Epac2 Mediates cAMP-Dependent Potentiation of Neurotransmission in the Hippocampus - PubMed (original) (raw)

Epac2 Mediates cAMP-Dependent Potentiation of Neurotransmission in the Hippocampus

Herman B Fernandes et al. J Neurosci. 2015.

Abstract

Presynaptic terminal cAMP elevation plays a central role in plasticity at the mossy fiber-CA3 synapse of the hippocampus. Prior studies have identified protein kinase A as a downstream effector of cAMP that contributes to mossy fiber LTP (MF-LTP), but the potential contribution of Epac2, another cAMP effector expressed in the MF synapse, has not been considered. We investigated the role of Epac2 in MF-CA3 neurotransmission using Epac2(-/-) mice. The deletion of Epac2 did not cause gross alterations in hippocampal neuroanatomy or basal synaptic transmission. Synaptic facilitation during short trains was not affected by loss of Epac2 activity; however, both long-term plasticity and forskolin-mediated potentiation of MFs were impaired, demonstrating that Epac2 contributes to cAMP-dependent potentiation of transmitter release. Examination of synaptic transmission during long sustained trains of activity suggested that the readily releasable pool of vesicles is reduced in Epac2(-/-) mice. These data suggest that cAMP elevation uses an Epac2-dependent pathway to promote transmitter release, and that Epac2 is required to maintain the readily releasable pool at MF synapses in the hippocampus.

Keywords: Epac2; cAMP; mossy fiber; readily releasable pool.

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Figures

Figure 1.

MF axon targeting is not affected by loss of Epac2. A, Representative Western blots of Epac2 expression from mossy fiber synaptosomal fractions from Epac2+/+ and Epac2−/− mice. Epac2 expression was not detected in samples obtained from Epac2−/− mice (n = 3 paired preparations). B, Immunofluorescence staining of mossy fiber marker synaptoporin (red) and principal cell nuclei (DAPI) in hippocampal sections from Epac2+/+ and Epac2−/− mice. Measurements of the SPB and IPB are indicated by dotted lines. C, Ratios of SPB to IPB bundle length were not significantly different between Epac2+/+ and Epac2−/− mice (n = 4 paired experiments, p > 0.05). H, homogenate; P2, small synaptosomes; P3, large synaptosomes/mossy fiber giant boutons.

Figure 2.

cAMP-mediated potentiation of transmitter release is impaired in Epac2−/− mice. A, Grouped data of time course of MF facilitation by diterpene FSK in hippocampal slices from Epac2+/+ and Epac2−/− mice. Shaded area represents application of 50 μ

FSK. B, Representative EPSC traces from Epac2+/+ (Bi) and Epac2−/− (Bii). Traces are shown for baseline period (1) and after potentiation (2). Calibration: Bi, 200 pA, 20 ms; Bii, 400 pA, 20 ms. C, Summary of all data of FSK potentiation calculated as the potentiation of the EPSC amplitude between 50 and 60 min compared with the baseline amplitude (0–10 min), *p < 0.05 by unpaired t test. D, Paired-pulse ratio of EPSCs (PP40 ms) for each recording during baseline and after FSK potentiation for Epac2+/+ (black) and Epac2−/− (gray) mice. E, Time course for FSK potentiation of MF transmission in the presence of the PKA inhibitor KT-5720 (1 μ

, pre-incubated for at least 1 h). F, Summary of all data of FSK potentiation calculated as the potentiation of normalized EPSC amplitude between 50 and 60 min (*p < 0.05 by unpaired t test).

Figure 3.

MF-LTP is impaired in Epac2−/− mice. A, Time course of MF-LTP in Epac2+/+ mice. Black bar represents application of group II mGluR agonist DCG-IV at the end of each experiment. Inset shows representative traces from one recording during baseline (1) and after 20–30 min after LTP induction (2). B, Average time course of LTP for all recordings from Epac2−/− mice. The magnitude of LTP measured as the potentiation 20–30 min after induction compared with the baseline amplitude (0–10 min) is significantly reduced in the knock-out recordings. C, Cumulative probability distribution of MF-LTP of all recordings. D, Paired-pulse ratios measured at 40 ms interpulse intervals during baseline and after LTP induction. Calibration for EPSC traces: 200 pA, 20 ms.

Figure 4.

MF STP and AMPA/NMDA ratios are unaffected in Epac2−/− mice. A, Paired-pulse facilitation over a range of interpulse intervals at MF-CA3 synapses in Epac2+/+ and Epac2−/− mice. A representative example of a recording from an Epac2−/− mouse is shown at the top. Calibration: 50 ms. B, Frequency facilitation at MF-CA3 synapses. Bi, Time course of FF from a single representative experiment in Epac2−/− mouse. Bii, Representative MF EPSC traces recorded at 0.05 and 1 Hz. Calibration: 200 pA, 10 ms. Biii, Summary of all frequency facilitation recordings. No significant difference was observed in 1 Hz FF between the two genotypes (n = 16 for both Epac2+/+ and Epac2−/−). C, Facilitation of EPSCs to short stimulus trains at 10 and 20 Hz. The facilitation ratio was calculated as the amplitude of the fifth EPSC in the train to the first EPSC in the train. No significant difference was observed in short-term facilitation between genotypes at 10 Hz or 20 Hz. D, Progressive block of synaptic NMDAR currents by 40 μ

MK-801 at MF synapses in slices from Epac2+/+ and Epac2−/− mice. Inset, Representative traces from a single experiment of the initial NMDAR current (sweep 1) and after block (sweep 60). Calibration: 100 pA, 10 ms. Ei, Representative MF EPSC traces recorded at −70 mV and +40 mV in Epac2+/+ (left) and Epac2−/− slices (right). The amplitude of the AMPA component was measured as the peak at −70 mV and the NMDA component was measured in a 2.5 ms window, 60 ms after the onset of the outward current at +40 mV. Calibration: Epac2+/+, 100 pA; Epac2−/− 200 pA, 20 ms. Eii, Grouped data for all AMPA/NMDA recordings. There was no significant difference between the two genotypes (p > 0.05, t test).

Figure 5.

Expression levels of Rab3A, CASK, and synaptoporin (Synprn) are significantly reduced in MF synaptosomes from Epac2−/− mice. A, Representative Western blots of P3 synaptosomal fractions from three independent synaptosomal preparations, run in parallel, from Epac2+/+ and Epac2−/− mice. B, Quantification of grouped Western blot data from MF synaptosomal preparations. Expression level of proteins of interest normalized to total protein. *p < 0.05, paired t test.

Figure 6.

MF synaptic ultrastructure in Epac2−/−. A, B, Representative electron micrographs of MF synapses from an Epac2−/− mouse brain, taken at 23,000× (Ai) and 13,000× (Bi); Aii and Bii are magnified areas from Ai and Bi, respectively, to illustrate AZ length measurements (between hashed red lines). Scale bars: Ai, Aii, 200 nm; Bi, 500 nm; Bii, 200 nm. Arrowheads indicate sites where AZs are apposed to PSDs. C, Analysis of AZ length from all experiments, *p < 0.05, unpaired t test. D, Measurement of vesicle diameter in MF bouton from Epac2+/+ and Epac2−/− mice. Sp, spine; MFB, mossy fiber bouton.

Figure 7.

Functional estimates of the total number of synaptic vesicles available for release in Epac2+/+ and Epac2−/− MF boutons. A, Time course of EPSC facilitation during trains in Epac2+/+ and Epac2−/− mice. B, Analysis of cumulative MF EPSCs in Epac2+/+ (black) and Epac2−/− (gray). Red lines indicate linear regression fit to the last 50 events and extrapolated to time = 0 to provide estimates of N*q. Ci, Cii, Measurement of quantal content (q) by evoking aEPSCs. Ci, Representative examples of evoked synchronous release events in normal extracellular Ca2+ and asynchronous release events (inset, indicated by gray arrows) in Sr2+ from Epac2−/− mice. Red arrows indicate time of stimulus. Calibration: 100 pA, 50 ms. Cii, Group data, all experiments for measurement of amplitude of quantal MF events (p > 0.05, unpaired t test). Ciii, Group data of measured N*q for Epac2+/+ and Epac2−/− (*p < 0.05, unpaired _t_ test). **_D_**, Analysis of cumulative EPSCs during trains at A/C synapses (CA3-CA3) in Epac2+/+ (black) and Epac2−/− (gray). Inset, Summary of _N_*_q_ product from A/C recordings (_p_ > 0.05, unpaired t test).

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Epac2 Mediates cAMP-Dependent Potentiation of Neurotransmission in the Hippocampus - PubMed (original) (raw)