Journal of Neuroscience Research zyx 30:92-104 (1991) NF-L and Peripherin Immunoreactivities Define Distinct Classes of Rat Sensory Ganglion Cells M.E. Goldstein, S.B. House, and H. Gainer Lab of Neurochemistry, NINDS, NIH, Bethesda, Maryland Double immunofluorescence studies using antibodies against NF-L and peripherin revealed three distinct subpopulations of neurons in rat dorsal root ganglia (DRG). In the adult rat, 46% of the DRG neurons were small and peripherin-positive (NF-L-negative), and 48 zyxwvutsrq % were large and NF-L-positive (peripherin- negative). About 6% were both peripherin- and NF- L-positive. All of the DRG neurons reacted with an- tibodies to NF-M and zyxwvut nonphosphorylation-dependent or phosphorylation-independent antibodies to NF-H. The neuropeptides were predominantly found in the peripherin-positive small cell population. Eighty- seven percent of the peripherin-positive small cell population contained substance P immunoreactivity , while 43% of this cell population contained CGRP. In contrast, only 18-24% of the NF-L-positive large-cell population contained neuropeptides, and these were primarily in a smaller sized subpopulation. Similar patterns of antigen representation were observed in neonatal (PN2) DRG cell populations. Tissue cultures of sensory ganglion cells from PN2 DRG, in serum- free medium, stably maintained exclusively periph- erin-positive neurons, with about zyxwvu 5% of these con- taining coexistent NF-L immunoreactivity. Very high levels of neuropeptide gene expression were exhibited by these postnatal neurons in culture. Key words: dorsal root ganglion, neurofilaments, cytoskeletal proteins, neuropeptides, calcitonin gene- related peptide, substance P INTRODUCTION Dorsal root ganglia (DRG) in the adult rat contain a heterogeneous population of sensory neurons that vary widely in their physiological and cytological properties. Classifications of DRG neurons based on cytological and ultrastructural features usually distinguish two types: large “light” neurons with myelinated axons or small “dark” neurons with unmyelinated axons (Lieberman, 1976; Duce and Keen, 1977; Lawson et al.. 1974; Ram- bourg et al., 1983; Sommer et al., 1985). Alternative classification schemes have attempted to correlate carbo- hydrate surface antigens, specific neuropeptide content, and cell size with relatively little success (reviewed in Jessell and Dodd, 1986). The first attempt to classify DRG neuron subpopulations based on their specific cy- toskeletal protein phenotype made use of a monoclonal antibody, RT97, that cross-reacted with the middle and high molecular weight neurofilament subunits in rats (NF-M and NF-H, respectively). Initial experiments with RT97 indicated that this antibody selectively labeled the large light neuronal population (Anderton et al., 1982; Lawson et al., 1984). However, subsequent studies us- ing this antibody showed that not all large light cells, even when they could be stained by NF-L-specific anti- bodies, could be stained by RT97 (Price, 1985), and the size distribution of RT97-positive cells extensively over- laps the distribution of the small dark cell population (Lawson, 1974; Price, 1985), thereby limiting the value of this marker. The discovery of the 57-kD type I11 intermediate filament protein peripherin provided a new potential marker to be used in the classification of DRG neurons (Portier et al., 1984a,b; Parysek and Goldman, 1987, 1988; Parysek et al., 1988; Leonard et al., 1988; Aletta et al., 1988; for review see Greene, 1989). Antibodies against peripherin have been used to show selective staining of small cells in the rat DRG as well as selective staining in various regions of the central nervous system (Portier et al., 1984a,b; Parysek and Goldman, 1988; Received April 26, 1991; revised May 13, 1991; accepted May 17, 1991. Address reprint requests to H. Gainer, Lab of Neurochemistry, Build- ing 36, Room 4D20, NINDS, NIH, Bethesda, MD 20892. Abbreviations: Dorsal root ganglion, DRG; high, middle and low molecular weight neurofilament subunits, NF-H, NF-M and NF-L, respectively; postnatal day 2, PN2; Earle’s balanced salt solution, EBSS; Hank’s balanced salt solution; HBSS; bovine serum albumin, BSA; serum free medium, SFM; phosphate buffered saline, PBS; calcitonin gene-related peptide, CGRP. Published 1991 zyxwvutsr by Wiley-Liss, Inc.