Extended Data Fig. 9 |. Estimating the overall spike conduction failure from the dorsal root to the motoneurons.

a, Experimental setup to indirectly measure sensory axon conduction failure following DR stimulation, by examining whether failed axon segments are relatively less refractory to activation after failure, using a double pulse method adapted from Wall³⁰. A tungsten microelectrode (12 MΩ) was placed in the ventral horn (VH) near the sensory axon terminals on motoneurons (S3 or S4 VH), to activate the branches/nodes of the axon projecting to the motoneuron that may have failed (VH stimulation). Spikes from VH or DR stimulation were recorded intracellularly in a proprioceptive axon penetrated in the dorsal columns. b, VH threshold in refractory period. Rapidly repeated VH stimulation (VH doublet; two 0.1 ms pulses) at an interval short enough to produce spike inactivation on the second stimulation (4 ms), with stimulus current adjusted to threshold for inactivation, T_VH2. This T_VH2 (~15 uA) was always higher than the threshold VH stimulation for evoking a spike with the first stimulation, T_VH1 (~10 uA). Recorded in sacral S4 afferent resting at - 72 mV, with doublets repeated at 3 s intervals to determine current thresholds. c, VH threshold after DR stimulation. Similar repeated activation of the axon in (b), but with the first activation from a DR stimulation (at 1.5x DR threshold) and the second from VH stimulation at the T_VH2 intensity from (b). In this case the VH stimulation readily activated the axon spike, likely because the DR- evoked spike did not propagate to the VH, leaving the silent portion of the axon non refractory. Thus, this VH stimulation evoked spikes with a lower current than T_VH2, with this lower threshold denoted T_DR,VH (~ 12 μA). This DR – VH stimulation interval was deliberately set too short for the involvement of PAD (which rises in > 4 ms; Fig. 4). d, Computation of spike failure based on changes in VH stimulation thresholds. If the DR-evoked spike entirely fails to propagate to the VH, then the threshold for subsequently activating the VH (T_DR,VH) should be the same as the threshold without any prior activation (T_VH1 = T_DR,VH), whereas if it does not fail then the threshold for activating the VH should be the same as with a VH doublet (T_VH2 = T_DR,VH). In between these two extreme scenarios, the DR evoked spike may only partially fail to propagate spikes to the VH; in this case T_DR,VH should be between T_VH1 and T_VH2, with the difference T_VH2 - T_VH1 representing the range of possible thresholds between full failure and conduction. Overall the % conduction failure can be thus quantified as: (T_VH2 - T_DR,VH)/(T_VH2 - T_VH1) * 100%, which is 100% at full failure and 0% with no failure. e, Average spike conduction failure to the VH in proprioceptive axons, and decrease following a DR conditioning stimulation that depolarized the axon (PAD). Box plots of failure estimated as in (b-d). Prior DR conditioning to produce PAD (via adjacent S4 or Ca1 DR stimulation at 3xT) reduced the failure estimated 20 ms later by the paired-pulse conduction testing (repeating DR – VH stimulations of b-d). DR conditioning itself lowered the thresholds for VH activation (n = 5 rats), as previously reported³². We studied two lengths of axons: long axons (intersegmental, n = 11 axons, from 5 rats) with the VH stimulation one segment away from the recording site, and short axons (segmental, n = 12, from 5 rats) with the VH stimulation near the recording site, in the same segment. Box plots show the interquartile range (box), median (thin line), mean (thick line), extremes, 10 and 90 percentiles (whiskers). + significantly less failure with PAD and * significantly less failure with short compared to long axons, two-sided paired and unpaired t-tests, respectively, P < 0.05.