The mechanism of cAMP-mediated enhancement at a cerebellar synapse

Abstract

Increases in cAMP have been shown previously to enhance the strength of the granule cell to Purkinje cell synapse. We have examined the mechanisms underlying this enhancement in rat cerebellar brain slices. Elevation of cAMP levels by forskolin increased synaptic currents in a dose-dependent manner. Fluorometric calcium measurements revealed that forskolin did not affect presynaptic calcium influx or resting calcium levels. The waveform of the presynaptic volley was also unaltered, indicating that changes in the presynaptic action potential did not contribute to synaptic enhancement. However, forskolin enhanced the frequency but not the size of spontaneous miniature EPSCs. There was a one-to-one correspondence between increases of spontaneous and evoked neurotransmitter release. These results suggest that forskolin increases release at this synapse via presynaptic mechanisms that do not alter calcium influx. The effect of forskolin on paired-pulse facilitation was examined to assess the relative contributions of changes in the probability of release (p) and changes in the number of functional release sites (n) to this form of enhancement. These experiments suggest that although small changes in n cannot be excluded, most of the enhancement arises from increases in p.

Fig. 1.

Forskolin enhances synaptic currents in a dose-dependent manner. A, Continuous application of 50 μm forskolin causes a rapid increase in the Purkinje cell EPSC. The time course of the peak current is plotted before and after the addition of forskolin (bar). Theinset shows averaged EPSCs before (thin line) and after (thick line) exposure to the drug. The time constant of decay of the EPSC in forskolin (6.1 msec) was not significantly changed from that of control (5.3 msec).Traces are the averages of 15–20 trials.B, Summary of the mean relative responses (± SEM) of synaptic currents to continuous application of 1 μm(circles; 207 ± 38%; n = 5), 20 μm (squares; 290 ± 42%;n = 10), and 50 μm(triangles; 390 ± 45%; n = 11) forskolin and of 50 μm 1,9-dideoxy-forskolin (diamonds; 115 ± 15%; n = 3). The time course of each experiment is normalized to the average control peak EPSC. Forskolin was added at time 0.

Fig. 2.

Forskolin enhances spontaneous mEPSCs.A, Purkinje cell mEPSCs recorded before (left) and after (right) application of 50 μm forskolin. Unprocessed traces shown in compressed time scale are taken from the last 5 sec of four consecutive 20 sec epochs. Below each side is a traceshown in expanded time scale. B, Time course of the average mEPSC frequency in 20 sec epochs during continuous exposure to 50 μm forskolin from time 0. The example is taken from the same experiment shown in A. C, Amplitude histograms from the same experiment for control (thin line) and 50 μm forskolin (bold line) at a holding potential of −70 mV. Insetshows that normalized cumulative amplitude distributions for control (thin line) and forskolin (bold line) are similar (P = 0.85 by Kolmogorov–Smirnov test).

Fig. 3.

Forskolin enhances mEPSC in a dose-dependent manner. Summary of the average relative responses of spontaneous mEPSCs to application of 1 μm (circles; 170 ± 5%; n = 4), 20 μm(triangles; 230 ± 6%; n = 6), and 50 μm (squares; 280 ± 8%;n = 4) forskolin as well as 50 μm1,9-dideoxy-forskolin (diamonds; 126 ± 12%;n = 3). Forskolin was applied continuously from time 0. The time course of each experiment was normalized to the average baseline frequency of control mEPSC. No significant change in the mEPSC amplitude distributions was detected at any dose of forskolin (e.g., at 50 μm forskolin, P = 0.88 ± 0.04; n = 4).

Fig. 4.

Effects of forskolin on presynaptic calcium transients. A, B, Simultaneous time courses of the amplitude of peak magnesium green fluorescence transients (A) and presynaptic volleys (B) during exposure to 20 μmforskolin (t = 0). Insets show superimposed average traces of control (thin line) and forskolin (bold line) ΔF/F (A) and volleys (B). Each trace is an average of 20 trials. The stimulus artifact, determined by the addition of 1 μm TTX at the end of the experiment, was subtracted off the averaged traces of presynaptic volleys.C, The same time courses shown in A(circles) and B (squares) are normalized to their respective mean control values. Forskolin enhancement of presynaptic volley (35%) is very similar to that of presynaptic calcium transients (30%). D, Plot of the relative magnesium green fluorescence transient as a function of the relative enhancement of presynaptic volley for (from left to right) 1 μm (n = 4), 50 μm (n = 8), and 20 μm(n = 4) forskolin. Thin linerepresents x = y.

Fig. 5.

Fura-2 fluorescence measurements of presynaptic calcium influx. A, Average traces of fluorescence changes produced by one stimulus superimposed on that of two stimuli in control conditions (left) and in the presence of 50 μm forskolin (middle).Right, The trace for the first stimulus of control is scaled to that of forskolin to emphasize that the relative fluorescence change between one and two stimuli is the same for the two conditions. Traces are the averages of 15–20 trials. B, The time course of peak fura-2 ΔF/F for one (lower curve) and two (upper curve) stimuli before and after the addition of forskolin (time 0). The relative change in ΔF/F for two versus one stimuli is plotted below. C, Summary of the fura-2 fluorescence changes for four experiments. The effects of forskolin on the relative mean ΔF/F values for one (lower curve) and two (upper curve) stimuli are plotted as a function of time. The average ratio of ΔF/F for two versus one stimuli, a direct measurement of the change in calcium influx, is plottedbelow.

Fig. 6.

Relationship of forskolin enhancement of evoked to spontaneous release. Plot of the average relative peak EPSC of evoked versus spontaneous responses for (left to right) 0 (theoretical point), 1, 20, and 50 μm forskolin. The relative peak EPSCs were corrected for small enhancements of presynaptic volley by dividing the mean relative evoked enhancement (see Fig. 1B) by the mean relative ΔF/F enhancement (see Fig.4D) for each forskolin concentration. Thin line represents x = y.

Fig. 7.

Effects of forskolin on paired-pulse facilitation.A, Peak EPSCs of two stimuli separated by 30 msec were plotted as a function of time before and after 50 μmforskolin application (time 0). The amount of facilitation, recorded as the ratio of the amplitude of the second peak (white) to that of the first peak (black) is shownbelow. The top right panel shows the average traces for control (thin line) and 50 μm forskolin (bold line) currents. Scaling of the first peak of the control trace to the first peak of the forskolin trace shows a decrease of facilitation in the presence of forskolin (bottom right panel). Traces are averages of 10–15 trials. Stimulus artifacts are blanked for clarity. B, Analogous experiment to that described in A except 750 μm kynurenic acid is present. C, Mean ± SEM of the relative facilitation before and after the continuous application of 50 μm forskolin (circles;n = 4) or 1,9-dideoxy-forskolin (squares; n = 4). Drug application occurred at time 0. The time course of the facilitated ratio of each experiment was normalized to its mean control value. Eachpoint in the time course of the facilitation ratio represents the average value of three consecutive time points.