Evoked and Spontaneous Synaptic Transmission

To test for a functional redundancy of α- and β-SNAP, I analysed glutamatergic synaptic transmission of double mutant autaptic neurons, where in addition to the absence of the β-SNAP isoform also α-SNAP expression is strongly reduced, leading to a ~70% decrease in total SNAP levels (Fig. 15B).

Figure 17 Evoked Glutamatergic Synaptic Transmission in Autaptic Double-Mutant Neurons: Smaller RRP and Increased Pvr. To determine the functional effects of a ~70% reduction in total SNAP levels, I analysed glutamatergic transmission in autaptic hippocampal neurons. The results indicate that, while evoked EPSCs in double mutant neurons were not significantly different from control neurons, the RRP size was significantly smaller (~25%) and Pvr was ~30% higher in double mutant neurons. (A) EPSC traces (left panel) and release induced by the application of 0.5 M sucrose solution for 9 s (right panel) in control (black) and double mutant (grey) neurons. (B) Mean EPSC amplitudes in control (black) and double mutant cells (grey). (C) Mean RRP size estimated by the charge integral measured after release induced by application of 0.5 M sucrose solution. (D) Mean Pvr, calculated by dividing the charge transfer during single EPSCs by the charge transfer measured during RRP release. Numbers in the bars indicate the number of cells. Error bars indicate standard error of the mean. Stars above two bars indicate a statistically significant difference.

Surprisingly, while evoked EPSCs in double mutant neurons were not significantly different from control values, the RRP size was ~25% smaller (Fig. 17A-B-C).

Unaltered evoked EPSCs together with smaller RRP sizes indicate an increase in Pvr, which was indeed ~30% higher in double mutant neurons (Fig. 17D).

I also analysed spontaneous synaptic transmission by recording spontaneous miniature currents (mEPSC) (Fig. 18) in the presence of Tetrodotoxin (TTX), a Na⁺ channel blocker, which prevents action potential generation. Under these conditions, evoked transmission is blocked and only spontaneously fusing quanta are recorded.

Double mutant neurons showed no statistically significant differences from controls with regard to the amplitude or the frequency of mEPSCs (Fig. 18B-C).

The effects of the mutations on both RRP size and Pvr are predicted to have significant consequences for short-term synaptic plasticity (Zucker and Reger, 2002).

I therefore monitored the stability of evoked EPSCs during trains of action potentials at 10 Hz frequency. As expected, double mutant neurons showed stronger and much faster depression during the simulation train (Fig. 19). Importantly and in accord with the increase in Pvr, depression in double mutant cells was already detectable with the second stimulus. A change in the ratio of the second to the first evoked EPSC in resonse to a stimulus pair (so called “Paired-Pulse Depression Ratio”) essentially reflects a change in Pvr, with an increased Pvr causing increased Paired-Pulse Depression and vice-versa (Zucker and Regher, 2002). Similarly, depression of evoked EPSCs during 40 Hz stimulation was faster in double mutant neurons (Fig.

Figure 18 Normal Spontaneous Glutamatergic Synaptic Transmission in Autaptic Double-Mutant Neurons. Spontaneous synaptic transmission was analysed by recording spontaneous miniature currents (mEPSC) at a holding potential of –70mV in the presence of the Na⁺ channel blocker Tetrodotoxin (TTX). Double mutant neurons showed no statistically significant difference in the amplitude or the frequency of mEPSCs. (A) Representative traces of mEPSC activity in control (black) and double mutant cells (grey). (B) Mean mEPSC amplitudes. (C) Mean mEPSC frequencies. (D) Similar current amplitude of responses to 10 μM Kainate application in double mutant and control neurons, indicating that the total pool of surface expressed glutamate (Kainate and AMPA-type) receptors is very similar between the two genotypes. Numbers in the bars indicate the number of cells tested. Error bars indicate standard error of the mean.

100 stimuli at 40 Hz are believed to deplete the RRP of hippocampal neurons (Moulder et al., 2005; Pyott and Rosenmund, 2002). Elmqvist and Quatsel (1965) developed a cumulative method to calculate the RRP size from the integral of the total synaptic charge being transferred during a stimulation train. In this method, the cumulative charge is plotted versus time and a linear fit to a steady-state phase is

extrapolated to a point on the y-axis, which reflects the RRP size. Like the RRP measure based on hypertonic sucrose application, this independent RRP measure yielded a similar relative estimate for the RRP change in double mutant neurons, with a ~28% reduction as compared to control data (Fig. 19D). Since the contribution of the tonic current is largely neglected by the linear fit, the RRP estimate obtained with this method mainly arises from the phasic component. Therefore, above data indicate that the RRP size and phasic release are decreased to the same extent in double mutant cells as compared to control neurons.

Figure 19 Short-Term Synaptic Plasticity is Impaired in Double Mutant Neurons. The effects of the mutations on both RRP size and Pvr are predicted to have significant consequences for short-term synaptic plasticity. As expected, double mutant neurons showed stronger and much faster EPSC depression during both 10 and 40 Hz stimulation trains. Interestingly, tonic release was reduced in double mutant neurons. (A) Normalised EPSC depression during 10 Hz (Ctrl, n = 234;

DMut, n = 281). (B) Normalised EPSC depression during 40Hz (Ctrl, n

= 151; DMut, n = 167). (C) Normalized average of continuous EPSC

marked section (white box) for double mutant and control cumulative traces. (D) Integral of the total synaptic charge being transferred during the 40 Hz stimulation train. An extrapolation method yielded similar relative estimates for RRP size changes in double mutant neurons (intercept double mutant / intercept control = 0.243/0.342 ~ 28%).

Numbers in the bars indicate the number of cells tested. Error bars indicate standard error of the mean. Stars above two bars indicate a statistically significant difference.

Surprisingly, the amount of tonic release elicited by the 40 Hz stimulation train was also drastically reduced in double mutant neurons, as shown in Fig. 19C by the shift in the steady-state current level after phasic release has decayed.

3.4.3.2 Unaltered Synaptic Release Probability and Munc-13-1 Dependent