Extended Data Fig. 6: Cutaneous driven trisynaptic circuits mediating PAD and assisting repetitive firing.

a, Cutaneous driven dorsal trisynaptic circuit mediating PAD. A minimally trisynaptic circuit is classically known to depolarize afferents via GABA_axo neurons. This circuit involves sensory afferents activating excitatory intermediary neurons (glutamatergic) that in turn activate GABA_axo neurons that return to innervate sensory axons^13,115. Even though GABA_axo neurons are small⁵ this circuit influences afferents over widespread regions of the spinal cord¹⁵. Specifically, the activation of a small group of sensory axons in just one DR or nerve causes this trisynaptic circuit to produce a widespread activation of many axons across the spinal cord, even many segments away and across the midline¹⁵. This allowed us to activate PAD from adjacent roots without directly activating an axon in a particular root, as detailed in Fig. 4 and the rest of this figure. One variant of this classic trisynaptic circuit specifically involves cutaneous stimulation activating dorsal intermediary neurons^13,14 that activates GABA_axo neurons (likely dI4 neurons¹¹⁵) that in turn innervate cutaneous afferents, which we term the cutaneous dorsal circuit. While this cutaneous dorsal circuit also synaptically innervates some proprioceptive afferents¹¹⁶ (a), its main action on proprioceptive afferents is to produce a pronounced extrasynaptic spillover of GABA that depolarizes these afferents tonically via α5 GABA_A receptors (termed: tonic PAD, L655708 sensitive), especially with repetitive cutaneous nerve stimulation (1–200 Hz) that leads to minutes of depolarization¹⁵, and we see similar tonic PAD here (detailed next). b, Intracellular recording from a proprioceptive axon branch in rat dorsal horn (sacral S4 axon, DR2). The axon branch spontaneously exhibited spike propagation failure when its was stimulated alone (denoted DR2 stimulation, repeated at 1 Hz, 1.1xT, 0.1 ms; T: afferent volley spike threshold), with only a small failure potential (FP) visible (lower pink traces). Activation of a largely cutaneous DR (caudal Ca1 DR, innervating the tip of the tail, stimulation at intensity for cutaneous afferents, 3xT, 0.1 ms; denoted DR1) evoked a slowly rising tonic PAD when repeated at 1 Hz (blue). When the axon stimulation (DR2 stimulation) was combined with the repeated cutaneous stimulation (DR1, 60 ms prior to each DR2 stimulation) the slowly building PAD prevented spike failure (black spikes), and this outlasted the cutaneous stimulation (after effect). Similar results obtained in n = 20/20 axons tested from 10 rats.