Purpose The crayfish (Procambarus clarkii and simulans) neuromuscular junction (NMJ) is an established model of synaptic transmission. Glutamate is its excitatory neurotransmitter. The purpose of this experiment is to confirm the validity of the crayfish NMJ as a viable model for follow-on experiments addressing the prevention or reduction of glutamate excitotoxicity secondary injury following spinal cord trauma. Specifically, the effects of DL-2-amino-4-phosphonobutyric acid (AP4) on the excitatory junctional potentials (EJP) of NMJs of the dactylopodite opener muscle of the first and second walking limbs of crayfish will be studied. Efficacy of known NMDA agonist/antagonist AP4 will verify the existence of ionotropic NMDA glutamate receptors in the crayfish NMJ and characterize the effects of the agonists at an invertebrate NMJ.
Methods The first or second walking limb was removed. The meropodite was dissected to expose the single excitatory axon to the opener muscle of the dactylopodite. The propodite was dissected to expose the opener muscle. The preparation was bathed continually in Van Harrevald's solution (Van H), pH 7.2 ± 0.1. For each preparation, control EJPs were obtained. AP4 was added and EJPs were recorded immediately and at 1 and 2 minutes post addition. AP4 was then washed out with Van H, using three complete exchanges of the bath. EJPs were recorded following three, six, and nine washouts. Serial dilutions were performed for concentrations of 1 mM to 3.75 μM AP4.
Results Based on the serial dilutions, AP4 acts as an antagonist at a concentration of 1,000 μM and an agonist at 3.75 μM. Results at intermediate values were equivocal. Qualitatively similar results were obtained in at least three additional preparations at each concentration.
Discussion The crayfish NMJ contains glutamate receptors that respond to NMDA agonist/antagonist AP4 in a concentration-dependent, biphasic manner. This study provides additional evidence of the presence of NMDA glutamate receptors at the crayfish NMJ. Further studies to construct a complete concentration-response curve are warranted.
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.
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