We next asked whether CaCC also affects spike duration in the axon terminals. If so, applying CaCC blocker should increase transmitter release from CA3 axon terminals that form synapses with CA1 neurons. First, we performed field recording of the pharmacologically isolated AMPA-fEPSP in the CA1 dendritic field, while stimulating Schaffer collaterals ten times at 10 Hz (Figure 5C). NFA did not alter the AMPA-fEPSPs (106% ± 8.4%, n = 7,

p = 0.8). 100 μM NFA also did not alter the pharmacologically isolated NMDA-EPSCs IOX1 supplier recorded from individual CA1 pyramidal neurons (101% ± 3.1%, n = 5, p = 0.2; Figure 5D) (external Mg2+ was removed to facilitate NMDA receptor activation, and 10 mM internal Cl− was used to minimize the driving force for Cl− ions when ECl is −64.4 mV and holding potential is −65 mV). Thus, blocking CaCC alters the action potential

waveform in the soma without altering transmitter release, indicating that functional CaCCs reside in somatodendritic regions find more but not the nerve terminals of CA3 pyramidal neurons. Next, we asked whether CaCCs are near NMDA-Rs to be activated during synaptic responses. To maximize the chance of detecting CaCC activation during voltage-clamp recording of isolated NMDA-EPSC in the presence of 20 μM CNQX, we replaced external Mg2+ with Ca2+ and increased the Cl− driving force by including 130 mM Cl− in the whole-cell patch pipette solution (ECl ∼0 mV) and holding the cell at −65 mV (65 mV driving force), so that CaCC activation would result in Cl− efflux thus enhancing the NMDA-EPSCs elicited from CA1 pyramidal neurons in acute almost slices (P14–21) by stimulating Schaffer collaterals every 20 s. As shown in Figure 5E, blocking CaCC with NFA reduced the NMDA-EPSC by 28% ± 4.3% (n = 10, p < 0.05), indicating that CaCC is in the vicinity of NMDA-Rs to be activated

by the Ca2+ influx through NMDA-Rs. Importantly, when 10 mM of BAPTA was included in the 130 mM Cl− internal solution, NFA no longer had effect on NMDA-EPSC (100% ± 1.4%, n = 10, p = 0.13; Figure 5F), providing further evidence that NFA has no presynaptic effect on transmitter release. In contrast, when we included 10 mM EGTA in the patch pipette solution with 130 mM Cl−, 100 μM NFA still reduced NMDA-EPSC by 32 ± 9% (n = 5, p < 0.01; Figure 5G). As summarized in the histogram (Figure 5H), Ca2+ influx through NMDA-Rs is capable of activating CaCCs that are in close proximity so that the slower Ca2+ chelator EGTA, but not the fast Ca2+ chelator BAPTA, allows CaCC activation for feedback modulation of NMDA-EPSCs. To explore the physiological contribution of CaCCs, first we performed field recording of the pharmacologically isolated NMDA-fEPSP in the CA1 dendritic field in the presence of 2.5 mM Ca2+ and 1.3 mM Mg2. During a 10 Hz stimulation of Schaffer collaterals, the NMDA-fEPSPs gradually increased (Figure S5, left). Bath application of NFA enhanced the third, sixth, and tenth (n = 8; third, 138.