Presynaptic mechanism underlying cAMP-dependent synaptic potentiation

Abstract

The adenylyl cyclase activator forskolin presynaptically facilitates synaptic transmission at many synapses, but the exact intracellular mechanism underlying this effect is not known. We studied this issue at the calyx of Held, where it is possible to make simultaneous presynaptic and postsynaptic whole-cell recordings. Bath application of forskolin or intracellular application of cAMP into presynaptic terminals strongly potentiated EPSCs. The forskolin-induced synaptic potentiation was associated with increases in release probability (P) and number of releasable synaptic vesicles (N). Forskolin had no effect on the peak amplitudes of presynaptic Ca2+ currents or K+ currents, suggesting that the main target of cAMP resides in downstream of Ca2+ influx. Intracellular application of the selective Epac agonist 8-(4-chlorophenylthio)-2'-O-methyl-cAMP into presynaptic terminals potentiated EPSCs, suggesting that Epac is the main target of cAMP-induced synaptic potentiation. We conclude that an increase in cAMP concentration in the nerve terminal facilitates transmitter release by increasing both release probability and number of releasable vesicles via activating the Epac pathway at the calyx of Held.

Figure 1.

Potentiation of EPSCs by forskolin. A, Bath application of forskolin (50 μm; ○) potentiated EPSCs, although the inactive analog Dd-forskolin (•) had no such effect. Hatched bars in A, D, and F indicate the period of drug application, which was started at time 0. Averaged EPSCs (n = 5) sampled before (a) and after (b) forskolin application are superimposed in the inset. The aCSF contained 1 mm Ca²⁺, 2 mm Mg²⁺, and 2 mm kynurenate. B, Mean magnitudes of potentiation (error bars; ±SEM) induced by forskolin (50 μm) in 1 mm Ca²⁺, 2 mm Mg²⁺, 2 mm kynurenate (n = 7; C, •), 2 mm Ca²⁺, 1 mm Mg²⁺, 5 mm kynurenate (n = 4; C, ○), 0.6 mm Ca²⁺, 3.8 mm Mg²⁺ (n = 4; C, ▾), 1 mm Ca²⁺, 2 mm Mg²⁺ (n = 5; C, ▿), and 2 mm Ca²⁺, 1 mm Mg²⁺ (n = 4, C, ▪). There was no significant difference (N.S.) between values in a bracket (ANOVA; p > 0.5). The magnitude of potentiation in 1 mm Ca²⁺, 2 mm Mg²⁺ was 121 ± 17% (see Results for values in other conditions). C, An inverse relationship between the initial EPSC amplitude (abscissa) and the magnitude of forskolin-induced potentiation (ordinate, r = –0.78) for data obtained from different conditions indicated in B. D, The concentration dependence of forskolin effect in the aCSF containing 1 mm Ca²⁺, 2 mm Mg²⁺, 2 mm kynurenate. Ordinate indicates the percentage of EPSC potentiation measured 15 min after forskolin application. A curve fit to data points represents the equation; the magnitude of potentiation: (%) = [maximal potentiation]/[1 + EC₅₀/forskolin concentration)ⁿ], where the maximal potentiation is 353%, EC₅₀ is 2.3μm, and Hill coefficient is 0.95. The time plots of EPSC potentiations induced by forskolin of different concentrations (4–7 cells each) are shown in the inset with the EPSC amplitude being normalized to control before forskolin application. E, Cumulative amplitude histograms of spontaneous mEPSCs recorded in the standard aCSF in the presence of TTX (1 μm). Sample traces are mEPSCs averaged from 300 events before and after forskolin (50 μm) application (completely superimposed). There was no significant difference in the distribution profile of mEPSC amplitude between the presence and absence of forskolin (Kolmogorov–Smirnov test; p > 0.5). F, EPSC potentiation induced by forskolin (50 μm) at different postnatal ages. Data are derived from four to seven cells. The potentiation at P21–P22 was significantly smaller than that at P8–P9 (p < 0.05; two sample t test). The aCSF contained 1 mm Ca²⁺, 2 mm Mg²⁺, and 2 mm kynurenate.

Figure 2.

Intracellular application of cAMP into the calyceal terminal potentiated EPSCs. A, EPSCs are evoked by presynaptic action potentials in simultaneous presynaptic and postsynaptic recording (a). After retracting a presynaptic pipette, whole-cell recording was made again from the same terminal with a new patch pipette containing cAMP (500 μm; hatched bars). This caused a potentiation of EPSCs (b, open circles). Sample records of presynaptic action potentials (V_pre) and EPSCs (I_post) are averaged, each from 10 consecutive records before (a) and after (b) intracellular cAMP application. The nerve terminal underwent a depolarization after cAMP application (filled circles). B, Data summarized from five experiments for the EPSC potentiations (open circles) and presynaptic depolarizations (filled circles) caused by cAMP. C, An example showing that presynaptic depolarization by 6.1 mV by passing currents caused only 19% increase in EPSC amplitude.

Figure 3.

Forskolin increases both the number of releasable vesicles and release probability. A, Depressions of EPSCs during trains (20 stimuli) of 100 Hz stimulation before (control; open circles) and after (filled circles) application of forskolin (50 μm). Sample records of the one-sixth EPSCs before and after forskolin application are shown (superimposed) in the inset. B, Cumulative amplitudes of EPSCs during the 100 Hz train before (open circles) and after (filled circles) forskolin application. Amplitudes of EPSCs from sixteen-twentieth were fitted with a linear regression line and extrapolated to time 0 for estimating the readily releasable pool size. C, Mean number of releasable vesicles (N) multiplied by q, estimated in the low-Ca²⁺ (1 mm) high-Mg²⁺ (2 mm) aCSF, significantly increased (p < 0.05; paired t test) after forskolin application (n = 6). D, The mean release probability (P), which was estimated from the ratio of the first EPSC amplitude divided by Nq, underwent a significant increase (p < 0.05) after forskolin application (n = 6).

Figure 4.

Forskolin accelerates the time course of block of NMDA-EPSCs by MK-801. NMDA EPSCs were evoked at 0.033 Hz at a holding potential of +40 mV in the presence of MK-801 (40 μm). Data are derived from a different group of cells, one group in the presence of forskolin (50 μm; filled circles; n = 5) and the other in its absence (open circles; n = 5). The numbers on sample traces (superimposed) indicate the sequence of stimulation (1–50). Ordinate indicates the amplitude of NMDA-EPSCs normalized to the initial amplitude. Abscissa indicates the time after starting stimulation in the presence of MK-801. Mean relative amplitudes derived each from five cells were fitted with double exponential functions.

Figure 5.

Effects of forskolin on presynaptic Ca²⁺ currents, K⁺ currents, and holding currents. A, The current–voltage relationships of Ca²⁺ currents before (circles) and after (triangles) forskolin application (superimposed). The Ca²⁺ current amplitude was measured at 1.5 msec from the depolarizing command pulse onset and normalized to the value at 0 mV. Forskolin (50 μm) had no effect on the voltage-dependent Ca²⁺ currents, as also shown in completely overlapped sample records before and after forskolin application. B, The current–voltage relationships of K⁺ currents before (circles) and after (triangles) forskolin application. The K⁺ current amplitude was measured at 10 msec from the depolarizing pulse onset. Sample records show the effect of forskolin accelerating the decay of K⁺ current amplitude during the depolarizing pulse. C, The effect of forskolin on the presynaptic holding current (n = 4; error bars merge into symbols) measured at the holding potential of –80 mV (after correcting the liquid junction potential of 11.6 mV).

Figure 6.

Potentiation of EPSCs by presynaptic loading of 8CPT-2Me-cAMP. In simultaneous presynaptic and postsynaptic recordings, 8CPT-2Me-cAMP (500 μm) was loaded into the calyceal terminal by replacing the presynaptic pipette. A, Averaged sample records (from 10 events each) of presynaptic action potentials and EPSCs before and after 8CPT-2Me-cAMP application. 8CPT-2Me-cAMP enhanced EPSCs and slightly depolarized presynaptic terminal. Lines indicate control resting potential level (top column) and EPSC amplitude (bottom column). B, Summary data of five experiments showing the potentiation of EPSCs by presynaptically loaded 8CPT-2Me-cAMP.

Figure 7.

Coapplication of forskolin and phorbol ester. A, Forskolin (50 μm) potentiated EPSCs (a,b). Addition of PDBu (1 μm) further potentiated EPSCs (c). Sample records are averaged EPSCs (from 5 events) taken from different epochs (a,b,c, superimposed). B, Mean magnitude of EPSC potentiation by PDBu alone (PDBu), forskolin alone (Forskolin), and PDBu plus forskoin (P+F). The magnitude of potentiation by PDBu plus forskolin (693 ± 132%; n = 7) is similar to the sum of potentiations by PDBu (410 ± 82%; n = 5) and forskolin (335 ± 64%; n = 7).