Developmental expression of spontaneous activity in the spinal cord of postnatal opossums, Monodelphis domestica: An anatomical study

Abstract

Using Sulforhodamine-101 (SR101) labeling and calcium imaging on in vitro preparations, we investigated the development of spontaneous activity in the spinal enlargements of a marsupial born more immature than eutherian mammals, the opossum Monodelphis domestica. Following the retrograde transport of Calcium Green dye from the limb nerves, we observed the occurrence of spontaneous calcium waves activating the motor columns of the cervical enlargement of opossums aged from P3 to P15 (day of birth: P0) and of the lumbar enlargement from at least P6 to P12. In other preparations, SR101 was added to the bath to identify the active cells. In P1 opossums, only a few SR101-labeled cells were observed in the cervical enlargement and none in the lumbar enlargement. At P5, their number increased cervically and they appeared in the lumbar enlargement. Motoneurons were the major cell type labeled by SR101 but dye leakage made their quantification inaccurate. SR101-labeled cells also occurred elsewhere in the ventral and dorsal grey. Their number increased until P12–14 in both enlargements and then decreased to disappear by P21, the last age examined. Thus in contrast to eutherian mammals, in which spontaneous activity is mostly prenatal, spontaneous activity occurs predominantly postnatally in opossums. It increases at the time when connections from the brain begin to impinge on spinal neurons and when the limbs, especially the hindlimbs, start moving and then decreases as the systems mature.

Introduction

Spontaneous activity, a rhythmical activity generated intrinsically by the networks through which it propagates, is a widespread phenomenon in the developing central nervous system (CNS) of vertebrates (reviews in Ben-Ari, 2001, O'Donovan, 1999). It has been particularly studied in late-maturing systems, such as the visual system of mammals where it plays a significant role in the formation and reinforcement of synapses and in the refinement of neural networks (see Katz and Shatz, 1996, Zhang and Poo, 2001), the hippocampus and the neocortex (Ben-Ari, 2001, O'Donovan, 1999).

In the mature spinal cord, the basic rhythmic activity underlying locomotion is generated by networks of interneurons defined as central pattern generators (CPGs) located within the cervical and lumbar (or lumbosacral) enlargements, where limb-innervating motoneurons are also located (reviews in Falgairolle et al., 2006, Goulding and Pfaff, 2005, Grillner, 2006, Kiehn, 2006). In rats, spontaneous activity can be recorded in vitro on ventral roots from embryonic day (E) 13.5 to E19.5 (Ren and Greer, 2003), and the spinal networks can produce an alternating rhythm similar to locomotor activity at E20.5 (Nishimaru and Kudo, 2000). The expression of spontaneous activity thus precedes and overlaps the maturation of motor networks, as also documented in mouse (Branchereau et al., 2000, Hanson and Landmesser, 2003, Yvert et al., 2004) and chick (O'Donovan et al., 1998) embryos. Pharmacological perturbations of spontaneous activity result in disturbance of motoneuron pathfinding (Hanson and Landmesser, 2004, Hanson and Landmesser, 2006) and synaptic drive (Gonzalez-Islas and Wenner, 2006), indicating that it plays a role in the establishment of motor circuitry. Spontaneous activity has been reported to disappear before birth in in vitro preparations of mice and rats (Ren and Greer, 2003, Yvert et al., 2004). However, some studies report the persistence of spontaneous activity in in vitro preparations of postnatal rats up to about one week of age (Fellippa-Marques et al., 2000, Pflieger et al., 2002).

Newborn rodents are able to locomote with the four limbs, at least when postural constraints are removed (swimming, “air walking”) or when they are strongly stimulated (Fady et al., 1998). In contrast, marsupials such as opossums Monodelphis domestica are born much more immature and their motor development occurs largely after birth (Pflieger et al., 1996). The main behavioral activity of a newborn opossum consists of automatic movements of the forelimbs which enable it to move towards a mother's nipple, where it attaches for about three weeks. The hindlimbs are immobile until the second week. In view of the immaturity of locomotor behaviors of neonatal opossums and the likewise immaturity of their spinal cords (Cabana, 2000), could spontaneous activity be present in the spinal cord of this species postnatally and affect maturation of the motor networks? To answer this question, we have developed in vitro preparations of postnatal opossum CNS in which the expression of spinal network activity could be recorded and modified experimentally. Nicholls et al., 1990, Stewart et al., 1991, Zou, 1994 had already demonstrated the viability of similar in vitro preparations.

In the present study, we have used calcium imaging and bath application of Sulforhodamine-101 (SR101), a neuroanatomical marker of activity, in in vitro preparations. Calcium imaging has been used to study physiological activity in neural networks (for example, Wenner and O'Donovan, 2001, Bonnot et al., 2005). SR101 is captured by active synaptic sites and retrogradely transported to cell bodies. It has been employed as a label of active neurons after induced locomotor rhythm in in vitro spinal cord preparations of neonatal rats (Kjaerulff et al., 1994, Cina and Hochman, 2000). The intensity of SR101 labeling has been shown to be proportional to the intensity of activity (Teng et al., 1999, Miller et al., 2001). Part of this work was presented in abstract form (Lavallée and Pflieger, 2007).