Methodological optimization of applying neuroactive agents for the study of locomotor-like activity in the mudpuppies...

We compared the effects of mode of delivery of neuroactive agents and the effects of dimethyl sulfoxide (DMSO), a vehicle for dissolving neuroactive agents, on locomotor-like activity in vitro. By superfusion, d-glutamate (0.3-0.9 mM) produced robust walking-like activity at superfusion rates 10-25 ml/min. In contrast, bolus application of the same or higher doses of glutamate (0.1-1.5 mM) failed to induce any rhythmic activity. Superfusion with AP-5, a NMDA receptor antagonist, produced dose-dependent inhibition of the ongoing walking-like activity induced by D-glutamate and completely blocked the activity at 20 microM. In contrast, bolus application of AP-5 did not block the walking-like activity at concentrations up to 120 microM. Similarly, superfusion of AP-5 inhibited the initiation of walking-like activity and completely blocked the initiation at 20 microM, while bolus application of AP-5 failed to do so at concentrations up to 120 microM. Superfusion of strychnine, a glycine receptor antagonist, blocked the walking-like activity at concentrations of 3-5 microM, while its bolus application altered NMDA-induced, but not glutamate-induced, walking-like activity to a synchronized pattern. DMSO significantly affected the walking-like activity in a dose-dependent manner at concentrations ranging 1-10% (v/v). These results demonstrate that the way by which the neuroactive agents are applied is a significant factor that determines the outcome of experiments on the neural control of locomotion. Also, the dose-dependent effects of DMSO on the activity of neural networks for locomotion should be taken into account in data interpretation.