Switching off and on of synaptic sites at aplysia sensorimotor synapses

Abstract

Using the highly plastic synapses between mechanoreceptor sensory neurons and siphon motor neurons of Aplysia as a model, we have investigated whether switching off and on of individual synaptic release sites is a strategy that is used by neurons in forms of short-term synaptic modulation with a time course of minutes to hours. We have modified some of the techniques of classical quantal analysis and examined the kinetics of synaptic depression under different stimulation protocols to answer this question. Our analysis shows that both synaptic depression caused by homosynaptic activity and synaptic facilitation induced by an endogenous facilitatory transmitter occur by means of the shutting off and turning on, respectively, of synaptic sites, without intermediate changes in the probability of release. Our findings imply that other forms of plasticity at these synapses, such as post-tetanic potentiation, long-term facilitation, and long-term potentiation, are also expressed by all-or-none changes in activity at individual sites. We thus show that in addition to the mechanisms of synaptic integration that are known to operate in single cells and networks, neurons can exercise a further layer of fine control, at the level of individual release sites.

Fig. 1.

Small variability of depressed EPSPs and large facilitation by 5HT. Records of successive groups of monosynaptic potentials from a sensorimotor pair in culture (A) and plot of the whole experiment (B). C, Magnified view of EPSPs 4–15 showing the small variation in amplitude. Stimulation was interrupted for 5 min after EPSP 15, and two more EPSPs (data not shown) were elicited before addition of 5HT. The interstimulus interval was 30 sec. Calibration bars: A, 2 mV, 10 msec; C, 1 mV, 10 msec.

Fig. 2.

Binomial fits of amplitude distributions of sensorimotor EPSPs. A, Examples of amplitude histograms and fits. In each column the top andbottom amplitude histograms represent miniature synaptic potentials evoked with hypertonic sucrose and evoked EPSPs from the same pair of cells, respectively. Fits (solid lines) to the mini distributions were performed using Equation 1 of Bekkers et al. (1990); fits to the EPSP distributions were performed on the assumption that evoked release follows a binomial distribution based on the quantal parameters derived from the mini distribution in the same pair of cells. The broken lines represent the underlying distributions of noise (peak at zero) and convolutions of the uniquantal distribution that resulted in the best fits, characterized by the binomial parameters n and p shown to the right of each graph. B,C, Correlation between estimates of pfrom histogram fits and from calculations. B, Estimates of p derived from Equations 2 (all quantal variability intrasite) and 3 (all quantal variability intersite) are plotted against estimates of p from the best fits to histograms. The line through the origin has a slope of 1, representing perfect agreement. C, Estimates ofp derived from Equation 1 (quantal variability from both intrasite and intersite sources), with the fraction of the total variability from intrasite variance equal to 0.65.

Fig. 3.

Comparison of derived p with “maximal” p predicted from uniform decline inp during homosynaptic depression. Binomialp estimated from the best fit to amplitude histograms of EPSPs (pfit) or from calculation by Equations 2 (p intra) and 3 (p inter) is generally much greater than that predicted if homosynaptic depression were caused by a uniform decrease inp at all release sites (maximal p).Line has a slope of 1. See Results for definition of maximal p.

Fig. 4.

Absence of change inp with synaptic depression or 5HT-induced facilitation. EPSP amplitude (left) and ratio of the variance to the mean (right) for stationary periods in individual experiments (open circles) at earlier and later times during progressive synaptic depression (29 experiments) and before and after application of 5HT (10 experiments); filled squares are means. Experiments on depression included 8 in intact ganglia, 12 in cultures in normal ASW medium, and 9 in cultures with high calcium medium. Because there was no difference among the three groups, the results were pooled. Experiments with 5HT included five in intact ganglia, three with normal ASW in culture, and two with high calcium medium in culture. The absence of a change in the average ratio of the variance to the mean in both homosynaptic depression and 5HT-induced facilitation implies no change in p.

Fig. 5.

Absence of change in sliding estimate ofp during homosynaptic depression. Plots inA and B are from one experiment and illustrate how the slopes of sliding m andp were derived and normalized. Sliding p(calculated from Eq. 1, with W = 0.65) andm were determined for groups of five responses, moving one response at a time. In A, the approximately linear decline in sliding m was fit by linear regression, as was the corresponding portion of the plot of sliding p.B illustrates normalization of the slopes of slidingm and p to permit direct comparison of the slopes; the slopes were normalized by setting the respectivey-intercepts equal to 1. C is a plot of the normalized slope of sliding p against the normalized slope of sliding m in nine experiments on cultures in normal ASW medium. The two filled circles represent experiments in which the average p was ∼0.3;p in all of the other experiments was >0.6. Theline represents a slope of zero for slidingp. D, For experiments without an independent estimate of the quantal amplitude and variance, the variance (V) and mean (M) of the EPSP amplitude were used to test for changes in p; no change in the ratioV/M implies no change in p(see Results). The line represents a slope of zero for sliding V/M.

Fig. 6.

Test of sliding p technique by application to simulations with different contributions of decliningp to depression. A, Slope of calculated sliding p as a function of different rates of decline inp in simulations (n = 10 for each condition). B, Slope of the relation betweenp and m in simulations. The slope of this relation varies with the contribution of declining p to homosynaptic depression, ranging from close to 1 when all of the depression is caused by a drop in p, to near 0 when depression is attributable exclusively to a decline inn. See Materials and Methods for details.

Fig. 7.

Decline in recovery from depression with progressive stimulation. Stimulation was stopped after 1, 3, or 10 stimuli in different experiments (each experiment was on a different pair of cells), and recovery was examined by presenting a single stimulus after 0.5, 10, 30, 60, 120, or 300 sec. Recovery relative to the first EPSP of the experiment proceeds with an exponential time course to a plateau that is below the level of the initial EPSP. The plateau decreases with increasing number of stimuli, suggesting that an increasing proportion of the sites is progressively switched off. Each point is the average of four to seven separate experiments (error bars indicate SD). The time constants of recovery based on the exponential fits are 11.63, 20.11, and 40.03 sec after 1, 3, and 10 stimuli, respectively. Each of these values is within the 99% confidence intervals of the other two.

Fig. 8.

Fits of homosynaptic depression and recovery with a model incorporating switching off of release sites. Shown are fits of a sample experiment with three different models of depression; only the switching model fits both the kinetics of depression and recovery after rest. Experiments were performed with interstimulus intervals of 30 sec, with rests indicated by bars. For the same experiment, EPSPs 1 to 15 were fit with three different models (see Results for details). The solid curves for stimuli 16 to 30 are predictions based on the parameters obtained from best fits of stimuli 1 to 15 (Table 2). Only the model incorporating switching off of synaptic sites is able both to fit the initial depression and to predict the recovery and depression after rest.

Fig. 9.

A, Fits of depression and of recovery after varying periods of rest with the switching model. Experiments (circles) with 30 sec interstimulus intervals and interspersed periods of rest of various durations were fit with the switching model (solid and dashed lines; see Table 2 for parameter values). Stimuli after 0.5 and 10 sec rests were followed by 60 and 50 sec rests, respectively, to keep the average interstimulus interval constant. B, Fits of experiments with changing interstimulus intervals. Eachplot represents a single experiment. The interstimulus interval, initially 90 sec, was changed to 30 sec after 10 stimuli, as indicated by the double lines. Rests after the first EPSPs were 0.5 and 60 sec, respectively, and a second rest of 5 min was introduced after EPSP 20 or 21. Fits are solid anddashed lines. See Table 2 for parameter values that gave the best fit.