Methodological optimization of applying neuroactive agents for the study of locomotor-like activity in the mudpuppies (Necturus maculatus)

Abstract

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.

Figure 1. Optimal superfusion flow rate and comparisons of delivery modes

A: Frequency (●) and duration (○) of walking-like activity were dependent on the superfusion flow rate of D-glutamate (0.5 mM) (mean ± SEM, n=5). The optimal range of superfusion rate was between 10 and 20 ml/min. In this range, peak frequency (0.8 Hz) was achieved and the du-ration of the activity upon termination of the superfusion was longest (90–100 seconds). B: Effects of mode of delivery on the walking-like activity (mean ± SEM, n=12). Superfusion of D-glutamate (▼) induced stable walking-like activity, the frequency of which was dose-dependent in the range of 0.3–0.9 mM. Beyond 0.9 mM, superfusion of glutamate caused tonic activity of flexor and extensor muscles. Superfusion of NMDA (●) induced walking-like activity in a small effective range of concentrations (40–80 µM), with the highest frequency of 0.6 Hz at a concentration of 60µM. Beyond 80 µM, superfusion of NMDA induced tonic activities. Bolus application of NMDA (○) was effective in a much wider range of concentrations (50–300 µM). The peak frequency was higher than that induced by superfusion.

Figure 2. Mode of application of AP-5 affects the efficacy of its inhibitory effects

A. Compared to bolus application (●), continuous superfusion (○) of AP-5 was more effective in inhibiting the ongoing walking-like activity induced glutamate (mean ± SEM, n=7). B: Continuous superfusion of AP-5 (○) was similarly more effective in inhibiting the initiation of walking-like activity by glutamate, compared to bolus application (●) (mean ± SEM, n=7).

Figure 3. Inhibitory effects of strychnine on walking-like activity

Bolus application of strychnine changed the rhythmic, alternating the pattern of walking-like activity (A) to a slow and synchronized pattern (B). In contrast, superfusion of strychnine blocked completely the walking-like activity induced by NMDA (C) or glutamate (D).

Figure 4. Effects of DMSO on locomotor-like activity

A: The mean frequency of the walking-like activity was decreased dose-dependently with superfusion of DMSO (mean ± SEM, n=8). The activity induced by glutamate (○) was more sensitive to the inhibitory effects of DMSO than that induced by NMDA (●). In contrast, the amplitudes of the dorsal root reflexes were not significantly affected by DMSO (top curve, mean ± SEM, n=8). B: The mean amplitude of the walking-like active was more profoundly depressed in a dose-dependent manner. Again, the activity induced by glutamate was more sensitive to the inhibition compared to that induced by NMDA (mean ± SEM, n=8). C: Examples of dose-dependent inhibition of the walking-like activity by DMSO. Note that depression of the amplitude was more profound than the inhibition of the frequency, consistent with the pooled data shown in panels A and B.