Reflexes, fictive respiration and cell division in the brain and spinal cord of the newborn opossum, Monodelphis domestica, isolated and maintained in vitro

Abstract

1. The entire central nervous system (CNS) was isolated from 1- to 4-day-old newborn South American opossums (Monodelphis domestica). At this stage the CNS has only an embryonic forebrain (two-layered) and no cerebellum and corresponds to a 14-day rat embryo. Its eyes, ears and hind-limbs are only at an early stage of formation. The isolated CNS preparations continue to develop and to produce electrical signals for up to 4 days in oxygenated Krebs' fluid at 23 degrees C. 2. The longitudinal axis of the CNS showed markedly different stages of development. More neuroblast cells were present in the proliferative zone in lumbosacral than in cervical or thoracic regions of the cord. 3. The progeny of dividing cells were labelled in isolated preparations by applying bromodeoxyuridine (BrdU) to the bathing solution for 2 h. Stained precursor cells were observed in CNS that had been left in Krebs' fluid for 4 days before applying BrdU and also in CNS that had been exposed to BrdU shortly after dissection and then left for 4 days. 4. Compound action potentials were evoked from the isolated CNS by stimulation with extracellular electrodes. Compound action potentials increased in amplitude with stronger stimulation and showed discrete peaks of conduction velocity. All electrical activity was eliminated reversibly by 0.1 mumol l-1 tetrodotoxin applied to the bathing solution. Block and recovery occurred with a half-time of approximately 5 min. High concentrations of magnesium (20 mmol l-1) reversibly blocked slower components of the volley. 5. Reflexes in cervical and thoracic segments of the spinal cord continued to function in isolated preparations. Stimulation of a dorsal root evoked bursts of impulses in the appropriate ventral root. Spontaneous and evoked activity in ventral roots was eliminated reversibly by 20 mmol l-1 magnesium. 6. In thoracic segments, spontaneous rhythmical bursts of action potentials were recorded. Burst activity was correlated with respiratory movements of the ribs in semi-intact preparations in which a few ribs and muscles were left attached to the isolated CNS. 7. At raised temperatures of 28 degrees C compared to 23 degrees C both spontaneous and evoked electrical activity were reversibly reduced. 8. Together these results show that the isolated CNS of the newborn opossum survives well in culture. The preparation offers advantages for pharmacological and physiological studies of spinal reflexes, for analysis of the mechanisms underlying rhythmical respiratory activity and for following the time course of CNS development in vitro.