The mechanisms of epileptogenesis, the expression of alpha-amino-3-hydroxy-5-methyl-4-isoxazoleopropionic acid (AMPA) receptor subunits GluR1 and GluR2 and of the GluR-associated protein Narp in the hippocampus and the inferior colliculus (IC) from AGS-susceptible P77PMC rats after a single AGS and audiogenic kindling. Western blotting and immunohistochemistry showed that Narp was rapidly induced in both the hippocampus and the IC by AGS. In the hippocampus, up-regulation of Narp was concomitant with GluR1 and GluR2 under both conditions of a single AGS and AGS kindling. In the IC, however, Narp was up-regulated, GluR2 down-regulated, and GluR1 unchanged after kindling. In comparison with kindling, neither GluR1 nor GluR2 was changed, while Narp significantly increased in the IC following a single AGS. Thet down-regulation of AMPA receptor GluR2 subunit in the IC may contribute to AGS-mediated epileptogenesis, and up-regulation of Narp in the IC may be involved in audiogenic seizures.

The GluR1 cDNA clone encodes a functional excitatory amino acid receptor (Hollmann et al., Nature 342: 643-648 (1989]. the pharmacological properties of this homomeric (single subunit) receptor expressed in Xenopus oocytes and compared these properties with those of receptors encoded by rat forebrain mRNA. (RS)-alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionate, quisqualate, and glutamate were partial agonists at both GluR1 and forebrain non-N-methyl-D-aspartate (-NMDA) receptors. The potency of the agonists kainate, domoate, and glutamate was higher, and that of the antagonists 6-cyano-7-nitro-quinoxalinedione and 6,7-dichloro-3-hydroxy-2-quinoxaline carboxylic acid lower, for GluR1 receptors as compared with forebrain non-NMDA receptors. The GluR1 receptor differed strikingly from forebrain-derived non-NMDA receptors, however, in that it exhibited slow, calcium-dependent desensitization. Thus, most properties of the GluR1 receptor are similar but not identical to those of non-NMDA receptors expressed from forebrain mRNA. the ligand recognition sites on GluR1 homomeric receptors are subtly different from those of non-NMDA receptors expressed from a mixture of forebrain mRNA.

Narp is a neuronal immediate early gene that plays a role in excitatory synaptogenesis. The native Narp in brain is part of a pentraxin complex that includes NP1. These proteins are covalently linked by disulfide bonds into highly organized complexes, and their relative ratio in the complex is dynamically dependent upon the neuron's activity history and developmental stage. Complex formation is dependent on their distinct N-terminal coiled-coil domains, while their closely homologous C-terminal pentraxin domains mediate association with AMPA-type glutamate receptors. Narp is substantially more effective in assays of cell surface cluster formation, coclustering of AMPA receptors, and excitatory synaptogenesis, yet their combined expression results in supraadditive effects. The support a model in which Narp can regulate the latent synaptogenic activity of NP1 by forming mixed pentraxin assemblies. The mechanism appears to contribute to both activity-independent and activity-dependent excitatory synaptogenesis.

GRIA2 / GLUR2 Antibodies

glutamate receptor, ionotropic, AMPA 2

Gria2/Glurb is one of four subunits of the glutamate receptor that is sensitive to alpha-amino-3-hydroxy-5-methyl-4-isoxazolpropionate (AMPA). Glutamate receptors are ligand-activated cation channels that mediate the fast component of excitatory postsynaptic currents in neurons of the central nervous system. These channels are assembled from 4 related subunits, with the Gria2/Glurb subunit rendering the channel almost impermeable to Ca(2+).

The AMPA Receptor (GluR 2) (E1L8U) Rabbit mAb Antibody from Cell Signaling Technology is a Rabbit Monoclonal antibody to GRIA2, GluR-K2, GluA2, GluR2, GLURB, HBGR2. This antibody recognizes Rat, Mouse, Human antigen. AMPA Receptor (GluR 2) (E1L8U) Rabbit mAb has been validated for the following 

AMPA Receptor (GluR 2) (E1L8U) Rabbit mAb recognizes endogenous levels of total GluR 2 protein. The antibody is not predicted to recognize other AMPA receptor subunits (e.g. GluR 1, GluR 3 or GluR 4) based on sequence homology of the antigen

Thanks so much Egidio! 

Dear Huong,

We have done AMPAR surface staining a lot (Mostly GluR2)  and my advice would be to use a N-tagged receptor (with HA or Flag or EGFP), transfected in your target cells under a pSYN promoter (not too strong, avoid over-expression). EGFP is especially good because there are some very good anti-GFP and you get both total population (EGFP) and surface (Alexa543-antiGFP). If you want inverse color, the anti-GFP also work with red variant and GFP and an excellent chicken IgY directly coupled to Alexa 488 is available. I hope this helps   

Thanks Thomas! It looks like you are recommending not to use antibody against the receptors? Using N-tagged receptor is definitely a good idea. I have not done since I am trying to look at the endogenous AMPA receptors. Do you have these constructs deposited on Addgene? Also, does the eGFP tag not interfere with the activity of the receptor? For visualizing them with tag and look at their live activities, I am thinking of the super ecliptic phluorin tagged GluA1 or GluA2. 

If you want to look at the abundance of surface GluR receptors, then you can use the biotinylation methods employed by Liang and Huganir. The method works well for surface and internalization measures.

Thank you Julie for the suggestion. We actually use this biotinylation method in our lab to study neurexin and neuroligin yet the labeling is quite tricky. :) I will see if I could design the system to look at AMPAR!