Fig. 1
From: Itch suppression in mice and dogs by modulation of spinal α2 and α3GABAA receptors
Inhibitory input onto spinal GRP positive itch relaying interneurons. a Neurons providing synaptic input onto GRP positive neurons were identified using transsynaptic rabies virus based tracing. GRP::cre;ROSA26TVA mice (n = 3) were first injected with AAV.flex.mCherry-RabG into the left lumbar spinal cord, and 14 days later with an EnvA pseudotyped glycoprotein deficient rabies virus (EnvA.RabiesΔG-eGFP). b–g Transverse sections of the injected dorsal horn. Cre positive GRP neurons infected with AAV.flex.RabG (red) primary and secondary Rabies virus infected neurons (green). Co-staining with Lmx1b and Pax2 revealed excitatory and inhibitory neurons, respectively. b primary rabies virus infected neurons express eGFP and mCherry, and appear yellow. Secondary rabies virus infected neurons are green only. c Co-staining with Lmx1b (blue) reveals the excitatory population of secondary infected (transsynaptically labeled) neurons (magenta; eGFP and Lmx1b positive but mCherry negative) d Classification of transsynaptically labeled neurons as excitatory (39%, Lmx1b positive) or inhibitory (45%, Pax2 positive). 16% of transsynaptically infected neurons (eGFP positive but mCherry negative) were neither stained with antibodies against Lmx1b or Pax2, and remained unclassified. e Co-staining with PKCγ (red) indicates border between lamina II and III. mCherry was not visible in e-g because it required signal amplification by an mCherry antiserum. f Co-staining against Pax2 revealed transsynaptically labeled inhibitory neurons (magenta; Pax2 and eGFP positive). Scale bar, 100 µm. We did not find any mCherry/eGFP/Pax2 triple positive neurons (not shown) consistent with the known excitatory phenotype of spinal GRP neurons22. g Higher magnification of the area indicated in f. Arrow heads indicate rabies virus infected (eGFP positive) inhibitory (Pax-2 positive) neurons. Of the inhibitory transsynaptically labeled neurons, 48% were in laminae I/II and 52% in laminae III/IV. Quantifications were performed on 9 sections obtained from 3 mice
