Extended Data Fig. 10: Axonal spike conduction estimated from extracellular recordings.

a, Experimental setup to directly record spike conduction failure in proprioceptive axon terminals in the ventral horn (VH) following DR stimulation. Extracellular (EC) recordings from axon terminals in VH, with glass electrode positioned just outside these axons, and for comparison EC recording in the dorsal horn (DH). b, EC field recorded in VH after DR stimulation (S4 DR, 1.1xT; T: afferent volley threshold), with a relatively large initial positive field (magenta arrows, pf) resulting from passively conducted axial current from sodium spikes at distant nodes (closer to the DR; outward current at electrode), some of which fail to propagate spikes to the VH recording site; thus, this field is a measure of conduction failure, as demonstrated in (c-f) below. Following this, a negative field arises (blue arrow, nf), resulting from spikes arising at nodes near the electrode (inward current); thus, this field is a measure of secure conduction. Reducing conduction failure by depolarizing the axon (+PAD) with a prior conditioning stimulation of an adjacent DR (Ca1, 2xT, 30 ms prior), decreased the positive field (pf) and increased the negative field (nf), consistent with increased conduction to the terminals, and in retrospect the same as Sypert et al. (1980)¹²⁰ saw in cat (their Fig. 4). Large stimulus artifacts prior to these fields are truncated. c, Control recordings from proprioceptive axons in dorsal horn (DH) to confirm the relation of the EC negative field (nf) to spike conduction. Intracellular (IC) recording from axon (sacral S4, resting at -64 mV) and EC recording just outside the same axon, showing the DR evoked spike (IC) arriving at about the time of the negative EC field (nf). There is likely little spike failure in this axon or nearby axons, due to the very small initial positive field (pf). EC fields are larger in DH compared to VH (G, 10x), and thus the artifact is relatively smaller. d, Locally blocking nodes with TTX to confirm the relation of the positive EC field to spike failure. EC recording from proprioceptive axon in the dorsal horn (S4), with an initial positive field (pf) followed by a negative field (nf), indicative of mixed failure and conduction. A local puff of TTX (10 µl of 100 µM) on the DR just adjacent to the recording site to transiently block DR conduction eliminated the negative field (nf) and broadened the positive field (pf), consistent with distal nodes upstream of the TTX block generating the positive field via passive axial current conduction, and closer nodes not spiking. Recordings were in the presence of synaptic blockade (with glutamate receptor blockers, kynurenic acid, CNQX and APV, at doses of 1000, 100 and 50 µM respectively), to prevent TTX spillover having an indirect action by blocking neuronal circuit activity, including GABA_axo neuron activity. This synaptic blockade itself contributed to some spike failure, consistent with a block of GABA_axo neuron activity, as there was a more prominent positive field (pf) compared to without blockade in (c). e, EC field recorded from terminals of proprioceptive axons in the ventral horn near motor neurons (S4), in the presence of an excitatory synaptic block that largely eliminates most neuronal circuit behaviour (with kynurenic acid, CNQX and APV, as in d). In this synaptic block negative fields were generally absent (nf = 0), and only prominent positive fields (pf) occurred (as with TTX block), suggesting that conduction to the VH often completely failed when circuit behavior is blocked, which likely indirectly reduces GABA_axo neuronal circuit activity and its associated facilitation of nodal conduction. f, Rescue of spike conduction to the ventral horn by increasing sodium channel excitability by reducing the divalent cations Mg ++ and Ca ++ in the bath medium⁸⁰. Same EC field recording as in e, but with divalent cations reduced (Mg⁺⁺, 0 mM; Ca⁺⁺, 0.1 mM). The positive field was largely eliminated (pf = 0) and replaced by a negative field (nf), consistent with elimination of conduction failure, and proving that the positive field is not a trivial property of axon terminals^78,79,120. g, Conduction index computed from positive (pf) and negative (nf) field amplitudes as: nf / (nf + pf) x 100%, which approaches 100% for full conduction (pf ~0; as in c) and 0% for no conduction (nf = 0; as in e). h-i, Summary of conduction index estimated from EC field potentials, shown with box plots. Without drugs present in the recording chamber, the axon conduction from the DR to the dorsal horn was about 70% (h, n = 17 axon fields, from 10 rats), consistent with Fig. 2, whereas conduction from the DR to the VH was only about 50% (i, n = 11, from 5 rats), suggesting substantial failure at the many branch points in the axon projections from the dorsal horn to the motor neurons. Increasing GABA_axo neuron activity with DR conditioning (PAD, 30–60 ms prior) increased conduction (+GABA, in both the DH and VH, n = 5 and 9 from 5 rats, as in b), whereas decreasing GABA and all circuit activity in a synaptic blockade decreased conduction (-GABA, in both DH and VH, n = 5 and 6 from 5 rats, as in d-e). TTX (n = 5 from 2 rats, h) or removal of divalent cations (Mg⁺⁺ and Ca⁺⁺, -Divalent, n = 5 from 2 rats in synaptic block, i) reduced or increased conduction, respectively (as in d and f). Box plots show the interquartile range (box), median (thin line), mean (thick line), extremes, 10 and 90 percentiles (whiskers).* significant difference from control pre-drug or pre-conditioning, two-sided paired t-test, P < 0.05.