Contribution of Ca2+-permeable AMPA/KA receptors to glutamate-induced Ca2+ rise in embryonic lumbar motoneurons in situ

Intracellular Ca2+ ([Ca2+](i)) was fluorometrically measured with fura-2 in lumbar motoneurons of acutely isolated spinal cord slices from embryonic rats. In ester-loaded cells, bath-applied glutamate (3 mu M to 1 mM) evoked a [Ca2+](i) increase by up to 250 nM that was abolished by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) plus 2-amino-5-phosphonovalerate (APV). CNQX or APV alone reduced the response by 82 and 25%, respectively. The glutamatergic agonists kainate (KA), quisqualate (QUI), and S-alpha-amino-3-hydroxy-5-methyl-4-isoxalone (S-AMPA) evoked a similar [Ca2+](i) transient as glutamate. N-methyl-D-aspartate (NMDA) was only effective to increase [Ca2+](i) in Mg2+-free saline, whereas [1S,3R]-1-aminocyclopentane-1,3-dicarboxylic acid([1S,3R]ACPD) had no effect. The glutamate-induced [Ca2+](i) rise was suppressed in Ca2+-free superfusate. Depletion of Ca2+ stores with cyclopiazonic acid (CPA) did not affect the response. Thirty-six percent of the [Ca2+](i) increase in response to membrane depolarization induced by a 50 mM K+ solution persisted on combined application of the voltage-gated Ca2+ channel blockers nifedipine, omega-conotoxin-GVIA and omega-agatoxin-IVA. In fura-2 dialyzed motoneurons, the glutamate-induced [Ca2+](i) increase was attenuated by similar to 70% after changing from current to voltage clamp. Forty percent of the remaining [Ca2+], transient and 20% of the concomitant inward current of 0.3 nA were blocked by Joro spider toxin-3 (JSTX). The results show that voltage-gated Ca2+ channels, including a major portion of R-type channels, constitute the predominant component of glutamate-induced [Ca2+](i) rises. NMDA and Ca2+-permeable KA/AMPA receptors contribute about equally to the remaining component of the Ca2+ rise. The results substantiate previous assumptions that Ca2+ influx through JSTX-sensitive KA/AMPA receptors is involved in (trophic) signaling in developing motoneurons.