© 2007 Nature Publishing Group http://www.nature.com/natureneuroscience Molecular genetic visualization of a rare subset of unmyelinated sensory neurons that may detect gentle touch Qin Liu 1 , Sophia Vrontou 2,3 , Frank L Rice 4 , Mark J Zylka 5 , Xinzhong Dong 1 & David J Anderson 2,3 C-fiber tactile afferents are a subpopulation of unmyelinated cutaneous sensory neurons activated by gentle stroking. Using a genetically encoded tracer, we found that Mas-related G protein–coupled receptor B4 marks a rare subpopulation of unmyelinated, nonpeptidergic sensory fibers that exclusively innervate hairy skin. These fibers terminate in large arborizations similar in size and distribution to C-fiber tactile afferent receptive fields, suggesting that MrgprB4 may provide genetic access to these elusive neurons in mice. Mas-related G protein-coupled receptors (Mrgprs), also called sensory neuron-specific receptors (SNSRs), are specifically expressed in subsets of small-diameter sensory neurons 1–4 . Neurons expressing MrgprD project exclusively to glabrous and hairy epidermis 5 . Other Mrgprs are expressed in different subsets of small-diameter neurons 1,4 , raising the question of whether these subsets show distinct target specificities. We mapped the projections of MrgprB4 + neurons by targeting placental alkaline phosphatase (PLAP) to the MrgprB4 locus in embryonic stem cells (Supplementary Fig. 1 online). Homozygous MrgprB4D PLAP mice were viable and fertile, and did not show obvious phenotypic abnormalities. Histochemistry showed that PLAP was expressed exclusively in a small subset of dorsal root (DRG) and trigeminal ganglion neurons (Fig. 1a,b), with a postnatal onset (Supplementary Fig. 2 online). Thoracic (T12/T13) DRG contained, on average, 119 ± 21 MrgprB4 + neurons per ganglion (Fig. 1a, n ¼ 6), corresponding to B2% of all DRG neurons. There were even fewer MrgprB4 + neurons in lumbar (L4) and cervical (C6) DRG (Fig. 1b; 45.57 ± 8.96, n ¼ 7 and 45.86 ± 8.89, n ¼ 8, respectively). MrgprB4 + neurons were small diameter (average 26.86 ± 1.93 mm), negative for T12 L4 Obverse Inverse Marker CGRP P2X3 VR1 MrgprD M M M M M B B 80 μm Merge MrgprB4Δ PLAP a b c d e f g h i j k l o p m n 1 μm Figure 1 Characterization of MrgprB4 + sensory neurons. (a,b) Whole-mount PLAP histochemistry of DRG from heterozygous MrgprB4D PLAP adult mice. (c–k) lumbar level 2 (L2) DRG neurons from MrgprB4D PLAP mice stained with antibodies to PLAP and the indicated markers. (l–n) DRG from MrgprB4D PLAP ;MrgprDD EGFPf mice stained with antibodies to PLAP and GFP. (o,p) Transverse electron microscopic sections of thoracic nerves from MrgprB4D PLAP (o) and wild-type (p) mice stained for PLAP activity. Note the dense precipitate on unmyelinated MrgprB4 + axons (B). * indicates unstained, unmyelinated axons. Myelinated axons (M) were unstained. Received 16 April; accepted 11 June; published online 8 July 2007; doi:10.1038/nn1937 1 The Solomon H. Snyder Department of Neuroscience, School of Medicine, Johns Hopkins University, 725 N. Wolfe St., Baltimore, Maryland, 21205, USA. 2 Division of Biology, 216-76, California Institute of Technology, 1201 E. California Blvd., Pasadena, California 91125, USA. 3 Howard Hughes Medical Institute, 1201 E. California Blvd., Pasadena, California 91125, USA. 4 Center for Neuropharmacology and Neuroscience, Albany Medical College, 43 New Scotland Ave., Albany, New York 12208, USA. 5 Department of Cell and Molecular Physiology, UNC Neuroscience Center, University of North Carolina, 105 Mason Farm Road, Chapel Hill, North Carolina 27599, USA. Correspondence should be addressed to D.J.A. (wuwei@caltech.edu) or X.D. (xdong2@jhmi.edu). 946 VOLUME 10 [ NUMBER 8 [ AUGUST 2007 NATURE NEUROSCIENCE BRIEF COMMUNICATIONS