Primary Sensory Afferent Innervation of the Developing Superficial Dorsal Horn in the South American Opossum Monodelphis domestica PETER D. KITCHENER, 1 * ELSPETH J. HUTTON, 2 AND GRAHAM W. KNOTT 3 1 Department of Anatomy and Cell Biology, the University of Melbourne, Parkville, Victoria 3010, Australia 2 Department of Anatomy and Physiology, University of Tasmania, Hobart, Tasmania 7001, Australia 3 Institut de Biologie Cellulaire et de Morphologie, Universite ´ de Lausanne, Lausanne 1005, Switzerland ABSTRACT The development of the primary sensory innervation of the superficial dorsal horn (SDH) was studied in postnatal opossums Monodelphis domestica by using DiI labelling of primary afferents and with GSA-IB 4 lectin binding and calcitonin gene-related peptide (CGRP) immuno- reactivity to label primary afferent subpopulations. We also compared the timing of SDH inner- vation in the cervical and lumbar regions of the spinal cord. The first primary afferent projections to SDH emerge from the most lateral part of the dorsal root entry zone at postnatal day 5 and project around the lateral edge of the SDH toward lamina V. Innervation of the SDH occurs slowly over the second and third postnatal weeks, with the most dorsal aspect becoming popu- lated by mediolaterally oriented varicose fibers before the rest of the dorsoventral thickness of the SDH becomes innervated by fine branching varicose fibers. Labelling with GSA-IB 4 lectin also labelled fibers at the lateral edge of the dorsal horn and SDH at P5, indicating that the GSA-IB 4 is expressed on SDH/lamina V primary afferents at the time when they are making their projections into the spinal cord. In contrast, CGRP-immunoreactive afferents were not evident until postnatal day 7, when a few short projections into the lateral dorsal horn were observed. These afferents then followed a pattern similar to the development of GSA-IB 4 projects but with a latency of several days. The adult pattern of labelling by GSA-IB 4 is achieved by about postnatal day 20, whereas the adult pattern of CGRP labelling was not seen until postnatal day 30. Electron microscopy revealed a few immature synapses in the region of the developing SDH at postnatal day 10, and processes considered to be precursors of glomerular synapses (and thus of primary afferent origin) were first seen at postnatal day 16 and adopted their definitive appearance between postnatal days 28 and 55. Although structural and functional development of forelimbs of neonatal Monodelphis is more advanced than the hindlimbs, we found little evidence of a significant delay in the invasion of the spinal cord by primary afferents in cervical and lumbar regions. These observations, together with the broadly similar maturational appear- ance of histological sections of rostral and caudal spinal cord, suggest that, unlike the limbs they innervate, the spinal regions do not exhibit a large rostrocaudal gradient in their maturation. J. Comp. Neurol. 495:37–52, 2006. © 2006 Wiley-Liss, Inc. Indexing terms: development; sensory afferent; synapse; marsupial; carbocyanine dye; synaptogenesis; spinal cord Grant sponsor: Australian Research Council. *Correspondence to: Peter D. Kitchener, Department of Anatomy and Cell Biology, the University of Melbourne, Parkville, Victoria 3010, Aus- tralia. E-mail: pkitc@unimelb.edu.au Received 27 January 2004; Revised 17 June 2005; Accepted 14 Septem- ber 2005 DOI 10.1002/cne.20864 Published online in Wiley InterScience (www.interscience.wiley.com). THE JOURNAL OF COMPARATIVE NEUROLOGY 495:37–52 (2006) © 2006 WILEY-LISS, INC.