Purpose To determine the utility of the crayfish walking limb opener muscle (OM) as a model for studying glutamate excitotoxicity, it is necessary to confirm NMDA receptor activity and identify agonist agents that are effective in this preparation. This experiment studies the effects of 1-aminocyclobutane-cis-1,3-dicarboxylic acid (ACDA), a potent NMDA glutamate receptor agonist, on excitatory junctional potentials (EJPs) of the crayfish walking limb OM.
Methods A first or second walking limb from a medium-sized crayfish was dissected to reveal the OM of the walking limb dactylopodite as well as the single excitatory axon that innervates it. Standard intracellular electrophysiologic recording methods were used to examine EJPs of OM fibers. The preparation was bathed in Van Harreveld's solution (VanH) for control data and washing. The resting membrane potential (RMP) was monitored. The preparation was then bathed in 100, 50, and 25 μM solutions of ACDA in VanH. The axon was stimulated at 30 Hz to evoke short-term facilitated EJPs. EJPs averaged over 8 seconds were recorded, at time immediately upon addition of ACDA and at 1 and 2 minutes postaddition. ACDA was then removed, the preparation was washed with at least three complete exchanges of the bath, and postwash data were collected.
Results The RMP in both VanH and ACDA was unchanged during the studies. One-hundred micrometer ACDA showed depression with EJPs ranging from 0 to 70% of control amplitude (CA), with partial recovery upon wash (75-94% of CA). Fifty-micrometer ACDA showed enhancement of EJPs (116-214% of CA), with partial to full recovery upon wash (70-115% of CA). Twenty-five-micrometer ACDA showed a transient enhancement of EJPs initially (110-118% of CA) followed by depression with nearly complete recovery or enhancement upon wash (86-136% of CA).
Conclusion ACDA has a concentration-dependent, biphasic effect on EJPs at the concentrations studied. Qualitatively similar results were observed with at least three different limb preparations at each concentration. ACDA enhances EJPs at relatively lower concentrations and depresses at higher concentrations. The effects confirm NMDA receptor activity at the crayfish neuromuscular junction and suggest that ACDA may be useful for future studies of NMDA glutamate receptor activity.
Acknowledgment: Arnold C., Barbara M., and Georgianna Fossa Spinal Cord Injury Research Fund and the Indiana University Brain and Spinal Cord Injury Research Program.
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