(A). Injection scheme for expressing the excitatory DREADD hM3Dq in spinally projecting Tph2-Cre neurons. Brain injection coordinates according to the mouse brain atlas (−6,+/-0.5, 5.9 from bregma). (B). Example of the injection site from the hindbrain of a mouse that received a spinal dorsal horn injection of AAV2retro.flex.FLPo.BFP followed by a bilateral hindbrain injection of AAV1.FRT.hM3Dq.mCherry 1 week later (scale bar = 200 μm). (C). Example of the ipsilateral spinal dorsal horn form an animal that had received the injections indicated in A. Note that most 5-HT-containing terminals are not labeled, but the majority of labeled terminals contain a detectable level of 5-HT (scale bar = 200 μm). (D). Sensory tests of the ipsilateral hindpaw: Hargreaves plantar assay; repeated measures one-way ANOVA, (F(3, 42)=16.93, p<0.0001), post hoc tests with Bonferroni’s correction detected differences between post-vehicle and post CNO, as well as pre-CNO and post-CNO (adjusted p values are p=0.007 and p<0.0001 respectively). Cold plantar assay; repeated measures one-way ANOVA, (F(3, 39)=12.41, p<0.0001) post hoc tests with Bonferroni’s correction detected differences between post-vehicle and post CNO, as well as pre-CNO and post-CNO (adjusted p values are p=0.0122 and p=0.0103, respectively). von Frey test; repeated measures one-way ANOVA, (F(3, 39)=0.5013 p=0.6836). Rotarod test for sensorimotor coordination/sedation; repeated measures one-way ANOVA, (F(3, 30)=0.8684, p=0.4683). Significance: *p<0.05, **p<0.01, ***p<0.001.