Making quantal analysis more convenient, fast, and accurate: user-friendly software QUANTAN

Abstract

Quantal analysis of synaptic transmission is an important tool for understanding the mechanisms of synaptic plasticity and synaptic regulation. Although several custom-made and commercial algorithms have been created for the analysis of spontaneous synaptic activity, software for the analysis of action potential evoked release remains very limited. The present paper describes a user-friendly software package QUANTAN which has been created to analyze electrical recordings of postsynaptic responses. The program package is written using Borland C++ under Windows platform. QUANTAN employs and compares several algorithms to extract the average quantal content of synaptic responses, including direct quantal counts, the analysis of synaptic amplitudes, and the analysis of integrated current traces. The integration of several methods in one user-friendly program package makes quantal analysis of action potential evoked release more reliable and accurate. To evaluate the variability in quantal content, QUANTAN performs deconvolution of the distributions of amplitudes or areas of synaptic responses employing a ridge regression method. Other capabilities of QUANTAN include the analysis of the time-course and stationarity of quantal release. In summary, QUANTAN uses digital records of synaptic responses as an input and computes the distribution of quantal content and synaptic parameters. QUANTAN is freely available to other scholars over the internet.

1. Focal recordings from the mouse diaphragm nmj

A. Recording site: an endplate stained with 2-Di-4-Asp. B. Examples of EPSCs recorded at physiological conditions (2 mM Ca²⁺) and at the reduced Ca²⁺ (0.5 mM). Recordings at 2 mM Ca²⁺ demonstrate multiquantal EPSCs followed by asynchronous quanta. Recordings at 0.5 mM Ca²⁺ demonstrate a unitary EPSC (top), a transmission failure followed by an asynchronous mEPSC (middle), and a double quantal EPSC (bottom).

2. General organization of QUANTAN software

A. Summary of the QUANTAN modules involved in different steps of quantal analysis B. Starting QUANTAN: the initial dialog window.

3. Visual quantal detection

A. A dialog window for the visual quantal detection. The recorded episode is displayed, the EPSC area is marked, arrowheads point to the detected events. Detection parameters are displayed in the top right corner, and they can be edited and saved. A user can inspect all the sweeps in succession (“Next” button), only non-failure sweeps (“Events” button), or enter a sweep number (“Sweep N” button). A click on a screen displays the corresponding time-point and the value of the signal (Units). B. An example of multiple quantal peaks (arrowheads) detected in a single EPSC recorded from the mouse neuromuscular junction (extracellular Ca²⁺ was reduced to 0.5 mM, stimulation frequency was 15 Hz). C. Gaussian filtering does not change the shape of the signal while reducing the noise. Thin line – recorded signal; thick line – the same signal after filtering with 1/f_c=75 time points at the digital resolution of 50 μs.

4. Average quantal and multiquantal EPSCs

A. A dialog window for the visualization of the average quantal and multiquantal EPSCs. The list of the files analyzed appears in the “File” window. The onset and the end points of the average EPSCs are determined in response to the “EPSC borders” button, and, thus, the time window for synchronous release is defined. The latencies of the action potential, the onset, and the end of the average EPSC appear in the windows “AP”, “B”, and “E”, respectively. These values can be edited and saved by a user (“Save” button). The graph (appears in response to “Average trace” button) shows the average single and double EPSCs (thin lines, color coded), as well as the average of all the EPSCs in a highlighted file (thick dotted line). The table (right bottom corner) shows the ratios of the sizes (integrated traces) of multiple EPSCs to the size of the unitary EPSC. In this experiment, EPSCs were recorded from the mouse nmj at 15 Hz stimulation frequency at the reduced (0.5 mM) extracellular Ca²⁺. The area of the average double EPSC was 2.17 times larger than the average area of the unitary EPSC; triple quantal EPSCs were not detected; the average failure ran above the baseline, its size being equal 0. Thus, in this example quantal detection could be considered accurate, since the EPSCs detected as doubles are approximately twice (2.17 times) larger than the EPSCs detected as unitary. The ratio of the area of the average EPSC to the unitary EPSC (0.522) can be considered a reasonable estimate of m. The amplitudes and the areas of the average EPSCs can be plotted in this window and saved in the *.pfl file (“Amplitude” and “Integral” buttons), as well as the amplitudes of the average unitary EPSCs (“Q” button) and the estimates of m (“m” button). B. The average unitary EPSC and the average mEPSC obtained from the same dataset are similar, which confirms that that multiple quantal EPSCs were not classified as single quanta. Note that the average mEPSC has a slightly larger amplitude than the average unitary EPSC, and it is slightly sharper. The reason is that mEPSCs were superimposed according to their peak latencies, while EPSCs were always superimposed according to the action potential latencies, thus some asynchrony of synaptic latencies was present in the superposition of unitary EPSCs.

5. Evaluation of quantal content m

In this experiment, EPSCs (300 sweeps per file) were recorded from the lobster nmj at stimulation frequencies of 5, 10, 20, 30, 40, and 50 Hz (points 0−5). Quantal content was evaluated by four methods (checked): direct counts (circles); the average EPSC amplitude divided by the average amplitude of the unitary EPSC from the highlighted file (light triangles); the average EPSC area divided by the average area of the unitary EPSC from the highlighted file (dark triangles); the area of the average superimosed EPSC divided by the area of the average superimposed unitary EPSC from the highlighted file (squares). At the points 0−2 (5−20 Hz stimulation frequency) the results of all the methods agree, thus direct counts can be considered accurate. At the points 3−5 (30−50 Hz) the area measurements (squares and dark triangles) give the highest quantal content. Since these two m estimates obtained by area measurements are in a good agreement, area measurements can be considered accurate. In contrast, amplitude measurements (light triangles) and direct counts (circles) underestimate quantal content at high stimulation frequencies (30−50 Hz, points 3−5). Other methods available for m evaluation (listed) are: 1) the amplitude of the average superimposed EPSC divided by the amplitude of the average superimposed unitary EPSC in the highlighted file; 2) the average EPSC amplitude or area divided by the average amplitude or area of mEPSCs collected from either the highlighted file or throughout the experiment.

6. Deriving the distribution of quantal content m by the deconvolution method

All the graphs were created within the “Deconvolution” dialog window. A. The distribution of sizes as areas (integrated current traces) of EPSCs (squares) and quantal events (mEPSCs, columns) recorded from the same site at the mouse nmj. B. The distribution of EPSC sizes (squares) is enclosed between simulated distributions of unitary and 11-quantal events (lines). C. Deconvolution performed with the maximal number of quanta released in a trial m_max=11. Negative frequencies were obtained for m=9, m=10, and m=11. D. Deconvolution performed with the maximal number of quanta released in a trial m_max =8, which corresponds to the maximal number of quanta in a trail which had a positive frequency at the previous step (panel C). E. Original (squares) and restored (bars) distributions of EPSC sizes. F. Fit of the distribution of m by binomial (black line) and Poissonian (gray line) statistical models.