5%, p < 0.01; sixth, 145% ± 6.6%, p < 0.01; tenth, 140 ± 6.4%, p < 0.01) but not the first NMDA-fEPSP (105% ± 0.5%, n = 8, p =

0.8) (Figure S5, right), indicating that only when consecutive synaptic responses cause sufficient Ca2+ buildup for CaCC activation does NFA exert an effect on the synaptic response. Next, we recorded the pharmacologically isolated NMDA-EPSPs in CA1 pyramidal neurons while stimulating Schaffer collaterals at 10 Hz and asked whether CaCC plays a role in NMDA-EPSP-spike coupling. In the presence of 10 mM internal Cl−, 100 μM NFA enhanced NMDA-EPSP-spike coupling; NMDA-EPSPs summate to spike much later (first spike occurring most frequently at the tenth synaptic response) when CaCC is intact than when CaCC is blocked by 100 μM NFA (first spike occurring most frequently at the fourth or fifth responses) (Figure S6A). Thus, when CaCC is blocked by NFA, neurons fire spikes more readily with selleck compound reduced average latency to first spike and increased average number of spikes per train (Figure S6B; n = 10, p < 0.001). The CaCC function depends on the Cl− concentration gradient because when we elevated the internal Cl− level to 130 mM (ECl ∼0 mV), reducing CaCC with 100 μM NFA delayed spike initiation, increased the average latency to first spike and reduced

the average number of spikes generated (Figures S6C and S6D; n = 10, p < 0.001). Thus, whereas CaCC normally acts as an inhibitory brake GABA receptor drugs on NMDA-EPSP to spike coupling, elevating internal Cl− concentration during neuronal activity or dysfunction could cause CaCC to provide positive feedback and enhance excitation. To further explore the physiological contribution of CaCCs to synaptic responses,

we stimulated Schaffer collaterals at 100–200 microns from the CA1 pyramidal cell body layer every 30 s and recorded from CA1 pyramidal neurons at 35°C in physiological solution plus picrotoxin to block GABAA receptors. Reducing CaCC with 100 μM NFA increased the amplitude of large but not small synaptic potentials (Figure 6A), most likely because the former involved NMDA receptor activation. Indeed, in the presence of 100 μM until APV to block NMDA receptors, the EPSP was no longer affected by 100 μM NFA (Figure 5I), regardless the stimulus intensity (Figure 5J). Under physiological condition with 10 mM [Cl−]in (Figure 6B, left panel), reducing CaCC with 100 μM NFA amplified EPSPs of large amplitude. In 130 mM [Cl−]in (Figure 6B, middle panel), however, reducing CaCC with 100 μM NFA dampened EPSPs of large amplitude. NFA had no effect on EPSP amplitude when BAPTA was included with 10 mM [Cl−]in to chelate Ca2+ (Figure 6B, right panel). These controls reinforce the notion that the NFA block of CaCC affects the large synaptic potentials that involve activation of NMDA-Rs (6 mV EPSP: 147% ± 2.9%, n = 10, p < 0.05). To test whether CaCCs also play a role in EPSP summation to regulate synaptic integration, we delivered 3 nerve stimuli at 40 Hz.