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.