[PubMed] [Google Scholar] 11. should be elevated. The results revealed that the percentage of intensely stained cells with the anti-C-terminus GluR1 antibody was indeed increased in the bFGF-treated cultures (8.6%4.3 in the control culture, 21.7%8.0 in bFGF-treated cultures n = 7; p = 0.007). Dense GluR1-like staining with the C-terminal antibody in bFGF-treated neurons was XMD8-87 cytoplasmic and appeared in the neuritic processes (Fig. 4, middle panels). In order to confirm that this observation is specific, we also stained neurons with an anti-N-terminus GluR1 antibody (Fig. 4, lower panels). It should be noted that, with the anti-N-terminus antibody, the percentage of intensely labeled cells in the bFGF-treated cultures did not differ from the control (63.9%5.9 in control culture, 58.8%4.8 in bFGF-treated cultures n = 5; p = 0.299). These observations suggest that the bFGF-dependent decrease in SAP97 protein might result in the exposure of the GluR1 C-terminal domain. Open in a separate window Fig. 4 Immunohistochemistry of the AMPA-type glutamate receptor subunit GluR1 in control (A) or bFGF-treated neuronal cultures (B). Neocortical neurons were dissociated and cultured in the absence or presence of bFGF (20 ng/ml; added daily) for 4 days, fixed with 4% paraformaldehyde in a phosphate buffered saline solution, and incubated with antibodies recognizing the C-terminus (upper and middle panels) or the N-terminus (lower panels) of GluR1. The results depict an increase in the number of strongly stained cells with the anti-C-terminus antibody without affecting the staining pattern with the anti-N-terminus antibody. Scale bar, 50 m. DISCUSSION Specific members of the PDZ family of proteins have recently been implicated in important and distinct aspects of brain function and synaptic plasticity16C18,20,22C25). However, very little is known as to whether these molecules respond differently to various cytokines or growth factors. Resolving such an issue is important since these proteins have overlapping patterns of expression and interaction23C26,38). For instance, any factor that affects the expression of a particular PDZ protein might lead to a unique modulation of the cellular response to an afferent input, ultimately resulting in a divergence of the response at synapses that have different combinations of PDZ molecules. The regulation of glutamatergic receptors by bFGF has received much attention as these receptors have been implicated in important aspects of brain Rabbit polyclonal to TIGD5 physiology such as the induction of LTP or LTD1,2,5,19,39,40) Few reports discuss whether bFGF affects the AMPA-type receptors in neurons, their subcellular distribution, or the mechanisms applied therein2,29). SAP97 and GRIP1 interact with the GluR1 and GluR2 subunits. They have also been implicated in the synaptic targeting and/or stabilization of AMPA receptors18,20,21). Such stabilization might increase neurotransmission and synaptic responsiveness to an incoming stimulus, whereas their removal could be crucial in avoiding the harmful effects of extended neuronal excitation that yields excitotoxicity and neuronal death1,39,40). To elucidate the effects of bFGF on such interactions, we used anti-N-terminal GluR1 or anti-N-terminal GluR2 antibodies to co-immunoprecipitate the interacting PDZ proteins (SAP97 and GRIP1, respectively) and compared their levels in both culture conditions. Our results show that bFGF down-regulated the PDZ proteins that interact with AMPA receptors (Fig. 1), which was reflected in the dissociation of GluR1 from its interaction with SAP97 (Fig. 3), rendering the GluR1 C-terminal XMD8-87 epitope exposed to bind the corresponding antibody (Fig. 4). While elaborate studies have been published on the expression of PDZ proteins and glutamate receptors29,30), most of the published data used 4% paraformaldehyde as a crosslinking reagent/fixative, and different, XMD8-87 sometimes opposing, results have been obtained. Such has been the case for the C-termini of XMD8-87 NMDA-type glutamate receptors and their interacting PDZ proteins PSD-95, PSD-93 and SAP102. This discrepancy has been resolved using pre-incubation with a protease or exposure to microwaves. Such denaturing procedures are known to reveal those epitopes that XMD8-87 had been masked by interaction with partner molecules30). Labeling of AMPA receptors in young neurons in culture where mature synapses have not yet formed does not require such denaturating conditions29C30). Our staining results showed labeling in peri-nuclear and cytoplasmic compartments and in dendritic shafts, suggesting that by fixation time, significant.
- Unique magnification 20, green Keratin 8/18 and reddish Keratin 5 immunostaining
- and Krainer,A