ANALYSIS OF GLUTAMATE RECEPTORS IN PRIMARY CULTURED NEURONS FROM FETAL-RAT FOREBRAIN

In order to further analyze the development of glutamatergic pathways in neuronal cells, the expression of excitatory amino acid receptors w as studied in a model of neurons in primary culture by measuring the s pecific binding of L-[H-3]glutamate under various incubation condition s in 8-day-old intact living neurons isolated from the embryonic rat f orebrain, as well as in membrane preparations from these cultures and from newborn rat forebrain. In addition, the receptor responsiveness t o glutamate was assessed by studying the uptake of tetraphenylphosphon ium (TPP+) which reflects membrane polarization. In the presence of a potent inhibitor of glutamate uptake, the radioligand bound to a total number of sites of 36.7 pmol/mg protein in intact cells incubated in a Tris buffer containing Na+, Ca2+, and Cl-, with a Kd around 2 mu M. In the absence of the above ions, [H-3]glutamate specific binding dimi nished to 14.2 pmol/mg protein with a Kd-value of 550 nM. Under both o f the above conditions, similar Kd were obtained in membranes isolated from cultures and from the newborn brain. However, Bmax-values were s ignificantly lower in culture membranes than in intact cells or newbor n membranes. Displacement studies showed that NMDA was the most potent compound to inhibit [H-3]glutamate binding in membranes obtained from cultured neurons as well as from the newborn brain, whereas quisquala te, AMPA, kainate and trans-ACPD were equally effective. According to these data and to the ionic dependence of glutamate binding, it was co ncluded that cultured neurons from the rat embryo forebrain express va rious glutamate receptor subtypes, mainly L-AP4 and NMDA receptors, wi th characteristics close to those in the newborn brain, and which disp lay functional properties since a transient cell exposure to glutamate led to a 70% inhibition of [H-3]TPP+ uptake.