Synaptic Vesicle Priming in SNAP Double Mutant Synapses

The best documented function for the SNARE complex disassembly machinery is to recycle “spent” cis-SNARE complexes after fusion. Experimental conditions which limit, impair or block the activity of the disassembly machinery have been shown to impair the priming process, that is, the rate at which new vesicles are recruited into the RRP after its depletion. Under these conditions, impaired SNARE disassembly limits the supply of free “active” neuronal SNAREs in the presynaptic terminal, leading to inefficient recruitment of vesicles into the RRP. In order to study the priming process in double mutant synapses in more detail, I analysed both Ca²⁺ -independent and Ca²⁺-dependent RRP refilling rates.

3.4.3.3.1 Ca²⁺-Independent RRP Refilling

To measure Ca²⁺-independent RRP refilling, double pulses of hypertonic sucrose solution were applied at variable time intervals. The first pulse was applied to deplete the RRP, and the second to monitor its recovery (Fig. 21A). The charge integral of the second response was then plotted as a percentage of the first. RRP recovery was very similar between double mutant and control neurons (Fig. 21A), indicating that vesicle priming is not impaired at resting conditions in double mutant synapses.

Both data sets were fitted with a double exponential function (τf = 2.4 s and τs = 10.1s).

Furthermore, in order to monitor basal synaptic vesicle cycling in the presynaptic terminal, I repetitively depleted the RRP by consecutive (every ~1 minute) hypertonic sucrose applications and monitored RRP recovery over time (Fig. 21B). Endocytosed synaptic vesicle have been shown to recycle back to the active zone within hundreds of milliseconds (Deak et al., 2004; Aravanis et al., 2003; Pyle et al., 2000; Stevens et al., 2000; Gandhi et al., 2003; Klingauf et al., 1998; Sara et al., 2002; Sun et al., 2002). Therefore, if recycling of synaptic vesicles is impaired in double mutant synapses, one would expect a stronger and faster decrease of responses to consecutive hypertonic sucrose applications over time, due to the progressive loss of the recycled synaptic vesicles of the RRP.

Figure 21 Normal Ca²⁺-Independent RRP Refilling and Basal Synaptic Vesicle Cycling at Double Mutant Synapses. Ca²⁺-independent RRP refilling was measured by applying two pulses of hypertonic sucrose solution at variable time intervals. The charge integral of the second response was then plotted as a percentage of the first (A). Double mutant and control neurons showed very similar kinetics of RRP recovery, indicating that vesicle priming is not impaired at resting Ca²⁺

concentrations. The recovery process was fitted with a double exponential function (recovery time constants, Tau1 = 2.4s; Tau2 = 10.1 s). To monitor synaptic vesicle cycling under resting Ca²⁺

conditions, responses to consecutive (every ~1 minute) hypertonic sucrose applications were monitored over time. Double mutant and control neurons showed similar responses to consecutive hypertonic sucrose applications (B), indicating that basal synaptic vesicle cycling occurs normally at double mutant synapses (Ctrl, n = 19; DMut, n = 14).

Data shown are means ± standard error of the mean.

As expected, responses of both double mutant and control neurons were slightly decreasing over time, due to a well documented run-down phenomenon. However, the time course of this run-down was similar in mutant and control neurons, indicating that basal synaptic vesicle cycling occurs normally at double mutant synapses.

3.4.3.3.2 Ca²⁺- and Activity-Dependent RRP Refilling

In a subsequent set of experiments, I analysed priming rates in mutant neurons in the presence of high intracellular Ca²⁺ concentration ([Ca²⁺]i.) High-frequency stimulation leads to an increase in [Ca²⁺]i, a condition under which refilling of the RRP is faster than at resting Ca²⁺ concentrations (Dittmann and Regher, 1998; Stevens and

paradigms to study the effect of Ca²⁺ on the recovery of the RRP and synaptic transmission (Fig. 22): (1) I continuously stimulated the neuron at 10 Hz and intermittently depleted the RRP with a hypertonic sucrose pulse, and (2) I depleted the RRP with a hypertonic sucrose pulse and monitored recovery of the RRP under 10 Hz stimulation in normal osmolarity. The main difference between the two experimental paradigms is that in the first case high [Ca²⁺]i is present before, during and after the hypertonic sucrose pulse, while in the latter case high [Ca²⁺]i is present only after the sucrose pulse. Both experiments however gave similar results. As expected, due to their smaller RRP size, EPSC recovery in double mutant neurons reached a lower steady-state level as compared to control cells (Fig. 22A-C). The difference in the absolute EPSC steady-state levels between double mutant and control neurons is similar to that of RRP sizes measured by hypertonic sucrose applications (Fig. 17). To measure recovery rates of the RRP, I normalized the EPSC responses to the last value of the 10 Hz train (Schlüter et al., 2006). In both experiments, the time course of recovery of normalized EPSC responses was very similar between double mutant and control neurons (Fig. 22B-D), indicating that refilling rates after hypertonic sucrose depletion of the RRP were not changed in the presence of high [Ca⁺²]i.

Next, I analysed recovery rates after electrical discharge of the RRP. Instead of using hypertonic sucrose, a Ca²⁺-independent method to trigger fusion, I used a high-frequency stimulation train (100 stimuli at 40 Hz) to deplete the RRP. EPSC recovery was then monitored by single action potential stimulations delivered at different times after RRP depletion. As shown in Fig. 23B, EPSC recovery after the 40 Hz stimulation train was markedly slower in double mutant neurons as compared to control cells. As previously reported, fitting of EPSC recovery rates with a double exponential function identified two kinetic components of RRP recovery (Silver et al., 1998; Sakaba and Neher, 2001; Schlüter et al., 2006). Both the fast and the slow component of EPSC recovery were significantly slower in double mutant neurons (Fig. 23A), indicating that refilling after high-frequency depletion of the RRP is strongly impaired in double mutant neurons.

Figure 22 Normal Ca⁺²-Dependent RRP Refilling in Double Mutant Neurons after Hypertonic Sucrose Mediated RRP Depletion. RRP refilling was analysed in the presence of high intracellular Ca²⁺ concentration.

Two stimulation protocols were used: Intermittent RRP depletion via hypertonic sucrose in the presence of continuous 10 Hz stimulation (50 stimuli at 10 Hz - 50 stimuli at 10 Hz plus hypertonic sucrose – 50 stimuli at 10 Hz) (A), and hypertonic sucrose RRP depletion followed by 10 Hz stimulation to monitor recovery (hypertonic sucrose – 40 stimuli at 10 Hz) (C). Only EPSC recovery is shown in (A) and (C). In both experiments the time course of recovered EPSC responses normalised to the last value was very similar between double mutant and control neurons, as indicated in (B) and (D), respectively. (A) and (C) EPSC recovery monitored at 10 Hz stimulation frequency. EPSCs are normalised to the initial EPSC response (A: Ctrl, n = 127; DMut, n = 153. C: Ctrl, n = 33; DMut, n = 42). (B) and (D) Same as in (A) and (C), respectively, but EPSC responses are normalised to the last response of the 10 Hz train to allow direct comparison of recovery kinetics between double mutant and control neurons. Data shown are Mean ± standard deviation of the mean.

I next wanted to analyse EPSC recovery after complete depletion of neurotransmitter release. 100 stimuli at 40 Hz frequency can deplete the RRP, but are not sufficient to completely deplete total release. In fact, tonic release is still

prominent at the end of the train (Fig. 19). Therefore, I applied a stronger stimulation protocol (900 stimuli at 100 Hz) and monitored recovery of single action potential evoked EPSCs at different times after the stimulation train (Fig. 23B) (Sakaba, 2006). Also in this case, EPSC recovery was significantly slower in double mutant neurons as compared to controls (Fig. 23B). However, the relative difference of EPSC recovery time courses between mutant and control cells after 100 Hz stimulation was less than after 40 Hz stiumulation.

Figure 23 Strong Impairment of RRP Recovery in Double Mutant Neurons after Electrical Discharge of the RRP. The RRP was depleted by 100 stimuli at 40 Hz frequency, and then RRP refilling was followed by measuring the relative recovery of synaptic responses after 0.25, 1.3, 6, 8, 10 and 30 seconds (A). (B) RRP refilling monitored after 900 stimuli at 100 Hz. % recovery of EPSCs after 0.25, 1.3, 6, 8, 10, 30 and 60 seconds is shown (Ctrl, n = 15; DMut, n = 16). After electrical discharge of the RRP, EPSC recovery was markedly slower in double mutant neurons as compared to controls. Recovery time constants after 40 Hz (τ1- τ2): Ctrl = 0.51s – 1.29s; DMut = 1.78s – 12.19s. Recovery time constants after 100 Hz (τ1- τ2): Ctrl = 6.51s – 30.43s; DMut = 16.89s – 40.22s (Ctrl, n = 17; DMut, n = 18). Data shown are mean ± standard error of the mean.

Taken together, above results indicate that in double mutant neurons, Ca²⁺ -dependent and -in-dependent refilling kinetics of the RRP are not impaired. On the other hand, RRP refilling is strongly impaired only when the RRP is completely depleted by a high frequency stimulation train (40-100 Hz).

3.4.3.4 Strong Reduction in Calcymicin-Induced Neurotransmitter Release