Tissue PO2 and the effects of hypoxia on the generation of locomotor-like activity in the in vitro spinal cord of the neonatal mouse

The neonatal mouse en bloc spinal cord-brainstem preparation used in combination with advances in mouse genomics provides a novel strategy for studying the spinal control of locomotion. How well the mouse en bloc preparation is oxygenated however, is unknown. This is an important consideration given that (a) other superfused mammalian en bloc preparations have anoxic cores and (b) hypoxia can have profound effects on neuronal activity. Here we measure the level of tissue oxygenation in the mouse preparation and determine how neuronal activity within the spinal cord is influenced by poor superfusion and/or low oxygen. To measure tissue oxygenation, oxygen depth profiles were obtained (P0-1 and P2-3; Swiss Webster mice). At P0-1, spinal cords were oxygenated throughout under resting conditions. When fictive locomotor activity was evoked (5-HT 10 μM, dopamine 50 μM, NMA 5 μM), there was a substantial reduction in tissue PO2 starting within 5 min of drug application. Following washout, the PO2slowly returned to control levels over a period of 30 min. The experiments described above were repeated using P2-3 preparations. In this older age group, the spinal cord preparations had a hypoxiclanoxic core that was exacerbated during metabolically demanding tasks such as drug-evoked rhythmic activity. To examine how an anoxic core affects neuronal activity within the spinal cord we either altered the flow-rate or manipulated superfusate PO2. When the flow-rate was reduced a transient disruption in the rhythmicity of drug-induced locomotion occurred during the first 15 min (P0-1 preparations). However, the motor output adapted and stabilized. During prolonged superfusion with hypoxic artificial cerebrospinal fluid on the other hand, both the motor bursts in spinal nerves and the activity of most neurons near the center of the tissue were abolished. Overall, this study suggests that while oxygenation of P0-P1 preparations is adequate for studies of locomotor function, oxygenation of older preparations is more problematic. Our data also show that neonatal spinal neurons require oxygen to maintain activity; and the spinal locomotor rhythm generator continues to function providing the peripheral tissue of the cord is oxygenated. Together, these results are consistent with the results of a previous study which suggest that the locomotor pattern generator is located close to the surface of the spinal cord.