Purpose The D3 stereoisomer of the malonic acid (carboxy) derivative of buckminsterfullerene (D3) has been shown to inhibit excitotoxic death of cultured cortical neurons of mice exposed to NMDA and AMPA. The effects of D3 have never been tested in this model. The purpose of this research was to determine the effects of D3 on excitatory junctional potentials (EJPs) of neuromuscular junctions (NMJs) of the dactylopodite opener muscle of the first and second walking limbs of Procambarus clarkii and simulans.
Methods The limbs were dissected to expose the opener muscle and its excitatory axon in the meropodite. Van Harreveld's solution (Van H, pH 7.3 ± 0.1) was used to bathe the preparation during dissection and initial elicitation of EJPs (pretreatment control) and following washout (post-treatment control) of the VanH/D3 test solution. Standard intracellular electrophysiological recording techniques were used to measure and record EJPs of the opener muscle. A 30 Hz stimulus was applied to the excitatory axon for 10 seconds prior to each data collection. VanH/D3 was applied directly over the opener muscle using a 30-gauge needle, using separate preparations for each concentration. D3 was then washed from the preparation using 3 complete bath changes.
Result Following application of 100 μM D3, EJP amplitude was completely suppressed from control at the control stimulation. EJPs returned to 43% of control value post wash. 25 μM solution displayed complete suppression of EJP from control. EJP returned to 55% of control value post wash. The 12.5 μM solution caused immediate, 12.5% potentiation of EJPs compared to control, followed by complete suppression at 1-minute post application. EJPs returned to control levels post wash.
Conclusions D3 appears to suppress EJPs at all concentrations tested. This suppression seems to be partially reversible at the two lower concentrations. Furthermore, at the lowest concentration, there may be an initial increase in EJP amplitude. Because of results seen in the mouse model, EJP suppression was expected as AMPA and NMDA receptors are both present in the crayfish NMJ. EJP potentiation was not expected and cannot be explained at this time.
Supported by the Arnold C., Barbara M. and Georgianna Fossa Spinal Cord Injury Research Fund and the Indiana University School of Medicine Brain and Spinal Cord Injury Research Program. D3 was provided by Dr. L. Dugan, UCSD.
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