Figure 3. A test of the basis for the increased magnitude of EPSCs with progressive depolarization of the presyanptic hair cell. A, The mean EPSC rate grows appreciably as a function of depolarization. B, The mean EPSC amplitude displays negligible dependence on the extent of depolarization. The error bars indicate 95% confidence intervals. The measured mean amplitude of about 2100 pA is consistent with that reported in Figure 4C of [11]. doi:10.1371/journal.pone.0038198.g003 sampled at 50 ms intervals. MiniAnalysis experienced difficulties in aligning complex EPSCs and performed well only when well- separated EPSCs were present in the amplitude sensitivity assay (Fig. 4E). In contrast, our algorithm retrieved the correct impulse- response function after only one or two iterations. Detection of Multiquantal Peaks Amplitude histograms of measured EPSCs are often used in electrophysiological studies to investigate the quantization of neurotransmitter release. To assess whether multiquantal events could be discerned with our algorithm, we performed simulations in which we fixed the quantities of neurotransmitter to be integer multiples of a smaller quantum and then calculated amplitude distributions at three stages between ligand binding and deconvolution. Stage one (Fig. 5A for a total of 150 receptors and Fig. 5B for 300 receptors) records the number of AMPA receptors open due to a burst of neurotransmitter. Stage two (Fig. 5C and D) includes filtering of the incoming current by the membrane time constant and the stochastic reclosure of AMPA receptors. Stage three (Fig. 5E and F) includes colored noise and subsequent deconvolution by our algorithm. The blurring evident in the simulated records is a result of several sources of noise: stochastic ligand binding, stochastic channel reclosure, and additive noise. Additional noise may arise at stage one because neurotransmitter released from presynaptic vesicles is imperfectly quantized. Stages two and three may be contaminated by signals originating from different presynaptic cells or active zones, whose waveforms may be filtered to different extents before reaching the recording electrode. The blurring of the EPSC amplitude distribution may be mitigated in at least two ways. First, increasing the number of AMPA receptors or the proportion of AMPA receptors that a single vesicle’s neurotransmitter opens will reduce the relative Blind Deconvolution of Postsynaptic Currents fluctuations in ligand binding and channel reclosure. Second, reducing the experimental noise will permit more accurate amplitude estimation by any deconvolution algorithm. If the simple model used here captures a minimum of the stochastic processes involved in generating EPSCs, we may conclude that intrinsic fluctuations provide a rationale for the inability to observe quantization peaks in EPSC amplitude histograms derived from complex physiological recordings. Discussion We have developed an automated self-consistent deconvolution algorithm for the analysis of challenging EPSC data. The statistical model on which the algorithm is based is simply stated and cleanly separated from the details of the approximations used in the implementation. Several numerical strategies allow the algorithm to run efficiently, requiring a temporal duration approximately proportional to the problem size. For instance, analyzing a 10 s recording sampled at 50 ms intervals (size N~2:105) requires less than one minute on a mid-range workstation. We have demonstrated the algorithm’s applicability by deconvolving complex postsynaptic recordings from an auditory synapse. The analysis reveals that increasing presynaptic depolarization leads to more frequent postsynaptic events, rather than larger ones. A series of tests using synthetic data shows that the algorithm performs favorably compared to the widely used MiniAnalysis program. Most algorithms require the noise level or the impulse response to be supplied by the user. Moreover, in algorithms that use a regularizer, for instance those based on Wiener filtering, the regularization constant must be adjusted empirically. Our algorithm automates the estimation of the impulse response, the noise, and the regularization constants. For users requiring manual control, however, these automatic steps may be selectively overridden. A switch changes the prior from QME to Gaussian, making the algorithm perform as a Wiener filter. The noise and regularization constants can be overridden and the impulse response need not be estimated from the data if it is known from previous experimentation. Integral to the study of synaptic dynamics through EPSC measurements is the kinetics of the EPSCs themselves. A fundamental challenge facing all algorithms is that of separating multiple overlapping EPSCs from individual EPSCs that simply have intrinsic variability in their kinetics. Highly sparse records seem to indicate that EPSC kinetics do not vary wildly, so it is parsimonious to introduce that assumption into our analysis and classify multiphasic EPSCs as being due to multiple overlapping EPSCs. The possibility cannot be excluded, however, that when multiple vesicles fuse in synchrony there are additional factors modulating the postsynaptic response and thus changing the EPSC kinetics. It remains unlikely that the analysis of postsynaptic records alone will adequately resolve this issue. The range of observed EPSC amplitudes and multiquantal release might emerge from a number of mechanisms, including synchronized exocytosis [13] and sequential fusion. A further possibility is prefusion, in which several vesicles combine to form a supervesicle prior to exocytosis. If the area of vesicle membrane is conserved during fusion, and if exchangers bring the concentration of neurotransmitter to some constant level, then a supervesicle originating from the fusion of q single vesicles experiences a volume increase–and hence an increase in transmitter content–from q-fold to q3/2–fold that of a single vesicle. This change prior to or during the exocytotic process PLoS ONE | www.plosone.org 6 June 2012 | Volume 7 | Issue 6 | e38198