L-Glutamic, L-aspartic acids and a number of their structural analogs, including quisqualic, kainic, ibotenic, quinolinic, and N-methyl-D- aspartic (NMDA) acids, increase inositol phospholipid hydrolysis when added to primary cultures of cerebellar granule cells, as is reflected by an enhanced formation of 3H-inositolmonophosphate (3H-IP1) in the presence of Li+. L-Glutamic acid also enhances the formation of the initial products of inositol phospholipid hydrolysis, 3H-inositol di- (3H-IP2) and triphosphate (3H-IP3). In the absence of extracellular Ca2+, L-glutamic acid fails to enhance 3H-IP1 formation, but still increases 3H-IP2 and 3H-IP3 formation. The stimulation of 3H-IP1 formation elicited by L-glutamic acid is reduced by DL-2-amino-5- phosphonovaleric acid (APV) and gamma-glutamylglycine and, to a lesser extent, by 2,3-cis-piperidindicarboxylic acid (PDA). The stimulation of 3H-IP1 formation by kainic acid is antagonized by PDA and gamma- glutamylglycine, but it is almost unaffected by APV. The increase in 3H- IP1 formation elicited by quisqualic acid is not reduced by any of the dicarboxylic amino acid receptor antagonists that we have tested. We conclude that different subtypes of excitatory amino acid recognition sites are associated with inositol phospholipid metabolism in primary cultures of cerebellar granule cells.

The role of a nuclear factor κB (NF‐κB) in NMDA receptor‐mediated neuroprotection is not known. A candidate sequence from the 5′ flanking region of exon 3 of the rat brain‐derived neurotrophic factor (BDNF) gene was used to show that exposure of rat cerebellar granule cells to 100 μm NMDA activated a specific DNA binding activity that was blocked by the NMDA receptor antagonist MK‐801. Anti‐p65 antibody or anti‐p50 antibody ‘supershifted’ the DNA binding activity, suggesting that the DNA–protein complex was composed of p65 and p50 subunits. NMDA receptor‐mediated neuroprotection was blocked when cerebellar neurons were transfected with a double‐stranded oligonucleotide containing the BDNF gene NF‐κB sequence. Furthermore, nuclear extracts prepared from neurons treated with NMDA and the double‐stranded NF‐κB oligonucleotide showed reduced DNA binding activity to the target sequence, supporting the idea that NF‐κB may be involved in the transcriptional activation of the BDNF gene. To address this issue, we quantified the level of exon 3‐specific BDNF mRNA. Relative to GAPDH mRNA levels and compared with untreated neurons, NMDA increased exon 3‐specific BDNF mRNA twofold. In contrast, pretreatment of neurons with the NF‐κB target DNA abolished the increase in BDNF mRNA following addition of NMDA. We also determined that BDNF itself induced an NF‐κB DNA binding activity. Taken together, these data support a mechanism where NF‐κB plays a critical role in NMDA‐mediated neuroprotection.

Primary cultures of cerebellar granule cells have been used in pharmacologically and functionally characterizing excitatory amino acid recognition sites coupled with guanylate cyclase. When granule cells were incubated in physiological culture conditions (Locke's solution, pH 7.4), only kainate and, to a lesser extent, L-glutamate increased cyclic GMP (cGMP) levels. Under these conditions, L-aspartate, N-methyl- D-aspartate (NMDA), and quisqualate were inactive. When granule cells were incubated in the absence of extracellular Mg2+ or in the presence of the depolarizing agent veratrine, L-glutamate, L-aspartate, and NMDA became as effective as kainate in enhancing cGMP formation. The action of kainate was preferentially antagonized by 2,3-cis- piperidindicarboxylate, whereas the action of L-glutamate was preferentially antagonized by (+/-)2-amino-5-phosphonovalerate. These data suggest that 2 different excitatory amino acid recognition sites (activated by kainate or by L-glutamate, L-aspartate, and NMDA, respectively) are coupled with guanylate cyclase in primary cultures of cerebellar granule cells: While the coupling of the recognition site for kainate with guanylate cyclase operates under resting conditions and in the presence of Mg2+, the coupling of the recognition site for L- glutamate, L-aspartate, and NMDA with guanylate cyclase requires depolarizing conditions or the absence of extracellular Mg2+.