Figure 5. Activation of VGLUT3⁺ Dorsal Horn Neurons Produces Mechanical Allodynia and Reveals a Polysynaptic Mechanical Allodynia Circuit.

(A) Schematic of the VGLUT3^Cre dorsal horn unilaterally injected at P10 with the Cre-dependent excitatory DREADDs virus (AAV8 hSyn-DIO- hM3Dq-mCherry).

(B) Lamina III, but not PKCγ (arrowheads) or DRG neurons express hM3Dq-mCherry. Scale bars = 100 μm except for inset (20 μm) and whole spinal cord (400 μm).

(C) Recording electrode filled with Alexa-488 targets a lamina III neuron expressing hM3Dq-mCherry. Second electrode used to puff on the specific hM3Dq ligand CNO can be seen in DIC picture. Scale bar = 50 μm.

(D) Direct application of 3 mM CNO to an hM3Dq-mCherry expressing neuron generates an inward current in voltage-clamp and series of action potentials in current-clamp. CNO applied to a cell lacking hM3Dq-mCherry does not elicit a change in membrane potential (bottom trace).

(E) PWTs for VGLUT3^Cre and VGLUT3^fl/fl;VGLUT3^Cre mice expressing hM3Dq-mCherry were normal at baseline. After CNO (5 mg/kg) injection, only PWTs of VGLUT3^Cre mice dropped dramatically. Hargreaves’ withdrawal latencies were unchanged for the two mouse lines (both tests n=7 and n=10 respectively).

(F) After CNO (5 mg/kg) injection, hM3Dq-expressing VGLUT3^Cre mice show lifting, fluttering and guarding of the ipsilateral hindpaw. In contrast, hM3Dq-expressing VGLUT3^fl/fl;VGLUT3^Cre mice rarely exhibit the behaviors.

(G) Quantification of c-Fos⁺ cells in laminae I–III and laminae IV–VI of VGLUT3^Cre (labeled WT) and VGLUT3^fl/fl;VGLUT3^Cre (labeled KO) mice expressing hM3Dq-mCherry. Hatched bars indicate number of c-Fos⁺ cells that also express mCherry for each condition.

(H) Induction of c-Fos cells in laminae I–III neurons of VGLUT3^Cre, but not VGLUT3^fl/fl;VGLUT3^Cre mice (both expressing hM3Dq-mCherry) after CNO injection followed by mechanical stimulation. Scale bars = 100 μm.

(I) Co-staining of c-Fos with PKCγ (21%), calretinin (28%) and Pax2 (14%) neurons in laminae I–III of VGLUT3^Cre mice expressing hM3Dq-mCherry after CNO injection followed by mechanical stimulation. Nearly half (48%) of the mCherry⁺ cells were also c-Fos⁺. Scale bars = 50 μm.

Data are mean ± SEM. *p<0.05, **p ≤ 0.01, ***p ≤ 0.001.