Figure 1. Enkephalinergic Neurons in the Dorsal Horn Modulate Mechanical Sensitivity and Receive Inputs from RVM GABAergic Neurons.

(A) Coronal section of spinal cord from Penk^Cre mice injected with AAV-FLEx-YFP (green) showing the distribution of Penk+ neurons in laminae I through V (FLEx Cre on).

(B) In situ hybridization shows Penk mRNA (red) in the great majority of YFP+ neurons (green) (88% ± 2.6%; n = 4 mice).

(C) Half of Penk+ neurons (green) coexpress the glutamatergic neuron marker TLX3 (54% ± 4.3%; n = 4) (red, left panel) and a third coexpress the GABAergic/glycinergic neuron marker PAX2 (30% ± 2.4%; n = 4) (red, right panel).

(D) Electrophysiological characterization of Penk+ neurons in Penk^Cre;Rosa26-LSL-tdTomato mice. Injection of depolarizing currents in tdTomato+ neurons shows that 58% (20/34 neurons) of Penk^Cre neurons presented a tonic (top panel, pie chart), 29% (10/34) a delayed (bottom panel, pie chart), and 8% (3/34) a gap firing pattern (pie chart).

(E) Injection of AVV-FLEx-hM4Di-mCherry into the right side of the spinal cord dorsal horn of Penk^Cre mice causes Cre-dependent expression of hM4Di-mCherry 4 weeks after injection.

(F) CNO generated spontaneous nociceptive behaviors 1 hr after administration (Mann-Whitney test, **p < 0.01; n = 5).

(G) CNO induced profound mechanical hypersensitivity in the von Frey test (two-way ANOVA, Bonferroni post hoc test, *p < 0.05, ****p < 0.0001; n = 9).

(H) CNO did not alter heat sensitivity (Hargreaves test).

(I) Strategy for identifying neurons presynaptic to enkephalinergic spinal neurons with rabies virus-mediated trans-synaptic retrograde tracing.

(J) Coronal section of spinal cord dorsal horn from Penk^Cre mice injected with AAV helpers (red) and RVdG (green) (top panel). Arrows indicate examples of co-infected starter cells (yellow). Bottom panels show a close-up view of the dashed box shown in the top panel.

(K) GFP expression in RVM neurons revealing that enkephalinergic spinal neurons receive input from brainstem descending neurons (left panel). Right panels show a close-up of the dashed box in the left panel. Arrows indicate RVM RVdG GFP+ neurons coexpressing GABA (left column). RVM RVdG GFP+ neurons are TPH-negative (middle column) and rarely Penk positive (right column). RMg, Raphe Magnus nucleus; py, pyramidal tract; ml, medial lemniscus; 4V, fourth ventricle.

All scale bars represent 50 μm. All bar graphs represent mean ± SEM.

See also Figures S1 and S2.