Extended Data Fig. 9: Characterization of Aβ fibre and CST inputs onto CCK–tdTomato neurons.
From: Touch and tactile neuropathic pain sensitivity are set by corticospinal projections
a, Schematic of the stimulation and whole-cell patch recording set-up for tdTomato-labelled CCK+ interneurons. CST axons labelled by AAV-ChR2-YFP were stimulated with a 473-nm laser. A single dorsal root (L4–L6) was stimulated with a glass suction electrode. b, c, Representative consecutive traces (n = 3) of Aβ fibre (left) and opto-CST (right) stimulation-evoked responses (b) and summarization (c) of whole cell patch-clamp recordings on CCK–tdTomato interneurons. Three recording conditions were used: first, to detect evoked EPSCs (eEPSCs), we held the membrane potential at −70 mV, which is the equilibrium potential of Cl− and therefore minimizes the flow of eIPSCs. Second, by holding the membrane potential at 0 mV, we examined the polysynaptic, inhibitory inputs (eIPSCs) onto CCK–tdTomato interneurons. Third, we used current clamp mode to examine whether the stimulation drove action potential firing at the resting membrane potential. Type 1: CCK–tdTomato neurons receive only excitatory inputs, and few of them generated AP outputs when Aβ or CST inputs were stimulated. Type 2: CCK–tdTomato neurons receive both excitatory inputs and feed-forward inhibitory inputs, with no AP output. Type 3: CCK–tdTomato neurons receive predominant feed-forward inhibition, with no AP output. Type 4: CCK–tdTomato neurons show no response at either voltage or current clamp recording. d, Left, representative recording of an Aβ (25 μA) dorsal root evoked EPSC at −70 mV. Latency and jitter properties (magnified in inset) with quantifications (n = 8 neurons) are consistent with monosynaptic sensory connectivity. Right, opto-stimulation evoked EPSCs (averaged traces) at −70 mV in the same cell as shown on the left. The evoked EPSC was blocked by AMPA and NMDA antagonists (NBQX (5 μM)/CPP (20 μM)). In addition, opto-stimulation evoked EPSCs were eliminated by TTX (0.5 μM), and reinstated by 4-AP (2 mM), indicating a monosynaptic connection between the CST and CCK–tdTomato interneurons. Bar graph, quantification of eEPSC amplitudes after administration of drugs as shown. **P < 0.0001; one-way ANOVA followed by Bonferroni correction. n = 8 neurons. Data shown as mean ± s.e.m.
