Pharmacological characterization of a GluR6 kainate receptor in cultured hippocampal neurons

Abstract

We have examined the pharmacology of kainate receptors in cultured hippocampal neurons (6–8 days in vitro (DIV)) from embryonic rats (E17). Cultured neurons were pre-treated with concanavalin A to remove kainate receptor desensitization and whole-cell voltage clamp electrophysiology employed to record inward currents in response to glutamatergic agonists and antagonists. N-methyl- d-aspartate (NMDA) and α-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA) receptor responses were blocked using MK801 (3 μM) and the 2,3-benzodiazepine, LY300168 (GYKI53655, 50 μM), respectively. Inward currents were recorded in hippocampal neurons upon application of kainate and the 2 S,4 R isomer of 4-methyl glutamic acid (SYM2081) with EC 50 values of 3.4±0.4 μM and 1.6±0.5 μM, respectively ( n=6 cells). The GluR5 selective agonists, LY339434 (100 μM) and ( RS)-2-amino-3-(3-hydroxy-5- tert-butyl-4-isoxazolyl) propionic acid (ATPA) (100 μM), did not evoke detectable inward currents in any cell responding to kainate. LY293558 and the selective GluR5 antagonist, LY382884, had weak antagonist effects on responses evoked by either kainate or (2 S,4 R)-4-methyl glutamate (IC 50 >300 μM). The quinoxalinedione, 2,3-dihyro-6-nitro-7-sulfamoyl-benzo( f)quinoxaline (NBQX), blocked both kainate and (2 S,4 R)-4-methyl glutamate-activated currents at much lower concentrations (IC 50 approximately 10 μM). These results provide pharmacological evidence that ion channels comprised of GluR6 kainate receptor subunits mediate kainate receptor responses in hippocampal neurons cultured 6–8 DIV.