6 ± 0.4 mV,

paired t test p > 0.11, n = 5, Figure 7B). Finally, the local perfusion of zero Na+ to the distal part of the AIS was sufficient to completely abolish axonal spike initiation, as indicated by the large shift in voltage threshold (+19.6 ± 1.7 mV, n = 7, p < 0.001, Figure 7B). Most strikingly, blocking nodal Na+ currents either abolished or significantly reduced high-frequency AP generation (TTX, block in 5/5 IB neurons, zero Na+, block in 2/3 IB neurons, Figure 7C). On average, the AP frequency in the Protein Tyrosine Kinase inhibitor first interval reduced from 242.6 ± 19.4 Hz to 36.1 ± 24.9 Hz (paired t test p < 0.0001, n = 11, Figure 7C). Consistent with the observations from acute axonal transections, the RS L5 neurons were not affected in firing frequency after nodal Na+ channel

block (control, 10.2 ± 0.8 Hz; TTX/zero Na+, 9.9 ± 1.4 Hz; paired t test p > 0.7, n = 8, Figure 7D). Single APs were further investigated for their axonal and somatic components using the second derivatives. learn more Blocking nodal Na+ channels significantly reduced the first axonal component of the AP rate of rise of IB neurons (control, 12.4 ± 1.5 MV s−2, TTX/zero Na+, 9.3 ± 1.2 MV s−2, paired t test p < 0.01, n = 5, Figure 7E), while the second peak remained unaffected (control, 9.6 ± 1.1 MV s−2, TTX/zero Na+, 9.2 ± 1.0 MV s−2, n = 5, p > 0.61). No change was observed in the second derivatives of APs from RS neurons (unpaired t test p > 0.19, n = 7). These changes in the d2V/dt2 of the AP resemble the observations in axons cut proximally to the first node ( Figures S1 and GBA3 S2). Thus, Na+ channels in the first node of Ranvier contribute to the generation of axonal APs in IB firing L5 neurons. AP bursts occur at preferred stimulus input frequencies (Golomb et al., 2006 and Kepecs et al., 2002). It was therefore important to test whether the findings, based primarily on constant signals, could be extended to more physiological type of input stimuli. To mimic in vivo-like synaptic activity, simulated EPSCs were applied as randomized patterns of current injections with realistic rise and decay times in the soma (2 s epochs, 10–30 repetitions). In control IB neurons, the simulated

EPSC injections were encoded into a wide variety of AP frequencies up to 450 Hz (Figure 8A). After application of TTX to the node, the high-frequency bursts were strongly attenuated (Figure 8A). The impact on firing was first quantified by calculating the mean firing rate (number of spikes/s), which reduced to ∼55% of the control rate (control, 16.1 ± 2.8 Hz, n = 5; TTX, 8.8 ± 2.1 Hz, n = 5; paired t test p < 0.05, Figure 8B). Subsequently, the frequency distribution of all instantaneous spike intervals was plotted using a normalized probability density histogram (sum of five experiments, Figure 8C). These data showed that the probability of an AP burst (f ≥ 100 Hz) was significantly reduced after TTX application (paired t test p < 0.