Postsynaptic application of a peptide inhibitor of cAMP-dependent protein kinase blocks expression of long-lasting synaptic potentiation in hippocampal neurons

Abstract

Multiple trains of high-frequency synaptic stimulation evoke long-term potentiation (LTP) of synaptic transmission in hippocampal area CA1, which has been correlated with hippocampal long-term memory and requires the activation of cAMP-dependent protein kinase (PKA). To assess whether postsynaptic PKA is necessary for the expression of LTP, we made prolonged whole-cell voltage-clamp recordings from CA1 pyramidal neurons in mouse hippocampal slices during postsynaptic infusion of cell-impermeant modulators of PKA. Repeated stimulation (four 100 Hz trains at 5 min intervals) of the Schaffer collateral pathway increased synaptically evoked EPSCs for up to 2 hr. The postsynaptic infusion of either a cell-permeant PKA inhibitor (Rp-cAMPS) or a cell-impermeant PKA inhibitor (PKI(6-22)) did not alter post-tetanic peak potentiation, but it caused significant decay of EPSCs to pretetanization amplitudes within 1.5 hr. In contrast, postsynaptic infusion of PKI(6-22) did not alter a more modest, decaying form of LTP evoked by a single 100 Hz train. Paired-pulse facilitation was unchanged during most of the duration of LTP, suggesting that postsynaptic mechanisms, including PKA activation, are involved in the expression of LTP induced by multitrain stimulation. The postsynaptic infusion of a constitutively active isoform of the PKA catalytic subunit (Calpha) into CA1 pyramidal neurons increased EPSC sizes to elicit long-lasting synaptic facilitation. Thus, mimicking the activation of PKA in postsynaptic CA1 pyramidal neurons is sufficient for inducing persistent synaptic facilitation. Activation of apostsynaptic PKA is necessary for the expression of LTP in CA1 pyramidal neurons and is sufficient for initiating persistent synaptic facilitation.

Fig. 1.

GABA_A currents reduce the depolarization in CA1 neurons resulting from high-frequency (100 Hz) stimulation of the Schaffer collaterals, but whole-cell currents in response to low-frequency test stimulation are mediated primarily by glutamate receptors. A, Extracellular bicuculline greatly amplified the postsynaptic depolarization measured under current clamp in response to 100 Hz synaptic stimulation, indicating that GABA_A activity reduces the depolarization that induces LTP. B, Postsynaptic currents evoked by low-frequency test stimulation were reduced by 81% after the application of the glutamate receptor antagonist DNQX. The NMDA antagonist APV and the GABA_A antagonist bicuculline (Bic.) blocked the remaining current. The GABA_A-mediated IPSC comprises only a small fraction (15%) of the total current. C, Postsynaptic currents evoked by test stimulation after LTP induction (4 trains of 1 sec and 100 Hz separated by 5 min) were almost completely blocked by the glutamatergic antagonist DNQX and APV (average, 89%), whereas the remaining current was blocked by bicuculline.

Fig. 2.

Postsynaptic application of a cell-permeant PKA inhibitor blocks the expression of long-lasting LTP. Four 100 Hz trains 5 min apart evoked potentiation of EPSCs in CA1 pyramidal neurons (circles). Inclusion of a PKA inhibitor, Rp-cAMPS, in the patch pipette caused EPSCs to decay to pretetanization amplitudes within 2 hr after tetanization (triangles). The postsynaptic application of Rp-cAMPS had no significant effect on EPSC amplitudes evoked at test stimulus strength (0.033 Hz;squares). Sample paired EPSC traces from two experiments (top) were measured 20 min before and 1.5 hr after the final 100 Hz train. Rp-cAMPS was applied for the duration of these experiments.

Fig. 3.

Expression of long-lasting LTP is impaired by the postsynaptic application of a cell-impermeant inhibitory peptide of PKA, PKI_6–22 (20 μm). A, Plot of LTP data from two cells, recorded in the absence (circles) and presence (triangles) of postsynaptically applied PKI_6–22. B, Averaged data from all experiments. In the presence of postsynaptic PKI_6–22, EPSC amplitudes after tetanization decayed to pretetanization values within 100 min after tetanization (triangles). Postsynaptic PKI_6–22 did not significantly affect EPSCs evoked at 0.033 Hz, elicited at test stimulus strength (squares). C, Digestion of PKI_6–22 with trypsin blocked its inhibition of long-lasting LTP. The average EPSC amplitude measured 40 min after tetanization in the presence of intact PKI_6–22 was significantly smaller than that measured with digested PKI_6–22 (*p < 0.05; post hoc Tukey–Kramer test).

Fig. 4.

PPF is transiently depressed immediately after tetanization. A, Measurements of PPF before and after tetanization (4 × 100 Hz, 5 min between trains). PPF is transiently depressed during the first 5–10 min after tetanization but recovered to pretetanization amplitudes within 15 min after tetanization. The dotted line indicates the level of PPF before tetanization. B, Sample EPSC traces from one cell showing PPF before (a) and 30 min after (b) induction of multitrain LTP.C, Comparison of post-tetanization PPF in the presence of bath-applied CTZ or vehicle (0.05% methanol). CTZ had no significant effect on PPF after repeated tetanization, compared with controls treated with vehicle (p > 0.05).

Fig. 5.

Activation of PKA in postsynaptic CA1 pyramidal neurons is sufficient to elicit persistent synaptic facilitation.A, Infusion of PKI_6–22 into the postsynaptic CA1 pyramidal neuron attenuates persistence of the increase in evoked EPSCs induced by bath application of FSK and IBMX. Under control conditions, FSK and IBMX caused a rapid increase in EPSC amplitude that was maintained after drug washout. The infusion of PKI_6–22 had no effect on the early increase, but it attenuated persistence of the facilitation of EPSCs. B, Postsynaptic application of constitutively active PKA catalytic subunits (Cα) caused a rapid increase in the amplitude of EPSCs (circles) that was not seen in the presence of 20 μm DNQX and 50 μm APV (squares), indicating that Cα augmented the EPSC and not the smaller underlying IPSC. Heat-denatured Cα had no significant effect on EPSC amplitudes (diamonds). The facilitation was independent of the strength of the test stimulation used to elicit EPSCs that were 20 or 40% of maximum evoked amplitudes (circles, triangles). The first point on the graph was measured 1 min after break-in, and the first four EPSCs measured thereafter were used as baseline control amplitudes. This was necessitated by the rapidity of onset of EPSC facilitation seen after break-in with pipettes containing active catalytic subunits. Each pair of sample EPSC traces was measured at 2 and 24 min. The last two pairs of traces share the same calibration bars: 200 pA, 20 msec.