Within approximately 20 min, the resting membrane potential of most neurons returned to their initial values (81%, 17/21). Near the site of high-intensity laser exposure, a gap of ∼5 μm became visible, and distal parts of the axon showed typical beading and degeneration (Figures 4A and S2). Axotomy proximal

to the node (P) was made either in the internode (P, 90–140 μm, n = 5, Figure 4A, right) or within the AIS (15–50 μm, C646 manufacturer n = 7). To isolate the impact of axotomizing the first branchpoint from nonspecific changes (asymmetric current flow at the sealed end, heat-related swelling, phototoxicity, etc.), a control group was included in which the axon was cut distal from the identified first node (D, 120–160 μm, n = 5, Figure 4A, selleck chemicals llc middle). Figures 4A and 4B show an example in which the same axon was axotomized at two different locations with an interval of

25 min. The intrinsically burst firing neuron (236 Hz in control) continued firing at high frequency (240 Hz) when axotomized distal to the branchpoint, but switched to RS mode after a second cut proximal to the branchpoint (9.6 Hz). Data from multiple recordings showed that axotomy of the first branchpoint in IB neurons (230 ± 3 Hz) led to a RS mode (10.2 ± 0.4 Hz, n = 6; unpaired t test p < 0.001; Figure 4C). In contrast, in RS cells the firing rates at steady current injections remained similar to control (control, 7.7 Hz versus cut, 5.6 Hz, paired t test p > 0.09, Figure 5C). Axotomy proximal to the branchpoint did not affect the input resistance at resting potential (1.9 ± 1.0 MΩ increase, paired t test p > 007, n = 12, Figure S2). Similar to axons cut in the slice preparation, axotomy proximal to the first node significantly increased the AP threshold during steady current injections (+6.8 ± 1.1 mV, p < 0.01, Figure 4D) and reduced the ADP amplitude (−3.8 ± 0.6 mV, n = 5, p < 0.05,

Figure 4E). However, single APs were not affected when the axotomy was made distal of the first node; the AP amplitude (+0.3 ± 1.7 mV change), ADP (−0.5 ± 0.5 mV change), and voltage threshold (+1.6 ± 0.5 mV change) remained similar to control values (for all, paired t test p > Monoiodotyrosine 0.3, n = 5). The only specific impact of cutting within the AIS (on average 35 ± 5.4 μm, n = 7) was a significantly larger reduction in the ADP (−12.0 ± 2.7 mV, compared to internodal axotomy p < 0.05, n = 7, Figure 4E). Interestingly, large negative ADP amplitudes observed after laser axotomy in the AIS were quantitatively similar to the ADP amplitudes associated with axons that were cut in the AIS during the slice-cutting procedure (acute axotomy: −12.0 mV for a 35 μm axon versus slice cut: −10.5 mV for a 32 μm axon), suggesting that the functional consequences of acute transections (30 min) are comparable to the lasting impact by slice cutting (2–8 hr).