Postnatal Changes in the Rexed Lamination and Markers of Nociceptive Afferents in the Superficial Dorsal Horn of the Rat LOUIS-ETIENNE LORENZO, 1 MICHELE RAMIEN, 1 MANON ST. LOUIS, 1 YVES DE KONINCK, 1–3 AND ALFREDO RIBEIRO-DA-SILVA 1,2,4 * 1 Department of Pharmacology and Therapeutics, McGill University, Montre ´al, Que ´bec, Canada H3G 1Y6 2 Alan Edwards Centre for Research on Pain, McGill University, Montre ´al, Que ´bec, Canada H3G 1Y6 3 Unite ´ de neurobiologie cellulaire, Centre Recherche Universite ´ Laval Robert-Giffard, Que ´bec, Que ´bec, Canada G1J 2G3 4 Department of Anatomy and Cell Biology, McGill University, Montre ´al, Que ´bec, Canada H3G 1Y6 ABSTRACT In this study, we investigated postnatal changes in Rexed’s laminae and distribution of nocicep- tive afferents in the dorsal horn of the rat lumbar spinal cord at postnatal days 0, 5, 10, 15, 20, and 60. Transverse sections of the L4 –L5 segments were processed for triple labeling with isolectin B4 (IB4)-binding as a marker of nonpeptidergic C-fibers, calcitonin gene-related peptide (CGRP) immunoreactivity to label peptidergic nociceptive afferents, and a fluorescent Nissl stain to visualize cells and lamination at different stages of postnatal development. The Nissl staining revealed that the thickness of lamina I (LI) and outer lamina II remained mostly unchanged from birth until adulthood. CGRP afferents terminated mostly in LI and the outer two-thirds of lamina II, whereas the termination area of fibers binding IB4 was centered on the middle one-third of lamina II at all ages studied. In absolute values, the overall width of the bands of intense CGRP and IB4 labeling increased with age but decreased as a percentage of the overall thickness of the dorsal horn with maturation. The overlap of CGRP termination area with that of IB4 afferents increased with age. The consequences of these findings are twofold. First, the size of the different laminae does not grow evenly across the dorsal horn. Second, CGRP and IB4 labeling cannot be considered per se to be reliable markers of lamination during development. These findings have implications for comparing data obtained in immature and mature tissues with respect to localization of structures in the dorsal horn. J. Comp. Neurol. 508:592– 604, 2008. © 2008 Wiley-Liss, Inc. Indexing terms: development; spinal cord; nonpeptidergic fibers; peptidergic fibers; IB4; CGRP; Nissl; NGF; BDNF; TrkA; Ret; confocal microscopy; image analysis Even though numerous anatomical and physiological studies performed on the dorsal horn of the spinal cord use its lamination to identify the population of cells studied, there is no consensus on which markers are the most reliable, some markers being preferred for physiological studies and others for anatomical investigations. In par- ticular, data are lacking on the cytoarchitectural changes that occur during postnatal development, how homoge- neous they are across the dorsal horn, and the accuracy with which the different markers used reflects the lami- nation throughout this period. For example, electrophysiological studies in live slices from the rat spinal cord are often carried out on immature tissue, obtained from animals in the perinatal or early postnatal periods (Takahashi et al., 1992; Araki and de Groat, 1997; Ahmadi et al., 2003; Baccei and Fitzgerald, 2004, 2005; Keller et al., 2004; Gonzalez-Forero et al., 2005). It has been assumed that the data so obtained will Grant sponsor: Canadian Institutes of Health Research (CIHR); Grant number: MOP-79411 (to A.R.S.); Grant number: MOP-12942 (to Y.DeK.); Grant sponsor: CIHR Strategic Training Program “Pain from Molecules to Community” (to L.E.L.); Grant sponsor: Fonds de la recherche en sante ´ du Que ´bec (to Y.DeK.). *Correspondence to: Dr. Alfredo Ribeiro-da-Silva, Department of Phar- macology and Therapeutics, McGill University, 3655 Promenade Sir- William-Osler, Montre ´al, Que ´bec, Canada H3G 1Y6. E-mail: alfredo.ribeirodasilva@mcgill.ca Received 24 August 2007; Revised 30 November 2007; Accepted 29 January 2008 DOI 10.1002/cne.21691 Published online March 26, 2008 in Wiley InterScience (www.interscience. wiley.com). THE JOURNAL OF COMPARATIVE NEUROLOGY 508:592– 604 (2008) © 2008 WILEY-LISS, INC.