (D) Postsynaptic response of an AII amacrine cell observed on stimulating the presynaptic RBC. The evoked EPSC display a fast transient component followed by an asynchronous component. (E) Amplitude histogram of mlEPSC during asynchronous component of release from 5 AII-RBC pairs before and after FALI. (F) Bar graph showing contribution of small (<Median+2SD) and large (>Median+2SD) mlEPSCs, during asynchronous component of release (# p<0.001; paired t-test). The median and SD were defined for each pair as asynchronous response measured upon depolarizing the RBC from a holding potential of -60 mV or -50 mV to 0mV before FALI. The fast transient component and release observed during depolarization pulse to the RBC was not included for analysis.

If ribbon damage inflicts preferential loss of multivesicular release at resting potentials, then it may also hold true for depolarization mediated multivesicular release, which has previously been described at the RBC:AII synapse (Singer et al., 2004). As described previously (Singer and Diamond, 2003; Singer et al., 2004; Snellman et al., 2011), a step depolarization to -10 mV to the presynaptic rod bipolar cell evoked a characteristic postsynaptic response with a transient component followed by an asynchronous or delayed component (figure 4D). The asynchronous component shows distinct multi-quantal events that have been described as a result of coordinated multivesicular release (Singer et al., 2004). The large events are particularly pronounced when the intracellular calcium buffer in the bipolar cell is reduced. Figure 4E compares the size of the mlEPSCs during the asynchronous phase of release measured before and after illumination of the ribbon-binding peptide, in pairs in which the RBC was filled with an internal solution with an EGTA concentration of 0.5 mM. In agreement to our earlier finding, ribbon damage resulted in preferential loss in the number of larger, presumably multivesicular, mlEPSCs (p<0.001; paired t-test comparing fraction of large events). Figure 4F summarizes the results obtained from 5 experiments.

Calcium channel independent release is insensitive to ribbon damage

In the presence of 5mM CoCl2 (fig. 2B-C) a reduced number of small mlEPSCs remain, indicating that the majority of mlEPSCs observed in AII Amacrine cells arise as a consequence of calcium influx through voltage gated calcium channels. We tested whether the residual mlEPSC could be reduced by damage directed to the ribbon and whether the effect of FALI required calcium entry through calcium channels. We found that illuminating the ribbon bound peptide had only modest effects on the frequency (figure 5) and no effect on the amplitude (p>0.05) of calcium-channel independent mlEPSCs in the presence of CoCl2 (fig 5A-C). However, after a 5 minute washout of CoCl2, the mlEPSC's exhibited little recovery, indicating that illumination during the blockade of calcium channels was sufficient to affect mlEPSCs. These results suggest that a result of acute damage to the ribbon causes the specific loss of mlEPSCs that are dependant on calcium influx through voltage-gated channels.

Fig. 5. Calcium channel independent release is insensitive to FALI.

Fig. 5

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(A) mlEPSCs recorded from the postsynaptic AII-amacrine cell in presence of 5mM CoCl2. The black bar indicates timing of 20 second blue light illumination to stimulate FALI. (B) 20 second records of mlEPSCs from the postsynaptic AII before CoCl2 perfusion, in CoCl2, after photodamge (PD) in CoCl2, upon CoCl2 washout and after killing the presynaptic RBC. (C) Bar graph showing the normalized mlEPSCs for 3 AII-RBC pairs displaying the effect of FALI in absence of calcium influx.

Increasing mlEPSC frequency after FALI does not rescue larger mlEPSCs

The preceding experiments described above indicate that acute damage to the synaptic ribbon reduces both the amplitude and frequency of mlEPSCs, but a key question is whether the change in amplitude is secondary to the effect on frequency. To dissociate the two effects, we took advantage of the relationship between membrane potential and mlEPSC frequency (figure 1F) to match the frequencies before and after FALI. To do so, RBCs were held at membrane potentials varying between -70 and -45 mV before damage induction and between -50 and -40 mV after damage in 5 mV increments, while mlEPSCs were monitored in a synaptically connected AII. Figure 6A shows the normalized frequency of mlEPSCs recorded across 6 of 8 RBC:AII pairs at a selection of potentials before and after the ribbon was damaged. From the normalized bar graph, the frequency of mlEPSCs at -55 mV before damage approximately matched that at -45 mV after damage for the same duration and the amplitude of mlEPSCs were compared. In 2 of the 8 RBC-AII pairs, depolarization of the RBC after FALI failed to increase the mlEPSC frequency to pre-FALI levels and were not used for this analysis. Figure 6b shows the comparison of amplitude of events for a representative RBC-AII pair for RBC holding potential at -55 mV before FALI to -45 mV after FALI. As can be seen from figure 6B, despite exhibiting similar mlEPSC frequencies, the amplitude of the events exhibited a dramatic shift toward smaller events. On plotting the normalized amplitude of events as small (<Median+2SD) and big (>Median+2SD) for 6 RBC-AII pairs, over 50 percent reduction in the number of big events was observed (6C). The mlEPSCs had an amplitude of 8.34 +/-0.11 pA before damage and 6.94+/-0.09 pA after damage at a matching frequency. These results support the notion that ribbons are important regulators of multivesicular release, independent of its influence on mlEPSC frequency.

Fig. 6. Increasing mlEPSC frequency after FALI does not rescue larger mlEPSCs.

Fig. 6

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(A) Bar graph of normalized mlEPSC frequencies recorded from AII amacrine cells plotted for different holding potentials of RBC, before and after FALI to ribbons (n= 6 RBC-AII pairs). FALI to ribbons was performed at while RBC was held at -70 mV. Dotted line indicates that mlEPSC frequency for -55 mV RBC holding potential before FALI is roughly equivalent to -45 mV RBC holding potential after FALI. (B) Histogram of mlEPSC amplitude before FALI at the holding potential of RBC, -55 mV and after FALI at a holding potential of -45 mV post FALI for a representative experiment. (C) Normalized mlEPSC counts plotted as small (<Median+2SD) and large (>Median+2SD) across 6 RBC-AII pairs at RBC holding potential of -55mV before FALI, and at -45 mV after FALI. For the purposes of this analysis, the median and SD was defined at a membrane potential of -55 before photobleach. Note that, a significant drop in the number of multivesicular events was observed after FALI (*p<0.01).

Recordings from hibernating ground squirrel indicate that small ribbons exhibit reduced mlEPSC frequency, but are sufficient for multivesicular release

The results above support the idea that functional synaptic ribbons support multivesicular release in the RBC:AII synapse. To test whether ribbon size influences mlEPSC properties, we investigated mlEPSC amplitude and frequency in the control and hibernating ground squirrel cone synapses. Upon hibernation, much of the synaptic ribbon material in ground squirrel cones is sequestered away from the plasma-membrane in aggregates, leaving the synaptic release site with smaller synaptic ribbons that protrude a much shorter distance into the interior of the cone (Reme and Young, 1977). We hypothesized that the shrinking of ribbons in this preparation may also have an effect either on the amplitude or frequency of mlEPSCs. To test this idea, we monitored mlEPSCs (mediated by AMPA receptors) in postsynaptic b2 Off cone bipolar cells in response to vesicle release from presynaptic cones in slices taken from hibernating and non-hibernating ground squirrel retinae. Figure 7 summarizes the results. The inset in figure 7A confirms the previous results of Reme and Young (1977) and shows depletion of the major synaptic ribbon protein, ribeye, from the outer plexiform layer in hibernating animals. Recordings from retinas of hibernating animals exhibited a pronounced decrease in mlEPSC frequency (figure 7A,D) and amplitude (figure 7B,C), with no change in resting membrane potential (-44.5 ± 3.5 mV in control, n=7; -43.0 ± 3.5 mV in hibernating animals, n=7; carried out in current clamp), nor an obvious change in the distribution of glutamate receptor localization (figure 7E,F). The cone calcium currents were found to be slightly smaller in the hibernating tissues (peak amplitude when stepped from -70 mV to -20 mV: 97.5 ± 18.7 pA in control, n=5; 81.2 ± 13.9 pA in hibernating animals, n=5), but not statistically significant and there is no change in current-voltage relationship.

Fig. 7. Hibernating animals show decrease in the size and frequency of spontaneous release.

Fig. 7

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(A) Sample traces of mlEPSCs recorded from b2 Off cone bipolar cells in retinal slices from awake and hibernating animals. Insets: immunolabeling of photoreceptor ribbons in wholemount of awake and hibernating tissues using Ctbp2 antibody. (B) The amplitude of the average mlEPSC from hibernating animals is significantly smaller than that from awake ones, but with strikingly similar rise and decay kinetics (normalized waveforms shown in the inset). (C) Bar graph showing the average mlEPSC amplitude recorded in awake (black; n = 1982 events) and hibernating (gray; n = 595 events) animals. Error bars represent standard deviation. (D) There is a significant reduction in the frequency of mlEPSCs from hibernating animals (n=4) compared to that from awake ones (n=5), especially the large, putative multi-vesicular events. Note the change of y-axis scale before and after the split. (E&F) In retinal sections, immunolabeling of AMPA receptor subunit (GluR4) reveals no significant difference in expression level and distribution pattern between awake and hibernating tissues (n=3 animals each). Dashed curves outline the location of exemplary cone terminals.

On average, the amplitude of the mlEPSCs were 60 % smaller in hibernating squirrel retinas (figure 7B,C), with no change in the kinetics of the individual events (figure 7B, inset). An amplitude histogram revealed that the decrease in amplitude is the result of the specific loss of large amplitude events (figure 7D, 8B). To test whether the effect on mlEPSC size was secondary to the change in frequency, we next depolarized cones in paired cone – bipolar recordings to raise the frequency of mlEPSCs in bipolar cells in the hibernating squirrel retinas. To bring the mlEPSC frequency of hibernating animals to that of awake animals, we first depolarized the cones of hibernating animals in cone-bipolar recordings. After depolarization, both the frequency and amplitude of the bipolar mlEPSCs in the hibernating animals increased. However, even with continuous depolarization of cones, the bipolar mlEPSC frequency remained significantly lower than that in the awake tissues (figure 8A; blue bar). Moreover, we were not able to further increase the frequency of mlEPSCs with presynaptic cone depolarization, higher extracellular Ca2+ concentration, or treatment of Bay K (10 μM), indicating that the large portion of the ribbon structure removed from the membrane during hibernation is critical for maintaining mlEPSC frequency. To match the frequencies of depolarized hibernating cones, we instead added 500 μM Co2+ to awake animals (Figure 8A; green bar). When compared with slices from awake animals that were treated with low concentration of Co2+ (500 μM) their amplitude distributions are essentially the same (figure 8D). Evidently, the remaining small ribbons, which are limited to the very bottom compartment, are sufficient to support multivesicular release, but fail to support normal frequencies of mlEPSCs.

Fig. 8. Increasing mlEPSC frequency does elicit larger mlEPSCs in hibernating animals.

Fig. 8

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(A) mlEPSC frequency of bipolar cells under four different conditions: awake (n=5; red), hibernating (n=4; black), hibernating with presynaptic cone depolarization (n=5; blue), and awake with low concentration (500 μM) of Co2+ (n=5; green). Manipulations aimed to increase presynaptic release probability (such as depolarization of the presynaptic cone in paired recordings) did increase the frequency of mlEPSCs, but failed to reach the level of the awake condition. Thus, low concentration of Co2+ was applied to awake tissues to achieve a similar mlEPSC frequency as in hibernating tissues. (B - D) Normalized histograms comparing mlEPSC amplitude distributions of different conditions. Larger events apparently can be elicited in hibernating tissues when the frequency of mlEPSCs is augmented. (E) I-V curves show that 100 μM Co2+ reduced the peak amplitude of the cone Ca2+ currents by ~25% (24.1 ± 3.8%, n=5) in retinal tissues from awake animals. The black dashed curve represents the I-V curve from hibernating tissues (normalized to the peak amplitude of the I-V curve from awake tissues). (F) Under this condition, the frequency of mlEPSC dropped to a much less extend (21.5 ± 7.0%, n=5) than that in hibernating tissues (dashed line). (G) The distribution of the mlEPSC amplitude remained largely unchanged (dashed profiles depict mlEPSC distributions in hibernating tissues).

Although we did not measure a significant difference in calcium currents in awake and hibernating animals, even a small difference in calcium current, below our level of detection in these experiments, could have large effects on neurotransmitter release rates (Dodge and Rahamimoff, 1967). To test whether calcium current underlies the effects of hibernation on mlEPSC frequency, we used 100 μM Co2+ to modestly reduce calcium channel currents in awake animals. Reduction of the calcium current by approximately 20% (Figure 8E) resulted in a reduction of mlEPSC frequency by approximately 25% (figure 8F) and a shift in the amplitude distribution of mlEPSC, as expected for the decrease in frequency (figure 8G). By contrast, hibernating animals exhibited mlEPSC frequencies that were 90% less than awake animals (Figure 8A and dashed line in figure 8F). Evidently, the reduction in calcium current cannot explain the reduction in mlEPSC frequency.

Discussion

Functional ribbons play a role in setting tonic mlEPSC release rates

Our results support the idea that the ribbon is important for setting rates of tonic release. The small ribbons of hibernating ground squirrels exhibit a dramatic decrease in mlEPSC frequency though calcium channels and resting potential remain largely unaffected, suggesting that tonic release is reduced with the loss of ribbon material from the synapse. Similarly, FALI directed damage to the mouse bipolar cell ribbon causes an immediate drop in frequency of mlEPSCs, while having little effect on spontaneous Ca-channel independent rates of release. Together, these results indicate that synaptic ribbons, in some way, play a critical role in setting tonic release rates.

Evidence for heterogeneity between vesicle pools on the ribbon

In previous work, we showed that fluorophore-conjugated peptides containing a ribeye-binding motif can be used to selectively label synaptic ribbons (Zenisek et al., 2004). FALI using a fluorescein-conjugated Ribeye-binding peptide, has little effect on the release of a pool of vesicles that presumably populate the ribbon prior to illumination, however it prevents the release of vesicles that repopulate the ribbon following the stimulus (Snellman et al., 2011). Here we show that FALI of bipolar cells loaded with the ribbon-binding peptide results in a rapid reduction of mlEPSC frequency at low membrane potentials (-60/-50mV), prior to a subsequent stimulus (figure 3B). These results suggest the existence of at least two ribbon-associated pools of vesicles: one pool that undergoes release at -50 to -60 mV, which is depleted within seconds of FALI and one that is released by depolarizing steps to 0 mV. Since both release at low and high membrane potential are blocked by ribbon-directed FALI, we believe both pools to be ribbon associated, consistent with bassoon mutants, which exhibit fewer membrane-anchored ribbons and the parallel loss of spontaneous and evoked release from hair cells (Buran et al., 2010). Of note, recent studies in conventional synapses have demonstrated that spontaneous and evoked neurotransmission arise from largely independent pools of vesicles, which can be distinguished molecularly (Hua et al., 2011; Kavalali et al., 2011; Ramirez and Kavalali, 2011; Ramirez et al., 2012; Sara et al., 2011). A similar mechanism, with two parallel pools of vesicles for spontaneous and evoked release, both resident on the ribbon, could explain our results here. Alternatively, the different effects of FALI on mlEPSCs at -60 mV and evoked EPSCs could arise from heterogeneities in vesicle release probabilities, arising from either differences in proximity to calcium channels or differences in sensitivity to calcium (Burrone and Lagnado, 2000), such that a subset of vesicles are released at more negative potentials, whereas others can only be released in response to stronger stimuli. Such an explanation is plausible, because the FALI driven loss of release appears to be use dependent (Snellman et al., 2011). A third model to explain our results is that the ribbon could act as a catalyst that facilitates vesicle exocytosis, by reducing the energy barrier in some way for neurotransmitter release. In this model, ribbon damage would increase the amount of calcium required to drive release and effectively prevent neurotransmitter release at low calcium concentrations, but have far less effect at more positive potentials, where calcium is sufficient to trigger the release process without aid of a functional ribbon. Further experiments will be necessary to distinguish between these mechanisms.

The ribbon's role in multivesicular release

Both AII amacrine cells in the mouse retina and Off cone bipolar cells in the ground squirrel retina show mlEPSCs with variable amplitude with strikingly similar waveforms and kinetics (e.g figure 1E and figure 7B; Singer et al., 2004; Veruki et al., 2003), similar to observations made postsynaptic to hair cell ribbon synapses (Glowatzki and Fuchs, 2002; Grant et al., 2010; Graydon et al., 2011; Li et al., 2009). Given the rapidity of the multivesicular events observed, these events are thought to represent the near simultaneous release of the contents of multiple vesicles. We observed a substantial decrease in the amplitude of mlEPSCs in the presence of the calcium channel blocker, cobalt (fig 2D), supporting the idea that opening of voltage gated calcium channels near the ribbon is important for multivesicular release. Moreover, our results show that tonic depolarization causes a shift in the amplitude of mlEPSCs toward larger events, reminiscent of the voltage-dependence observed post-synaptic to hair cells of the amphibian papilla (Li et al., 2009). These results are consistent with the idea that multivesicular release could be a means of distinguishing synaptic noise generated by spontaneous release from tonic light-driven release (Singer et al., 2004), which may be important for signaling luminance levels by rod bipolar cells (Oesch and Diamond, 2011).

Evoked multivesicular release has been described in several conventional synapses, arising likely as a result of high release probability at some synapses in response to single action potentials (Auger et al., 1998; Biro et al., 2006; Christie and Jahr, 2006; Kirischuk et al., 1999; Tong and Jahr, 1994; Wadiche and Jahr, 2001). The multiquantal events described at ribbon-type synapses differ in that event synchrony persists in the absence of an action potential (Glowatzki and Fuchs, 2002; Li et al., 2009; Singer et al., 2004). Two theories have been proposed to explain multivesicular release at ribbon synapses- compound fusion of synaptic vesicles prior to exocytosis with the plasma-membrane (Matthews and Sterling, 2008); and coordinated fusion of several docked vesicles at the plasma membrane (Singer et al., 2004). In some hair cells, multivesicular events have been suggested to arise from calcium nano-domains near open calcium channels (Graydon et al., 2011). In rodent rod-bipolar cells, however, multi-quantal events can persist for hundreds of milliseconds after calcium channels are closed (Singer et al., 2004), suggesting some other mechanism for synchronizing events. We show here that damage directed to the entire ribbon by FALI reduces the amplitude of mlEPSCs and that this reduction in mlEPSC amplitude persisted even after accounting for changes in mlEPSC frequency. In contrast, the residual ribbons in hibernating ground squirrel cones remain capable of generating multivesicular events (Figure 8), while exhibiting a dramatic decrease in mlEPSC frequency. Hibernating animals retain a small ribbon that is dramatically reduced in height, but continues to be attached to the plasma membrane (Reme and Young, 1977). We suggest that longer ribbons that penetrate deeper into the cell are important for supporting mlEPSC-rates, perhaps by ensuring a large number of pre-primed vesicles are available for release, but that multi-vesicular release requires only the base of the ribbon that associates with the plasma-membrane.

It should be noted that after FALI some large events were still observed, albeit infrequently, which could reflect incomplete damage to the ribbon (Snellman et al., 2011) or suggest that the ribbon supports multi-vesicular release, but is not required for it. The effects of FALI on mlEPSC size could alternatively be explained by a role for the ribbon in packing synaptic vesicles with neurotransmitter or with the coordination of multivesicular release. While our results cannot distinguish between these two mechanisms, we favor the idea that the ribbon coordinates multi-vesicular release, based on the abundance of literature supporting multivesicular release from ribbon type cells and the lack of evidence suggesting a role for the ribbon in vesicle packing.

Methods

Mouse Retinal Slice Preparation and solutions

Retinal slices were prepared from 4-6 week old C57/BL6 mice (Harlan). All procedures were approved by the Yale University Animal Care and Use Committee. Mice were anaesthetized with halothane (Sigma), sacrificed by cervical dislocation, and their eyes removed and enucleated. Whole retinas were isolated and placed on a 0.45 micron cellulose acetate/nitrate membrane filter (Millipore), which was secured with vacuum grease to a glass slide adjacent to the recording chamber. Slices were cut to a thickness of 150 μm using a tissue slicer, and transferred to the recording chamber while remaining submerged. The recording chamber was immediately attached to a perfusion system, and the slices were perfused at a rate of 5 ml/minute with Ames media bubbled with 95% O2 and 5% CO2. The Ames media was supplemented with 100 μM picrotoxin and 50 μM 1, 2, 5, 6-Tetrahydropyridin-4yl methylphosphinic acid (TPMPA) to block GABAA and GABAC receptors, 10 μM strychnine to block glycine receptors and 4uM L-AP4 to hyperpolarize On bipolar cells. The standard recording solution for rod bipolar cells was composed of (in mM): 108 gluconic acid, 2 EGTA, 10 CsCl, 10 TEA, 4 MgATP, 1LiGTP. For FALI experiments, 1 mM of the free radical scavenger glutathione was included in the recording solution. For experiments looking at asynchronous release following step depolarizations to -10 mV, the EGTA concentration was reduced to 0.5 mM. The amacrine cell internal solution was composed of (in mM): 100 CsCH3SO3, 10 EGTA, 20 TEA, 10 HEPES, 4 MgATP. 2 mM QX314 was added to block action potentials. The pH was adjusted to 7.4 with CsOH. The osmolarity of both extracellular and intracellular solutions was 289-297, with a pH of 7.35–7.40. All chemicals were obtained from Sigma, except for L-AP4, TPMPA (Tocris) and ribbon-binding peptides (Genscript). Glutathione was stored as a powder at 4°C, and was added to the pipette solution immediately before use. L-AP4 was stored as a stock solution at 4°C and was added to the bath solution at the day of experimentation. All other drugs were aliquoted, stored at -20°C, and dissolved in the pipette solution on the day of use.

Ground squirrel retinal slice preparation

All animal procedures were approved by the Animal Care and Use Committee of the National Eye Institute. The procedures for making ground squirrel retinal slices have been described (Li and DeVries, 2006). During recording, the tissue was superfused with bicarbonate buffered Ame's media (Sigma-Aldrich) containing picotoxin (50 μM) and Strychnine (10 μM). The pipette solution contained (in mM): CsCH3SO, 120; MgSO4, 2; HEPES 10; TEA 20; EGTA 5; ATP 3; GTP 1; and Neurobiotin (Vector Laboratories), 0.1. pH was adjusted to 7.4 with CsOH. Slices were mounted on a Zeiss Examiner 1D microscope and superfused continuously at room temperature. Recordings were obtained with Axopatch 200B amplifiers (Molecular Devices), and currents low–pass filtered at 1 or 5 kHz using the 4 pole Bessel filter on the amplifier. The input/series resistances of the cone and bipolar cell recordings were approximately 1 GΩ/<20 MΩ, and 0.8 GΩ/<20 MΩ, respectively. Data were digitized at 10 kHz using an ITC-18 interface (HEKA) controlled by a Dell computer running IgorPro 6.0 (WaveMetrics). Data were analyzed with custom-made software (IgorPro 6.0) and MiniAnalysis (Synaptosoft). In voltage clamp experiments, cone, bipolar, and amacrine cell membrane voltages were maintained at –70 mV unless otherwise indicated.

Immunocytochemistry

Retinal tissues were fixed and processed for immunocytochemistry as previously described(Li and DeVries, 2006). Antibodies to the following were used: GluR4 (1:100; Millipore) and Ctbp2 (1:500; BD Biosciences). Tissues were mounted and imaged in the vertical or flatmount orientation. Images were acquired with a LSM-510 confocal microscope (Zeiss) and edited with Zeiss Zen software and Photoshop CS4 (Adobe Systems). An α-Plan-Apochromat 63x/1.40 oil immersion lens was used.

Data acquisition

Patch pipettes of resistance 8-11 MΩ were fabricated from borosilicate glass (TWF150-4, WPI) using a two-stage vertical puller (Narishige). Pipettes were coated with Sticky Wax to reduce noise (Kerr Corp). Whole-cell recordings were obtained using a dual EPC10 amplifier (HEKA Instruments). The input/series resistances of the RBC and AII amacrine cell recordings were approximately 3 GΩ/15-20 MΩ, and 1 GΩ/<40 MΩ, respectively. Cells were discarded if the holding current changed suddenly. Holding potentials were corrected for the liquid junction potential, which was measured to be approximately –12 mV depending on solutions. Slices were viewed with a Zeiss Axioskop 2FS plus equipped with a water-immersion 40X DIC objective. Rod bipolar and AII amacrine cells were confirmed by their shape and position in the slice, as determined by fluorescent imaging of dye filled cells. FALI was performed using X-Cite 120Q (EXFO, Ontario, Canada) with a 488 nm band pass excitation filter (Chroma). Data were acquired using PatchMaster software (HEKA Instruments). mlEPSCs were recorded for 60 - 120 s for each presynaptic holding potentials. Currents were elicited at 60 second intervals, collected at 20 kHz, and low-pass filtered at 1 kHz. Rod bipolar cell calcium currents were leak subtracted using a p/4 protocol.

Analysis

Analysis was performed using IgorPro (WaveMetrics), MiniAnalysis (Synaptosoft) and Origin 7.5 (Microcal). Candidate mlEPSCs and evoked asynchronous mlEPSCs were detected automatically using MiniAnalysis using a threshold of 3 - 8 pA depending on the noise level and then subsequently confirmed by eye. Data are presented as means ± S.E.M. (n = number of cells or events). mlEPSCs and asynchronous EPSCs are categorized as small or big based on the number of events less than or greater than the median of events plus two standard deviation (median + 2SD). Statistical significance was determined using a Student's t test, and differences were considered significant at the P < 0.05 level. The current traces shown in the figures represent individual traces.

Highlights.

  • Acute ribbon damage reveals different vesicles for evoked release and tonic mlEPSCs

  • Functional synaptic ribbons are necessary for multi-vesicular release

  • Small ribbons in hibernating ground squirrels exhibit low tonic release rates

  • Cones from hibernating animals retain capacity for multi-vesicular release

Acknowledgments

This work was funded by National Institute of Health grant EY014990 (D.Z.) and the National Eye Institute Intramural Research Program (W.L.).

Footnotes

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Supplemental Information: none

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