Calcium-Permeable AMPA Receptors Mediate the Induction of the Protein Kinase A-Dependent Component of Long-Term Potentiation in the Hippocampus

Abstract

Two forms of NMDA receptor (NMDAR)-dependent long-term potentiation (LTP) at hippocampal CA1 synapses can be distinguished based on their sensitivity to inhibitors of protein kinase A (PKA). The PKA-dependent form requires multiple episodes of high-frequency stimulation (HFS) or theta burst stimuli (TBS) with a spacing between episodes in the order of minutes. To investigate the mechanism by which spaced episodes induce the PKA-dependent form of LTP, we have compared, in interleaved experiments, spaced (s) and compressed (c) TBS protocols in the rat CA1 synapses. We find that LTP induced by sTBS, but not that induced by cTBS, involves the insertion of calcium-permeable (CP) AMPARs, as assessed using pharmacological and electrophysiological criteria. Furthermore, a single TBS when paired with rolipram [4-(3-(cyclopentyloxy)-4-methoxyphenyl)pyrrolidin-2-one], to activate PKA, generates an LTP that also involves the insertion of CP-AMPARs. These data demonstrate that the involvement of CP-AMPARs in LTP is critically determined by the timing of the induction trigger and is associated specifically with the PKA-dependent form of LTP.

Significance statement: Long-term potentiation is a family of synaptic mechanisms that are believed to be important for learning and memory. Two of the most extensively studied forms are triggered by the synaptic activation of NMDA receptors and expressed by changes in AMPA receptor function. They can be distinguished on the basis of their requirement for activation of a protein kinase, PKA. We show that the PKA-dependent form also involves the transient insertion of calcium-permeable AMPA receptors. These results have implications for relating synaptic plasticity to learning and memory and suggest a specific linkage between PKA activation and the rapid synaptic insertion of calcium-permeable AMPA receptors during long-term potentiation.

Keywords: NMDA receptor; PKA; calcium-permeable AMPA receptor; hippocampus; long-term potentiation; rectification.

Figure 1.

Defining the PKA dependence of LTP. A, A schematic of the protocol for studying LTP. Two inputs were stimulated alternately and TBS delivered to one input with the second input serving as a control for stability. Traces are the averages of five successive records of baseline and LTP, obtained at the times indicated by numbers in E for a vehicle (Veh) control (black) and a KT experiment (red). B, The lack of effect of KT on LTP using a cTBS protocol (inter-episode interval of 10 s). Data are pooled (mean ± SEM from 5 experiments) and illustrate the effects of treated (colored) and interleaved controls (n = 8, black) for the test (filled symbols) and control (open symbols) input. The timing of TBS is shown by the blue arrows. KT (1 μm) was applied for the duration indicated by the gray bar. C–G, The effects of KT on a sTBS with an inter-episode interval of 2 min (C; n = 5 and 3 for the KT and vehicle experiment, respectively), 5 min (D; 4 and 3), 10 min (E; 5 and 5), 20 min (F; 4 and 6), and 60 min (G; 4 and 3). H, The time course of inhibition (percentage) of LTP by KT. Each value was calculated by comparing the level of LTP in KT with its interleaved control experiment. The line is a log Gaussian curve with 95% confidence intervals (shaded).

Figure 2.

A role for CP-AMPARs in LTP. A, The lack of effect of IEM (30 μm) on LTP using the cTBS protocol (n = 6 and 8 for the IEM and vehicle groups, respectively). B–F, The effects of IEM on LTP induced with sTBS, with an inter-episode interval of 20 min. IEM was applied immediately after the first TBS (B; n = 7), second TBS (C; n = 6), third TBS (D; n = 10), or 1 h after the third TBS (E; n = 5). A single set of interleaved vehicle controls (n = 21) are shown. F, Quantification of the effects of IEM on LTP. The histograms plot the level of inhibition (percentage) of LTP, measured 2 h after the last TBS, for the conditions illustrated in A–E. *p < 0.05, **p < 0.01, and ***p < 0.001 versus control.

Figure 3.

Additional evidence for a role of CP-AMPARs in LTP. A, The lack of effect of PhTx (5–10 μm) on LTP using a cTBS protocol (inter-episode interval of 10 s, n = 5). B, Inhibitory effect of PhTx on LTP induced by sTBS protocol (inter-episode interval of 20 min, n = 8). C, The lack of effect of NASPM (30 μm) on LTP using a cTBS protocol (inter-episode interval of 10 s, n = 6). D, Inhibitory effect of NASPM of LTP induced by sTBS protocol (inter-episode interval of 20 min, n = 7).

Figure 4.

Stopping stimulation after the delivery of TBS reduces the magnitude of LTP. A, The effects of stopping stimulation after the second and third episodes of TBS on sLTP (n = 9). Initial test responses were collected after the delivery of the second and third TBS, so as to estimate the level of sLTP induction. B, Interleaved control experiments (n = 7). C, Example traces from representative experiments. D, A comparison of the levels of LTP, quantified 2 h after the delivery of the third TBS episode, for the data presented in A (Stop) and B (Con). **p < 0.01 versus control.

Figure 5.

Rolipram enhances LTP via the insertion of CP-AMPARs. A, The effects of a wTBS. B, Enhancement of LTP induced by a wTBS in the presence of rolipram (0.1 μm). C, The lack of effect of IEM on LTP induced by a wTBS. D, Inhibition of the rolipram-enhanced LTP by IEM. In B–D, the vehicle dataset is replotted for illustrative purposes. E, Traces from representative experiments to illustrate rolipram-enhanced LTP (red) and the effects of rolipram plus IEM (purple), which were obtained at the times indicated by numbers during baseline and 1 h after the delivery of wTBS. F, Quantification of the effects measured 1 h after LTP induction. **p < 0.01 versus vehicle. Rol, Rolipram; Veh, vehicle.

Figure 6.

Alterations in AMPAR rectification associated with LTP. A1, A representative example of LTP recorded extracellularly induced by a cTBS. A2, The simultaneous whole-cell recording from a neuron in the same region of the slice. d-AP-5 (100 μm) plus L-689,560 (5 μm) were applied immediately after the delivery of TBS, as indicated by the gray bar. After 10 min, the holding current was varied to obtain an RI. A3, The traces are averages of five successive EPSCs recorded at 40, 0, and −70 mV for the LTP pathway (top), the control pathway (middle), and the two inputs normalized at 40 mV and superimposed (bottom). Note the absence of any alteration. The histogram plots the RI calculated for 21 neurons from 10 animals. Data from the individual neurons are shown in the plot to the right. Dotted lines connect the RI measurements for the control and tetanized inputs for each neuron. B1–B3, Equivalent records for the LTP induced by sTBS. Note that the whole-cell recording (WC) was obtained after the second TBS. This was necessary because of the lability of LTP washout and to enable the RI measurements to be made after a similar length of whole-cell dialysis. Note that there is a small but discernible alteration in AMPAR rectification (quantification of 23 neurons from 11 animals, **p < 0.01, paired Student's t test). Con, Control.

Figure 7.

Alterations in AMPAR rectification associated with rolipram-enhanced LTP. A1, Extracellular recordings were performed as described in Figure 6 except that wTBS was used for LTP induction. A2, The simultaneous whole-cell recording from a neuron in the same region of the slice. After 10 min, the holding current was varied to obtain the RI. A3, The traces are averages of five successive EPSCs recorded at 40, 0, and −70 mV for the LTP pathway (top), the control pathway (middle), and the two inputs normalized at 40 mV and superimposed (bottom). Note the absence of any alteration. The histogram plots the RI calculated for 20 neurons from 10 animals. Individual data points are shown in the plot on the right. B1–B3, Equivalent records for the LTP induced by wTBS in the presence of rolipram. Note that there is a small but discernible alteration in AMPAR rectification (quantification of 24 neurons from 10 animals, **p < 0.01, paired Student's t test). Con, Control.