Application of an Epac activator enhances neurotransmitter release at excitatory central synapses

Abstract

cAMP regulates secretory processes through both PKA-independent and PKA-dependent signaling pathways. Their relative contributions to fast neurotransmission are unclear at present, although forskolin, which is generally believed to enhance intracellular cAMP levels by stimulation of adenylyl cyclase activity, was shown to increase vesicular release probability (p) and the number of releasable vesicles (N) in various neuronal preparations. Using low-frequency (0.2 Hz) electrophysiological recordings in the presence of the Epac-selective cAMP analog 8-pCPT-2'-O-Me-cAMP (ESCA(1)), we find that Epac activation by this analog accounts on average for 38% of the forskolin-induced increase in evoked EPSC amplitudes and for 100% of the forskolin-induced increase in miniature EPSC (mEPSC) frequency in dissociated autaptic neuronal cultures from mouse hippocampus. From paired-pulse facilitation experiments, and considering the enhancement of mEPSC frequency, we conclude that ESCA(1)-induced Epac activity is presynaptic in origin and increases p. In addition, preapplication of ESCA(1) augmented a subsequent enhancement of evoked EPSC amplitudes by phorbol ester (PDBu). This effect was maximal when ESCA(1) application preceded the PDBu application by 3 min. Because the PDBu response was abolished after downregulation of intracellular PKC activity, we conclude that ESCA(1)-induced Epac activation leads to presynaptic changes involving Epac-to-PKC signaling.

Figure 1.

Expression of Epac in neuronal cell cultures. A, Cultured autaptic neurons express both Epac isoforms. Lanes, Rat cerebellum lysate, positive control (+), murine dentate gyrus neurons (m-DG), murine CA1 neurons (m-CA1), rat dentate gyrus neurons (r-DG), rat CA1 neurons (r-CA1). Protein bands were detected at the following molecular weights: 126 kDa for Epac2, 100 kDa for Epac1, and 43 kDa for β-actin. Epac1 experiments were performed in triplicate. B1, Test for the specificity of the Epac-activating cAMP analog, ESCA₁. A Western blot analysis of cAMP- or cAMP-analog-mediated phosphorylation of synapsin I at its PKA phosphorylation site (Ser9) is shown. For drug treatment procedure, see Materials and Methods. β-Actin controls for equal protein loading. B2, Quantitative change in the phosphorylation level of the PKA site of synapsin I. To correct for nonspecific effects (unrelated to drug treatment), which might have been introduced by variability in protein loading, P-Ser9 synapsin I chemiluminescence intensities were first normalized to the intensities of the corresponding β-actin values. These values were further normalized to the control intensity value to determine the increase or decrease in P-Ser9 levels after drug treatment. Values are shown as mean ± SEM based on the number of different Western blot performances. Statistical significance was tested by an unpaired two-tailed Student's t test assuming unequal variances (see Materials and Methods). Numbers within histogram bars represent the number of measurements.

Figure 2.

ESCA₁ enhances neurotransmitter release at excitatory autaptic neurons from dentate gyrus. A, Average EPSC amplitude potentiation (mean ± SEM) by 50 μm ESCA₁ (EPSC₀ = 5.43 ± 0.51 nA, mean ± SEM) and 50 μm forskolin (EPSC₀ = 4.95 ± 0.48 nA, mean ± SEM). Statistical significance was analyzed by using the paired t test. The statistical significance of the difference between the ESCA₁ and the forskolin effect was tested by the KS test. B1, Average time course of EPSC amplitudes in the presence and absence of ESCA₁ (mean ± SEM). ●, ESCA₁; N = 27; n = 5 (EPSC₀ = 7.01 ± 0.94 nA, mean ± SEM); □, average EPSC amplitude rundown under control condition; N = 17; n = 2 (EPSC₀ = 9.2 ± 1.5 nA, mean ± SEM). The black bar represents the onset and duration of drug application. B2, Average time course of EPSC amplitudes in the presence and absence of forskolin (mean ± SEM). Markers were used in analogy to the top panel. N = 13; n = 1 (EPSC₀ = 4.76 ± 0.81 nA, mean ± SEM). C, Analysis of the changes in the AP-related Na⁺- and K⁺-current peaks (mean ± SEM). AP-related sodium and potassium currents were observed before every EPSC. Top left, A typical waveform, indicating negative and positive peaks as values representative for Na⁺ currents and K⁺ currents. Peak current changes were analyzed for the control rundown condition (top right) and for both drug conditions (middle, ESCA₁; bottom, forskolin). The changes both during the steady-state phase of drug application and 3 min after drug removal are shown. In the control rundown experiment, the time intervals for analysis were chosen to match those used in the drug experiments. Significance was tested by a paired t test. The numbers within the histogram bars are N values, representing the number of cells. For the control rundown condition, 13 cells were analyzed.

Figure 3.

The magnitude of the drug-induced EPSC amplitude potentiation can be correlated to the size of the EPSC₀ amplitude. Electrophysiological recordings of excitatory DG neurons. A, Left, cAMP-related EPSC amplitude potentiation versus the EPSC₀ amplitude (mean ± SD). ■, ESCA₁ (50 μm); N = 60; n = 13; △, forskolin (50 μm); N = 46; n = 3. Individual data points were binned in classes of 1 nA for EPSC₀ > 0.5 nA. A monoexponential fit is applied to the ESCA₁ values. The forskolin data were fitted by a biexponential function. Right, Representative EPSC sample traces before and after ESCA₁ or forskolin application for both small (a) and large (b) EPSC₀, as marked by arrows in the left panel. AP-related currents were blanked out. B, Left, EPSC amplitude potentiation induced by PDBu (1 μm) versus the EPSC₀ amplitude (mean ± SD). ○, Murine DG; N = 35; n = 7; ◇, murine CA1; N = 5; ♦, rat CA1; N = 16; n = 3. Data binning and layout of the figure are as in A. The PDBu data were fitted by a biexponential function. Right, The time course of the PDBu effect on EPSC amplitudes (mean ± SEM) depends on the hippocampal region used for the preparation of neuronal cultures. ○, Murine DG; N = 7; n = 1 (EPSC₀ = 3.33 ± 0.51 nA, mean ± SEM); ♦, rat CA1; N = 6; n = 1 (EPSC₀ = 3.37 ± 0.7 nA, mean ± SEM). C1, Drug-induced EPSC amplitude potentiation as a function of culture age (DIV; mean ± SEM). A, DIV 8–10; B, DIV 11–15. The numbers within the histogram bars are N values, representing the number of cells. Significance was tested by a KS test. C2, Dependency of the EPSC₀ on the age of the culture (DIV; mean ± SD). ●, Mean control EPSC₀ for a given culture age. The number in brackets indicates the number of cells measured for a given culture age. Gray open circles indicate individual EPSC₀ data points measured at a certain culture age; white open squares, triangles, and circles indicate mean EPSC₀ values of the cells from C1.

Figure 4.

ESCA₁ application enhances mEPSC frequency. For every individual cell, mEPSC events were recorded for at least 1 min from murine DG neurons in the absence or presence of cAMP-related pharmacological agents. To prevent AP-related calcium influx, sodium channels were blocked by coapplication of 300 nm TTX. A, Representative mEPSC sample traces for the control (DIV 10 and DIV 14) condition and the corresponding ESCA₁ (50 μm) or forskolin (50 μm) treatment. B, mEPSC amplitude versus the EPSC₀ amplitude. Black circles, Control; squares, ESCA₁; N = 47; n = 3; gray circles, forskolin; N = 11; n = 2. Inset, Mean mEPSC sample traces are shown for control and ESCA₁ treatment. Arrows indicate their corresponding data points in the curve. C, mEPSC frequency versus the EPSC₀ amplitude. Significance was tested by a paired t test. Black circles, Control; N = 101; n = 9; squares, ESCA₁; N = 47; n = 3; gray circles, forskolin (50 μm); N = 11; n = 2. In B and C, mEPSC data are pooled over the following EPSC₀ intervals: from >0 to <1 nA, from 1 to <2 nA, from 2 to <4 nA, from 4 to <10 nA, and ≥10 nA. The values are presented as mean ± SEM. The number of cells in the EPSC₀ classes is given in parentheses.

Figure 5.

The number of sucrose-releasable vesicles is not affected by ESCA₁ application. A, Representative control sucrose sample traces (a–e) for selected EPSC₀ amplitudes (see B). Arrowheads indicate onset and end of sucrose (500 mm) application. B, Linear dependency of vesicle numbers on the EPSC₀ amplitude (mean ± SD). The vesicle number was calculated as described in Materials and Methods. ●, Control; N = 50; n = 6; □, ESCA₁ (50 μm); N = 44; n = 3. Data points were binned for the following EPSC₀ intervals: <0.5 nA, from 0.5 to 1 nA, from >1 to <4 nA, from 4 to <10 nA, from 10 to <15 nA, and >15 nA. The number of cells in the EPSC₀ classes is given in parentheses. The arrows (a–e) indicate the EPSC₀ values for the control sample traces shown in A.

Figure 6.

ESCA₁ application enhances a subsequently induced PDBu response. A, Stimulation protocol and sample traces. Before PDBu (1 μm) application for 2 min, cultured neurons were stimulated at various IAIs for 1.2 min with ESCA₁ (50 μm). EPSCs were recorded at 0.2 Hz. Average traces of one complete experiment (numbered 1–4) are shown for IAI = 2 min. AP-related currents were blanked. The reference PDBu trace was calculated from the basal EPSC (3) according to the fit of the PDBu reference curve shown in Fig. 3B (left). B, Dependency of the PDBu effect on EPSC₀ in the presence (+) or absence (−) of neuronal prestimulation with ESCA₁ for both murine DG neurons [DG₍₊₎ or DG₍₋₎] and rat CA1 neurons [CA1_(±)]. ○, Murine DG(−), control value; ●, murine DG(+), ESCA₁ prestimulation; N = 21; n = 5. ◇, Rat CA1(−), control value; ♦, rat CA1(+), ESCA₁ prestimulation; N = 11; n = 2. In the case of the murine DG neurons, the PDBu effects for the IAIs 0 and 1 min were pooled to increase the number of data points. Similarly, for rat CA1 neurons, the PDBu effects corresponding to the IAIs 1 and 2 min were pooled. For all curves, individual data points were binned in classes of 1 nA for EPSC₀ > 0.5 nA. The PDBu-generated EPSC potentiation values are shown as mean ± SEM for murine DG neurons and as mean ± SD for rat CA1 neurons. C, Time dependency of the ESCA₁-induced enhancement of the PDBu effect. Black circles, murine DG; gray circles, murine CA1; diamonds, rat CA1. Values from different age and EPSC₀ classes were pooled by averaging for each IAI the relative difference of PDBu-induced EPSC amplitude potentiations (“rel. EPSC Potentiation diff.”): for the definition of “EPSC Potentiation,” see Materials and Methods. Values are presented as mean ± SEM. Statistical significance was tested by a KS test. The number of cells for distinct IAIs is given in parentheses.

Figure 7.

Inhibition of PKC activity by bisindolylmaleimide I reduces the PDBu response. A, Time course of the EPSC amplitude potentiation of dentate gyrus autapses by 1 μm PDBu in the absence or presence of 1.2 μm Bis I during 0.2 Hz stimulation (mean ± SEM). Horizontal bars show the onset and the duration of drug application. ●, Reference PDBu response (−Bis I); N = 24; n = 6 (EPSC₀ = 4.35 ± 0.51 nA, mean ± SEM); ◇, PDBu response in the presence of Bis I (+Bis I); N = 31; n = 3 (EPSC₀ = 4.69 ± 0.49 nA, mean ± SEM). B, PDBu-mediated EPSC amplitude potentiation in the absence (−) or presence (+) of Bis I versus EPSC₀ (mean ± SD). Individual data points were binned in classes of 1 nA for EPSC₀ > 0.5 nA, and mean drug effects were calculated for each class. The continuous line is the fit to the values from Figure 3B (left); ▼, expected reference PDBu response calculated from the EPSC₀ amplitudes of the cells used in the Bis I experiment and the curve fit of the reference PDBu “potentiation curve” (continuous line) (Fig. 3B, left); ♦, mean PDBu response measured in the presence of Bis I; ◇, individual data points measured in the presence of Bis I.

Figure 8.

Downregulation of PKC activity by prolonged preincubation of neuronal cultures with PDBu abolishes the PDBu response. Dentate gyrus neurons were incubated either with PDBu (1 μm) or with DMSO (control) for 16 h before electrophysiological recordings. A, EPSC amplitude potentiation ± preincubation with PDBu for 16 h. Individual data points were binned in classes of 1 nA for EPSC₀ >0.5 nA. ●, Mean PDBu response reference values from Figure 3B (left); N = 21; n = 6 (EPSC₀ = 2.18 ± 0.28 nA, mean ± SEM); ▲, mean PDBu responses after preincubation with PDBu; ◇, individual PDBu responses after preincubation with PDBu. B, Average (ave.) potentiation of the EPSC amplitudes by PDBu in the presence or absence of preincubation with PDBu (mean ± SEM, significance tested by a KS test). The recordings for both conditions were not obtained from identical cells. Therefore, we generated three different control values for the mean PDBu effect. m, Potentiation values measured in the absence of PDBu preincubation (N = 21; n = 5). c, Potentiation values generated from the biexponential curve fit of the PDBu reference curve (Fig. 3B, left) using the individual EPSC₀ amplitudes measured after preincubation (N = 19; n = 2). d, Potentiation values after preincubation with DMSO for 16 h (volume of application corresponded to the one used for pretreatment with PDBu; N = 11; n = 3). The average EPSC₀ ± SEM (2.10 ± 0.59 nA) for the initial amplitude distribution used for d in B is indicated by a light gray bar in A. C, Top two traces, Sample traces for control and preincubation condition. Lowest traces, Superimposed traces of detected mEPSC events (24 events for control; 29 events for the preincubation condition). D, Dependency of the mEPSC frequency on the preincubation condition. ♦, Mean control frequency; N = 6 (EPSC₀ = 0.83 ± 0.48 nA, mean ± SD); ◇, individual control data points; gray circle, mean frequency after 16 h preincubation with PDBu; N = 5 (EPSC₀ = 0.81 ± 0.82 nA, mean ± SD); ○, individual preincubation data points.