Purpose Crayfish (Procambarus clarkii) neuromuscular junctions (NMJs) use the neurotransmitter glutamate to elicit excitatory junction potentials (EJPs) using NMDA and non-NMDA receptors. The D3 stereoisomer of the malonic acid (carboxy) derivative of buckminsterfullerene (D3) has been shown to have a reversible inhibitory effect on NMDA receptors at very low concentrations. The purpose of this experiment is to determine the lowest concentration of D3 that is effective for suppressing crayfish NMJ EJPs.
Materials/Methods The crayfish's first walking limb is removed and dissected at the meropodite to expose the excitatory nerve and at the propodite to expose the opener muscle that controls the crayfish's dactylopodite. The limb is then submerged in Van Harreveld's (Van H, pH 7.3 ± 0.1) solution and a nerve stimulator electrode is applied to the opener muscle's excitatory nerve. Standard intracellular electrophysiological techniques were used to measure EJPs. To evoke short-term EJP facilitation, a 30 Hz stimulus was applied for 10 seconds prior to each data collection. 5, 7.5, 10, 12.5 μM D3 was then added directly to the exposed opener muscle. EJPs were again recorded and compared to control. The experiment is concluded by changing bath with Van H 3 times to washout the D3, allowing a post-control EJP measurement.
Results Following application of 12.5 μM D3, EJPs were suppressed to 23% of control level with 76% EJP recovery after wash-out. At 10 μM, EJPs were suppressed to 79% of control level with full EJP recovery after washout. At 7.5 μM, EJPs were suppressed to 82% of control level with full EJP recovery after washout. At 5.0 μM, no EJP suppression was noted.
Discussion 7.5 μM appears to be the lower limit of D3's effectiveness in EJP suppression at the concentrations studied. The effects of D3 on crayfish EJPs appear to be fully reversible at 7.5 and 10 μM. There appeared to be a large drop in suppressive ability of D3 between 12.5 μM and 10 μM. No evidence of EJP enhancement was found at any of the concentrations tested. Further study of D3's effects on EJPs between the concentrations of 5 and 10 μM is warranted to determine the absolute lower limit of D3's effectiveness.
Supported by the Arnold C., Barbara M. and Georgianna Fossa Spinal Cord Injury Research Fund and the IU School of Medicine Brain and Spinal Cord Injury Research Program. D3 was provided by Dr. L. Dugan, UCSD.
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