Organotypic culture of embryonic rat lumbar spinal cord and dorsal root ganglia

Previous studies of the development of primary afferent innervation in the rat spinal cord have primarily focused on the time course of afferent innervation, the onset of reflex activity and the development of transmitter activity. However, the factors that contribute to the development of the dorsoventral termination pattern of morphologically and functionally distinct classes of primary afferent neurons are not known. The dorsoventral termination pattern of dorsal root ganglion (DRG) primary afferents in the adult spinal cord is well known (Brown, 1981; Willis and Coggeshall, 1991). Primary afferents have been classified according to both their fibre diameter and their physiological response properties. Briefly, small diameter myelinated and unmyelinated afferents, many of which mediate noxious mechanical, thermal and chemical cutaneous sensory information, terminate predominantly in the most superficial regions of the dorsal horn, in laminae I-II (laminae designations according to Rexed, 1952). Large diameter low threshold cutaneous afferents terminate in deeper laminae III-V and group Ia afferents that mediate muscle monosynaptic reflexes terminate deeper still in the ventral horn. In the rat spinal cord, the bundle of His, which contains central projections of DRG cells, is well developed by embryonic (E) day 14, and collaterals first enter the spinal cord grey matter on E14-15 (Windle and Baxter, 1936; Vaughn and Grieshaber, 1973; Smith, 1983; Altman and Bayer, 1984; Fitzgerald et al., 1991). By birth at around E21.5, dense innervation is present in all areas of the grey matter. On the basis of electron microscopic and electrophysiological evidence, the earliest afferents appear to make functional connections during E15-17 in intermediate regions of the spinal grey matter. These afferents are likely to be collaterals of Aβ cutaneous afferents and Group II muscle afferents that mediate polysynaptic spinal reflexes (Vaughn and Grieshaber, 1973; May and Biscoe, 1975a,b; Saito, 1979; Ziskind-Conhaim, 1990; Kudo and Yamada, 1987). Monosynaptic reflex activity does not occur until 12 days following the onset of polysynaptic activity (Saito, 1979; Kudo and Yamada, 1985, 1987; Ziskind-Conhaim, 1990), which is consistent with the fact that afferent axons do not innervate the lumbar ventral horn until about E1718 (Smith, 1983; Kudo and Yamada, 1987; Ziskind-Conhaim, 1990). Afferent innervation of superficial regions of the dorsal horn by small diameter myelinated and unmyelinated fibres occurs relatively late in embryonic development during E19-21 (Smith, 1983, Fitzgerald, 1987). Evidence from studies of axon guidance suggest that a 1377 Development 117, 1377-1384 (1993) Printed in Great Britain © The Company of Biologists Limited 1993