The objective was to test if sustained excitatory synaptic input to

a target neuron changed its intrinsic excitability. This is distinct from short-term depression of synaptic responses observed following Ipatasertib short periods of conditioning spontaneous activity (Hennig et al., 2008 and Hermann et al., 2007) in that our studies focused on how sustained synaptic inputs can influence postsynaptic voltage-gated conductances rather than synaptic strength. The conditioning synaptic stimulation lasted 1 hr and consisted of evoked EPSPs at a mean frequency of 10 Hz (with interstimulus intervals [ISIs] generated by a Poisson process, giving a total of 34,875 stimuli/1 hr). We stimulated the trapezoid body calyceal projection to the MNTB or mossy fiber/commissural projections (which were DCG-IV insensitive; see Figure S1C available online) to CA3 pyramidal neurons. Stimulation

at 10 Hz induces neither LTP nor LTD (Dudek and Bear, 1992) and provided a sustainable stimulation rate that did not deplete transmission to subthreshold levels (Figure S1A, stimulus recordings at 55 min) and was comparable with physiological firing rates for the MNTB (Kopp-Scheinpflug ATM inhibitor et al., 2003) and hippocampus (Fenton and Muller, 1998 and Klyachko and Stevens, 2006). In naive slices under current clamp recording, evoked EPSP trains at moderate frequencies securely triggered APs in principal neurons of the MNTB (<400 Hz). The illustrated example in Figure 1 shows single AP responses to each presynaptic stimulus at a frequency of 100 Hz (Figure 1A, Naive, upper black). But transmission failure occurred rapidly at 800 Hz or above (Figure 1A, Naive, lower black), consistent with previous reports (Taschenberger and von Gersdorff, 2000).

After synaptic conditioning (post-conditioning, PC: 1 hr stimuli), the response of MNTB neurons to moderate frequency stimuli was robust and unchanged (Figure 1A, upper red trace; 100 Hz, PC), but high-frequency stimuli now triggered APs with greater reliability (Figure 1A, PC, lower red trace; 800 Hz). The conditioning reduced evoked synaptic currents (Figure S1B), consistent with nitrergic suppression of AMPARs reported previously (Steinert et al., 2008). Comparison of the mean output (MNTB APs) to input (at Edoxaban 100, 800, or 1000 Hz) for naive (Figure 1B, black bars) and PC slices (red bars) showed increased reliability of transmission for high-frequency stimulation after conditioning. The synaptic conditioning also increased AP threshold (Figure 1C), consistent with reduced postsynaptic excitability. AMPAR and NMDAR antagonists (50 μM AP-5, 10 μM MK801, 10 μM CNQX applied for the 1 hr conditioning period) blocked these changes, whereas perfusion of NO donors (NO: sodium nitroprusside, SNP or PapaNONOate, each 100 μM for 1 hr) mimicked the threshold increase (Figure 1D).