A quantitative description of excitatory amino acid neurotransmitter responses on cultured embryonic Xenopus spinal neurons.
We have performed a quantitative analysis of excitatory amino acid neurotransmitter receptors on cultured embryonic Xenopus spinal neurons using the whole-cell patch-clamp technique. Neuroblasts and underlying mesodermal cells isolated from spinal regions of neural plate-stage embryos were placed into dissociated cell culture, and responses were studied soon after the appearance of neurites on embryonic neurons. Glutamate (Glu) receptors were separated into two general classes based on responses to the characteristic agonists quisqualate (Quis), kainate (Ka) and N-methyl-D-aspartate (NMDA); these were NMDA receptors (those activated by NMDA) and non-NMDA receptors (those activated by Ka and Quis). Half-maximal responses to Glu and other agonists on NMDA and non-NMDA receptors were determined from Hill analysis of dose response relations. The order of sensitivities observed was: GluNMDA (ED50 = 5.1 microM) greater than Glunon-NMDA (ED50 = 28 microM), and for Glu receptor agonists, Quis (ED50 = 1.5 microM) greater than NMDA (ED50 = 41 microM) greater than Ka (ED50 = 58 microM). The order of response amplitudes recorded at concentrations near the appropriate ED50s was GluNMDA greater than Glunon-NMDA, and Ka greater than NMDA greater than Quis. A 10-fold decrease in external [Na+] shifted the reversal potentials for Glunon-NMDA, Ka, and Quis to more negative voltages. Increasing external [Ca2+] shifted the reversal potential for NMDA responses to more positive potentials, an observation consistent with Ca2+ permeation of the embryonic NMDA-activated channel. NMDA-evoked currents could not be recorded in nominally glycine (Gly)-free media. Addition of Gly to external solutions potentiated NMDA responses (ED50 = 644 nM). NMDA responses were blocked by DL-2-amino-5-phosphonovaleric acid (APV; ED50 = 1.9 microM) and by Mg2+ at negative potentials. In their sensitivities to agonists and antagonists, and ionic dependences, amino acid neurotransmitter responses on embryonic Xenopus neurons closely resembled those previously observed for mature Xenopus and mammalian central neurons. The GluNMDA receptors present on these immature neurons were sufficiently sensitive to be activated by endogenous concentrations of extracellular Glu, suggesting a possible role for receptor activation in modulating early neural development.
Sands
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Department of Physiology and Biophysics, California College of Medicine, University of California, Irvine 92717.
Barish
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D000818:Animals; D001224:Aspartic Acid / Q000494:pharmacology; D004625:Embryo, Nonmammalian; D007608:Kainic Acid / Q000494:pharmacology*; D016202:N-Methylaspartate; D010069:Oxadiazoles / Q000494:pharmacology*; D016318:Quisqualic Acid; D017470:Receptors, Glutamate; D017981:Receptors, Neurotransmitter / Q000378:metabolism*; D014982:Xenopus laevis / Q000378:metabolism* / Q000502:physiology
D010069:Oxadiazoles; D017470:Receptors, Glutamate; D017981:Receptors, Neurotransmitter; D001224:Aspartic Acid; D016202:N-Methylaspartate; D016318:Quisqualic Acid; D007608:Kainic Acid