Figure 2.

Morphology and MrgprA3⁺ primary sensory input of GRP-cre-expressing neurons. A, Examples of two GRP central neurons form a spinal cord of GRP::cre ROSA26^lsl-TVA mice infected with SAD.RabiesΔG.eGFP (EnvA) virus. The morphology of the same cells is shown in the dorsoventral (left) and mediolateral view (right). Scale bars, 20 μm. B, Quantification of the morphological analysis of GRP neurons. C, Immunofluorescence staining of a lumbar spinal cord section of GRP::eGFP mice showing GRP-eGFP colocalization with the excitatory marker Lmx1b in lamina II. Arrowheads indicate examples of colocalization of GRP-eGFP and Lmx1b immunoreactivity. Scale bar, 100 μm. D, Quantitative analysis verified that GRP-eGFP neurons and GRP-cre neurons exhibit similar neurochemical characteristics (compare Fig. 1G). In bar charts, data are mean ± SEM. E, Overview of the spinal dorsal horn of an MrgprA3-cre; GRP::eGFP; ROSA^lsl-tdTom mouse. The termination area of MrgprA3-positive primary sensory neurons largely overlaps with the location where the GRP-eGFP cells are located. Scale bar, 100 μm. F, High-magnification images of a GRP-eGFP cell receiving direct synaptic input from MrgprA3-positive fibers. Arrowheads indicate MrgprA3-positive excitatory synaptic contacts onto a GRP-eGFP cell. Scale bar, 20 μm. G, Schematic showing optogenetic activation of MrgprA3-expressing primary afferent terminals (MrgprA3-ChR2) and targeted recordings from eGFP-positive GRP neurons (GRP-eGFP). H, Superposition of 20 consecutive light-evoked EPSCs traces recorded from GRP-eGFP neurons. Blue area represents 473 nm, 4 ms, 0.1 Hz. Gray represents individual responses. Black represents average response. I, J, Dot plots illustrate latency (I) and jitter (J) of light-elicited EPSCs recorded from GRP-eGFP cells (n = 6, from 5 animals). Circles represent individual cells. Error bars indicate SEM.