Figure 1.

Monosynaptic connections from sensory afferents to V3 neurons. A: V3 neurons in the spinal cord intermediate laminae decorated with VGLUT1⁺ contacts from afferents, some of which are Ia afferent contacts labeled by an intracellular injection of neurobiotin (blue). Spinal cord from Sim1//tdTom mouse. B: expanded image from A with 3-D reconstructed VGLUT1⁺ afferent contacts on V3 neurons labeled in yellow. Top: VGLUT1 contacts; middle: neurobiotin contacts; bottom: merge of above two panels. C: schematic of putative trisynaptic circuit where Ia afferents (blue) innervate V3 neurons, which in turn innervate GABAergic neurons, which return to innervate Ia afferents, ultimately producing PAD. Experimental setup indicated where Ia afferents are activated by dorsal root (DR) stimulation (0.1 ms pulse, 2xT, sensory threshold) and response recorded with a sharp intracellular electrode in or near V3 neurons or afferents. D: monosynaptic EPSP recorded intracellularly in V3 neuron in response to dorsal roots stimulation (S4 root of in vitro sacrocaudal cord; average of 10 trials at 0.3 Hz). E: same V3 EPSP as in D, but population response recorded nearby, extracellularly to V3 neurons (EC field). F: intra-axonal recording from Ia afferent during same DR stimulation, where the afferent is directly activated, as evident by an orthodromic spike, and following this a primary afferent depolarization (PAD) arises at a polysynaptic latency consistent with a circuit like in C. G: central latencies and durations of V3 EPSPs, EC fields, and PAD, where that latter was recorded either intracellularly in Ia afferents as in F (though in afferents of not directly activated by the DR stimulation, so the PAD onset can be judged) or by grease-gap recordings from DR (dorsal root potential, DRP). Central latency measured relative to arrival time of the orthodromic spike at the spinal cord, as in F, though measured from the extracellular afferent volley (not shown, though see Ref. 22). V3 EPSP latencies were minimally near 1 ms (0.8–3 ms), which is monosynaptic as the in vitro adult spinal cord has a synaptic delay of ∼1 ms at 23°C. PAD latencies were 2–4 ms, with an average of 3 ms, consistent with the trisynaptic circuit of C, though possibly with also some disynaptic innervation. *Significantly longer latency of PAD (Ia PAD or DRP) compared with V3 EPSP or its EC field latency, P < 0.05, n = 6 V3 neurons, 6 EC fields, 44 DRPs, and 5 Ia afferents, in 3–8 mice each. PAD, primary afferent depolarization.