Ca(2+) permeable AMPA receptor induced long-term potentiation requires PI3/MAP kinases but not Ca/CaM-dependent kinase II

Abstract

Ca(2+) influx via GluR2-lacking Ca(2+)-permeable AMPA glutamate receptors (CP-AMPARs) can trigger changes in synaptic efficacy in both interneurons and principle neurons, but the underlying mechanisms remain unknown. We took advantage of genetically altered mice with no or reduced GluR2, thus allowing the expression of synaptic CP-AMPARs, to investigate the molecular signaling process during CP-AMPAR-induced synaptic plasticity at CA1 synapses in the hippocampus. Utilizing electrophysiological techniques, we demonstrated that these receptors were capable of inducing numerous forms of long-term potentiation (referred to as CP-AMPAR dependent LTP) through a number of different induction protocols, including high-frequency stimulation (HFS) and theta-burst stimulation (TBS). This included a previously undemonstrated form of protein-synthesis dependent late-LTP (L-LTP) at CA1 synapses that is NMDA-receptor independent. This form of plasticity was completely blocked by the selective CP-AMPAR inhibitor IEM-1460, and found to be dependent on postsynaptic Ca(2+) ions through calcium chelator (BAPTA) studies. Surprisingly, Ca/CaM-dependent kinase II (CaMKII), the key protein kinase that is indispensable for NMDA-receptor dependent LTP at CA1 synapses appeared to be not required for the induction of CP-AMPAR dependent LTP due to the lack of effect of two separate pharmacological inhibitors (KN-62 and staurosporine) on this form of potentiation. Both KN-62 and staurosporine strongly inhibited NMDA-receptor dependent LTP in control studies. In contrast, inhibitors for PI3-kinase (LY294002 and wortmannin) or the MAPK cascade (PD98059 and U0126) significantly attenuated this CP-AMPAR-dependent LTP. Similarly, postsynaptic infusion of tetanus toxin (TeTx) light chain, an inhibitor of exocytosis, also had a significant inhibitory effect on this form of LTP. These results suggest that distinct synaptic signaling underlies GluR2-lacking CP-AMPAR-dependent LTP, and reinforces the recent notions that CP-AMPARs are important facilitators of synaptic plasticity in the brain.

Figure 1. GluR2-lacking mice are capable of robust long-lasting LTP in the presence of the NMDA antagonist D,L-AP5.

(A, B) D,L-AP5 completely inhibited NMDAR-dependent LTP induced by 2 trains of 100 Hz (as indicated by arrow) in (A) GluR2+/+slices (vehicle, n = 5; D,L-AP5, n = 5; P<0.001) but not CP-AMPAR-dependent LTP in (B) GluR2−/−slices (D,L-AP5, n = 6; P<0.001). (C, D) Robust LTP induced in GluR2−/−slices by TBS (as indicated by arrow) in the presence of D,L-AP5 in (C) field EPSP recordings (D,L-AP5, n = 6; P<0.001) and (D) whole-cell recordings (D,L-AP5, n = 6; P<0.001). All field EPSP recordings of CP-AMPAR-dependent LTP in GluR2 mutants involved the addition of 100 µM D,L-AP5 to perfusate 15 minutes prior to induction, lasting until 5 minutes post-induction, while all whole-cell studies of CP-AMPAR dependent LTP involved 100 µM D,L-AP5 being perfused during the entire recording period. Error bars represent SEM.

Figure 2. CP-AMPARs are capable of inducing long-lasting and protein-synthesis dependent forms of L-LTP.

(A, B) D,L-AP5 completely blocked NMDAR-dependent L-LTP induced by 4 trains of 100 Hz (as indicated by arrow) in (A) GluR2+/+slices (vehicle, n = 5; D,L-AP5, n = 5; P<0.001) but not CP-AMPAR-dependent L-LTP in (B) GluR2−/−(D,L-AP5, n = 5; P = 0.002) and GluR2+/−slices (D,L-AP5, n = 5; P<0.001). (C, D) L-LTP induced by 4 trains of 100 Hz (as indicated by arrow) is dependent on protein synthesis in both (C) GluR2+/+slices (vehicle, n = 5; anisomycin n = 5; P = 0.006) and (D) GluR2+/−slices (vehicle, n = 5; D,L-AP5+anisomycin, n = 5; P = 0.002). CP-AMPAR-dependent L-LTP recordings in GluR2 mutants involved the addition of 100 µM D,L-AP5 to perfusate 15 minutes prior to induction, lasting until 5 minutes post-induction. 25 µM anisomysin was added to perfusate 15–20 minutes prior to L-LTP induction and washed away 5 minutes post-induction. Error bars represent SEM.

Figure 3. Induction of CP-AMPAR-dependent plasticity is blocked by the CP-AMPAR inhibitor IEM-1460.

(A, B) Administration of IEM-1460 in (A) GluR2−/−slices significantly reduced basal synaptic response as well as completely attenuated CP-AMPAR-dependent LTP (D,L-AP5+IEM-1460, n = 6; P = 0.44) induced by 2 trains of 100 Hz (as indicated by arrow). (B) In a similar fashion, CP-AMPAR-dependent L-LTP induced by 4 trains of 100 Hz (as indicated by arrow) in GluR2+/−slices was also completely blocked by IEM-1460 (D,L-AP5, n = 5; D,L-AP5+IEM-1460, n = 5; P<0.001). All CP-AMPAR-dependent LTP field EPSP studies in GluR2 mutants involved adding 100 µM D,L-AP5 to ACSF perfusate 15 minutes prior to induction until 5 minutes post-induction. 100 µM IEM-1460 was added to the ACSF perfusate 25 minutes prior to LTP induction in GluR2−/−slices and was present throughout the entire recording period, while 100 µM IEM-1460 was added to the ACSF perfusate 15–20 minutes prior to L-LTP induction in GluR2+/−slices up until 5 minutes post-induction. Error bars represent SEM.

Figure 4. Plasticity induced through CP-AMPARs is completely dependent on Ca²⁺ influx.

(A, B) Paired pulse facilitation (PPF) revealed no significant difference in presynaptic involvement in CP-AMPAR-dependent plasticity induced by 2 trains of 100 Hz (as indicated by arrow) in (A) GluR2−/−slices (D,L-AP5, n = 3; P = 0.91) and by 4 trains of 100 Hz (as indicated by arrow) in (B) GluR2+/−slices (D,L-AP5, n = 5; P = 0.97) in a manner similar to NMDAR-dependent potentiation induced in wild-type controls. (C, D) Potentiation induced by TBS (as indicated by arrow) in whole cell recordings (as indicated by arrow) is completely dependent on Ca²⁺ influx in both NMDAR-dependent LTP in (C) GluR2+/+slices (untreated, n = 5; BAPTA, n = 5; P<0.001) and CP-AMPAR-dependent LTP in (D) GluR2−/−slices (D,L-AP5, n = 6 ; D,L-AP5+BAPTA, n = 8; P = 0.001). CP-AMPAR-dependent LTP field EPSP studies in GluR2 mutants involved adding 100 µM D,L-AP5 to ACSF perfusate 15 minutes prior to induction until 5 minutes post-induction. Whole-cell recordings of CP-AMPAR dependent LTP involved the presence of 100 µM D,L-AP5 throughout the recording period. 30 mM BAPTA was included in the intracellular solution for Ca²⁺ studies. Error bars represent SEM.

Figure 5. CaMKII is not involved in long-term potentiation induced through CP-AMPARs.

(A, B) Contrasting effects of the broad spectrum inhibitor staurosporine where NMDAR-dependent LTP induced by 2 trains of 100 Hz (as indicated by arrow) was significantly attenuated in (A) GluR2+/+slices (vehicle, n = 6; staurosporine, n = 5; P = 0.002) but not CP-AMPAR-dependent LTP in (B) GluR2−/−slices (vehicle, n = 5; D,L-AP5+staurosporine, n = 5; P = 0.97). (C, D) Contrasting effects of the CaMKII-specific inhibitor KN-62 where LTP induced by 2 trains of 100 Hz (as indicated by arrow) was completely blocked in (C) GluR2+/+slices (vehicle, n = 5; KN-62, n = 5; P<0.001) but not in (D) GluR2−/−slices (vehicle, n = 5; D,L-AP5+KN-62, n = 5; P = 0.42). (E) However, KN-62 significantly inhibited LTP induced in GluR2−/−slices (vehicle = 6; KN-62 = 5; P = 0.02) in the absence of D,L-AP5. (F) Summary graph of the means of the last 10 minutes of potentiation seen in KN-62 treatment studies. All CP-AMPAR-dependent LTP recordings in GluR2 mutants involved the administration of 100 µM D,L-AP5 to perfusate 15 minutes prior to induction, lasting until 5 minutes post-induction. 100 nM staurosporine and 15 µM KN-62 were added to perfusate 15–20 minutes prior to LTP induction and washed away 5 minutes post-induction. Error bars represent SEM. * denotes P<0.05.

Figure 6. PI3-kinase is required for CP-AMPAR-dependent long-term potentiation.

(A) The specific PI3-kinase inhibitor LY294002 significantly attenuated NMDAR-dependent LTP induced by 2 trains of 100 Hz (as indicated by arrow) in GluR2+/+slices (vehicle, n = 6; LY294002, n = 5; P<0.001). CP-AMPAR-dependent LTP elicited by 2 trains of 100 Hz (as indicated by arrow) in (B) GluR2−/−slices was also strongly suppressed in the presence of the structurally unrelated PI3K inhibitors LY294002 (vehicle, n = 5; D,L-AP5+LY294002, n = 5; P<0.001) and wortmannin (vehicle, n = 5; D,L-AP5+wortmannin, n = 5; P<0.001). (C) Summary graph of the means of the last 10 minutes of potentiation seen in LY294002 and wortmannin treatment studies. CP-AMPAR-dependent LTP recordings in GluR2 mutants involved the addition of 100 µM D,L-AP5 to perfusate 15 minutes prior to induction, ceasing at 5 minutes post-induction. 20 µM LY294002 and 1 µM wortmannin were added to ACSF perfusate 15–20 minutes prior to LTP induction up until 5 minutes post-induction. Error bars represent SEM. * denotes P<0.05.

Figure 7. The MAPK signaling cascade plays an essential role in long-term potentiation induced through CP-AMPARs.

(A) The specific MEK inhibitor PD98059 attenuated LTP elicited by 2 trains of 100 Hz (as indicated by arrow) in GluR2+/+slices (vehicle, n = 5; PD98059, n = 6; P<0.001). CP-AMPAR-dependent LTP induced by 2 trains of 100 Hz (as indicated by arrow) was also significantly inhibited in (B) GluR2−/−slices by the structurally unrelated MEK inhibitors PD98059 (vehicle, n = 6; D,L-AP5+PD98059, n = 5; P<0.001) and U0126 (vehicle, n = 6; D,L-AP5+U0126, n = 5; P = 0.02). (C) Summary graph of the means of the last 10 minutes of potentiation seen in PD98059 and U0126 treatment studies. CP-AMPAR-dependent LTP recordings in GluR2 mutants involved the addition of 100 µM D,L-AP5 to perfusate 15 minutes prior to induction, ceasing at 5 minutes post-induction. 50 µM PD98059 and 35 µM U0126 were added to ACSF perfusate 15–20 minutes prior to LTP induction lasting until 5 minutes post-induction. Error bars represent SEM. * denotes P<0.05.

Figure 8. The MAPK signaling cascade plays a role in the induction but not maintenance of plasticity induced through CP-AMPARs.

(A, B) Administration of the MEK inhibitor PD98059 during the induction phase of L-LTP significantly attenuated potentiation induced by 4 trains of 100 Hz (as indicated by arrow) during both NMDAR-dependent L-LTP in (A) GluR2+/+slices (vehicle, n = 5; PD98059, n = 6; P = 0.002) and CP-AMPAR-dependent L-LTP in (B) GluR2+/−slices (vehicle, n = 5; D,L-AP5+PD98059, n = 5; P = 0.009). (C, D) However, administration of PD98059 during the maintenance phase of L-LTP induced by 4 trains of 100 Hz (as indicated by arrow) had no significant effect on both (C) GluR2+/+slices (vehicle, n = 5; PD98059, n = 5; P = 0.9) and (D) GluR2+/−slices (vehicle, n = 5; D,L-AP5+PD98059, n = 5; P = 0.83). All CP-AMPAR-dependent L-LTP recordings in GluR2 mutants involved adding 100 µM D,L-AP5 to perfusate 15 minutes prior to induction until 5 minutes post-induction. For MAPK L-LTP induction studies, 50 µM PD98059 was added to ACSF perfusate 15–20 minutes prior to induction lasting until 5 minutes post-induction. For MAPK L-LTP maintenance studies, 50 µM PD98059 was added to the ACSF perfusate 20–30 minutes post-induction. Error bars represent SEM.

Figure 9. Receptor trafficking plays an important role in plasticity induced through CP-AMPARs.

(A) Synaptic plasticity induced by TBS (as indicated by arrow) was significantly attenuated in the presence of the exocytosis-inhibiting tetanus toxin (TeTx, 75 nM) during NMDAR-dependent LTP in GluR2+/+slices (inactive toxin, n = 6; 75 nM TeTx, n = 6; P = 0.003). (B) CP-AMPAR-dependent LTP elicited by TBS (as indicated by arrow) in GluR2−/−slices was also significantly reduced in the presence of 75 nM TeTx (D,L-AP5+inactive toxin, n = 7; D,L-AP5+75 nM TeTx, n = 5; P<0.001) and 250 nM TeTx (D,L-AP5+inactive toxin, n = 7; D,L-AP5+250 nM TeTx, n = 5; P<0.001) respectively to statistically similar levels (D,L-AP5+75 nM TeTx, n = 5; D,L-AP5+250 nM TeTx, n = 5; P = 0.62). (C) Summary graph of the means of the last 5 minutes of potentiation seen in tetanus toxin treatment studies. Whole-cell recordings of CP-AMPAR dependent LTP involved the presence of 100 µM D,L-AP5 throughout the recording period. 75 nM and 250 nM TeTx were included in the intracellular solution for exocytosis inhibition studies. Error bars represent SEM. * denotes P<0.05.