Supplementary Figure 14: Schematic of spinal pathways mediating dynamic versus punctate mechanical hypersensitivity

VT3^Lbx1 neuron-dependent pathway 1 could mediate brush-evoked dynamic allodynia, whereas VT3^Lbx1 neuron-independent pathways 2 and 3 could potentially mediate punctate allodynia preserved in VT3^Lbx1-ablated mice. VT3^Lbx1 neuron-dependent pathway 4 transmits acute light mechanical information. All these pathways are dependent on SOM^Lbx1 neurons. “V1” and “V2” indicate type 1 and type 2 VT3^Cre-tdTomato⁺ neurons. “SOM?” indicates _vII_i neurons receiving C fiber inputs, and these neurons either represent SOM^Lbx1 neurons or neurons connected with SOM^Lbx1 neurons based on loss of such inputs in SOM^Lbx1-ablated mice. “In” indicates inhibitory neurons. “PN”: projection neurons. Solid arrows for monosynaptic inputs, and dashed arrows indicating uncertainty of mono- or poly-synaptic inputs. A series of genetically labeled inhibitory neurons have been shown to play a role in gating LTMR inputs to dorsal horn neurons [Duan, B., et al. Cell 159, 1417-1432 (2014); Foster, E., et al. Neuron 85, 1289-1304 (2015); Petitjean, H., et al. Cell Rep. pii: S2211-1247(15)01134-1(2015); Cui, L. et al. Neuron 91, 1137-1153 (2016).], but their relative contributions to the gating of pathways 1-3 remain to be determined. It should be pointed out that all these three pathways could potentially relay afferent inputs to projection neurons located laminae IV-VI as well (to be determined).

Peirs et al. reported that activation of spinal neurons marked by their transgenic VT3::Cre was able to develop punctate mechanical sensitivity [Peirs, C. et al. Neuron 87, 797-812 (2015)], whereas ablation of adult spinal neurons marked by coexpression of our VT3^Cre and Lbx1^Flpo (VT3^Lbx1 neurons) did not affect the transmission of filament-evoked punctate hypersensitivity induced by nerve injury or strong inflammation (see Fig. 2). We envision several possibilities. Firstly, VT3^Lbx1 neurons have a redundant, but non-essential, role in mediating this form of mechanical hypersensitivity. Secondly, there are 14% of VT3^Cre-marked spinal neurons that are not ablated (see Fig. 1) and these neurons might mediate punctate mechanical hypersensitivity. Thirdly, considering that their VT3::Cre poorly labeled Calb2⁺ neurons in comparison with our knock-in mice (Supplementary Fig. 1), it remains to be determined if VT3::Cre might conversely mark spinal neurons not marked by our VT3^Cre, and if activation of these neurons generated punctate mechanical hypersensitivity. Future intersectional ablation of spinal neurons coexpressing transgenic VT3::Cre and Lbx1^Flpo, as we have done for VT3^Lbx1 neurons here, would be informative for distinguishing these possibilities.

Peirs et at. also reported that mechanical hypersensitivity evoked by von Frey filaments is eliminated in Vglut3 conditional knockout mice [Peirs, C. et al. Neuron 87, 797-812 (2015)], which again contrasts with the preservation of this form of hypersensitivity following ablation of adult VT3^Lbx1 neurons. Because VGLUT3 is expressed transiently in the dorsal spinal cord during postnatal development, we speculate that maturation of spinal substrates mediating VT3^Lbx1 neuron-independent punctate mechanical hypersensitivity might be non-autonomously influenced by VGLUT3-dependent glutamate release during development. Scale bars represent 50 μm.