NMDA receptors, a class of glutamate-gated cation channels with high Ca2+ conductance, mediate fast transmission and plasticity of central excitatory synapses. We show here that gene-targeted mice expressing NMDA receptors without the large intracellular C-terminal domain of any one of three NR2 subunits phenotypically resemble mice made deficient in that particular subunit. Mice expressing the NR2B subunit in a C-terminally truncated form (NR2B(deltaC/deltaC) mice) die perinatally. NR2A(deltaC/deltaC) mice are viable but exhibit impaired synaptic plasticity and contextual memory. These and NR2C(deltaC/deltaC) mice display deficits in motor coordination. C-terminal truncation of NR2 subunits does not interfere with the formation of gateable receptor channels that can be synaptically activated. Thus, the phenotypes of our mutants appear to reflect defective intracellular signaling.

We have characterized the genomic response of astroglial cells to excitatory amino acids by using selective agonists and antagonists for the various receptor subtypes and by analyzing different primary response genes, such as members of the Fos (c-fos and fosB) and Jun (c-jun, junB, and junD) families, zif/268, and c-myc. A rapid and transient elevation of mRNA levels for c-fos, fosB, c-jun, junB, and zif/268 was observed after addition of glutamate to cultured astrocytes, whereas junD and c-myc expression was not affected. The level of AP-1 DNA binding activity, as measured by the electrophoretic mobility shift assay, also increased after addition of glutamate to cultured astrocytes. Glutamate-induced c-fos expression was not affected by the N-methyl-D-aspartate receptor antagonists MK-801 and D-2-amino-5-phosphonopentanoate, by the kainate/alpha-amino-3-hydroxy-5- methylisoxazole-4-propionate (AMPA) receptor antagonist 6,7-dinitroquinoxaline-2,3-dione (DNQX), or by the broad-spectrum antagonist kynurenate. Kainate and AMPA were also effective in inducing primary response gene expression, and their actions were antagonized by kynurenate and DNQX but not by MK-801. 1S,3R-1-Aminocyclopentane-1,3-dicarboxylic acid, a selective agonist for the metabotropic glutamate receptor, induced primary response gene expression, but its action was not antagonized by different glutamate antagonists, including L-2-amino-3-phosphonopropionate. In conclusion, our data suggest that cultured astrocytes express both kainate/AMPA ionotropic receptors and metabotropic receptors coupled to the rapid and coordinated activation of different classes of transcriptional factor genes.

Excitatory amino acid (EAA)-induced polyphosphoinositide (PPI) hydrolysis was studied during the development in culture of cerebellar granule cells. The developmental pattern was similar using metabotropic glutamate (Glu) receptor (mGluR)

We have used primary neuronal cultures prepared from fetal cerebral hemispheres to investigate the effects of different glutamate receptor agonists and antagonists on the expression of transcription factor encoding genes, such as c-fos, fosB, c-jun, junB, junD, c-myc, and zif/268. The addition of glutamate (100 microM) to the culture medium rapidly activated c-fos, fosB, c-jun, junB and zif/268 gene expression, reaching the maximal level at 30-60 minutes for zif/268 and at 60 minutes for the other genes. The onset of fosB mRNA accumulation was slightly delayed in comparison to the other genes. No clear induction was found for junD and c-myc. Different glutamate receptor agonists, such as NMDA, kainate, quisqualate, trans-(+/-)-1-aminocyclopentane-1,3-dicarboxylic acid (t-ACPD) and alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate (AMPA) were able to increase c-fos, c-jun, and zif-268 mRNA levels with rapid and transient kinetics similar to those observed after glutamate treatment. Similar results were obtained for junB and fosB after kainate and quisqualate stimulation. Pretreatment with MK-801, a non competitive NMDA antagonist, produced an almost complete inhibition of glutamate-driven expression of transcription factor genes, thus suggesting that NMDA receptor plays a major role in glutamate induced-gene expression. On the contrary the kainate/AMPA receptor antagonist, DNQX, did not influence glutamate induced-gene expression. Under the conditions used in the present study, NMDA was effective in inducing the simultaneous activation of several IEGs even when added to the culture medium containing millimolar concentration of magnesium. When experiments were performed in Krebs solution, NMDA was effective in stimulating zif/268 and c-fos mRNAs only in the absence of Mg2+, while glutamate activated c-fos and zif/268 both in the presence and absence of magnesium ions. As expected, NMDA effect was fully inhibited by MK-801. The level of AP-1 DNA binding activity, as measured by electrophoretic mobility shift assay, increased after addition of glutamate and NMDA to cultured neurons and such increase was antagonized by the pretreatment with MK-801.

Tear lactoferrin, mainly secreted by the lachrymal glands, exerts a protective effect on the ocular surface, and an abnormal decrease of its production may lead to an increased risk of infection and pathological alterations of ocular surface epithelia. In this study we analyzed whether corneal and conjunctival epithelia could be an additional source of tear lactoferrin, and whether conjunctival epithelial cells in culture could be a suitable model system to address regulation of lactoferrin gene expression. Real-time PCR and Western immunoblotting showed that in bovines lactoferrin is indeed produced by these epithelia, and that the human lactoferrin promoter can direct the expression of a CAT reporter gene, thus indicating that these cells are a true source of lactoferrin, and may be used in vitro to study the regulation of lactoferrin expression.