Fase II: Construyendo el baúl de los recuerdos

The transition from the completely rested state to the in vivo state has already been sparsely described in chapter 3. EPSC amplitudes in answer to stimulation with Poisson-distributed pulse trains were plotted against time and fitted with double ex- ponential functions. Infigure 5.1 Aan example of a cell stimulated with a 20 Hz train is displayed. The double exponential fit clearly shows a slow tail with a characteristic time constant of 19 s. As it can be seen in figure 5.1 B all 15 cells in which this stimulation protocol was recorded show a double exponential decay with a slow time constant of on average 85 s.

According to the theory of the basic vesicle release model presented in chapter 4

the decrease in amplitude should not show a double exponential decay with a very slow time constant. For stimulations with regular spike trains the calculations for the time course leading from an arbitrary amplitude to the steady state amplitude of a given stimulation frequency predicted a single exponential decay. In the case of Poisson-distributed activity the time course is in theory more complex and cannot be described analytically. Nevertheless, a slow time constant of more than 10 s does not at all comply with the predictions of the vesicle release model as it is far slower than both time constants of the model (stimulation frequency and recovery time constant). Therefore, an additional effect has to be present. One possible explanation for this slow decay might be a change of the model parameters over time. To address this issue, the model was fitted to short intervals of the whole EPSC amplitude trace. The intervals had a length of 10 s and the first interval started 1 s after the beginning of the conditioning period. The first second was eliminated from this analysis to exclude the

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Figure 5.1:A: Development of EPSC amplitudes after starting the conditioning. Each gray dot corresponds to a single EPSC. The cell was stimulated with a 20 Hz Poisson-distributed activity train. The black line represents a double exponential fit to the gray dots. The parameters are given in the figure, Rf ast is the amplitude fraction of the exponential term with the time constantτf astdivided by the sum of the amplitudes of both exponential terms. The inset shows a magnified view of the first second to illustrate the initial steep decline. B: Summary plot of 15 cells. All cells were stimulated with the same activity pattern. For clarity only the exponential fits are shown. The parameters and the inset are presented in the same way as in A.

effects of the initial rapid decay phase. The following intervals were shifted by steps with a length of 2 s until the end of the 120 s long conditioning period was reached. The integration time of 10 s was chosen as a compromise between time resolution

Figure 5.2: A: development of model parameters over time. A1 shows the correlation coefficient, A2 - A4 show the three free model parameters. The model was fit to intervals of 10 s and the interval was shifted in steps of 2 s. Responses from the first second were eliminated from this analysis (see text). Each dot corresponds to an independent fit. The time interval of data used for each fit is restricted by the x value of the plotted dot and the x value plus 20 s. B: Comparison of the first and last interval for each of the four considered variables. Significant changes are marked with asterisks.

and signal to noise ratio. This period provides for a stimulation frequency of 20 Hz an average of 200 data points. This is enough input for the model to produce stable parameter sets. On the other side the time interval is still short enough to give a reasonable time resolution.

The graphs in figure5.2A show the development of the correlation coefficient and the three free model parameters over time at the beginning of the conditioning protocol. The displayed cell is the same that can be seen in figure5.1 A. As the minimum time interval that is needed to calculate a stable fit is 10 s, the time resolution of these plots is rather low. Therefore, no function could be fitted to the data points to quantify the time course of the parameter developments. To test the significance of the parameter changes the first and last time intervals were compared for all 15 cells. The results of this evaluation is displayed in figure 5.2 B. The most obvious change is the decrease in vesicle pool size to 0.64 of the value at the beginning of the conditioning. This parameter does not reflect the momentary pool size but the maximal pool size as defined by the model. On the other side this pool size does also not correspond to the

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full vesicle pool of a completely recovered synapse. Instead it reflects only the fraction recovered with the fast time constant of the vesicle recovery process. A change in this parameter influences the vesicle recovery time course. In this regard the vesicle pool size is directly related to the changed recovery time constant. Both parameters together define the dynamics of the vesicle pool refilling. Apart from this also the release probability shows a slight increase over time which suggests an accumulation of calcium in the presynaptic terminal.