br Results br Discussion Postsynaptic

Results
Discussion Postsynaptic trafficking of AMPA receptors, a process responsible for maintaining the steady-state level of AMPA receptor synaptic transmission, is believed to also actively partake in glutamatergic synaptic plasticity in principal neurons, such the hippocampal CA1 cells. Here we show that the AMPA receptor subunit GluR-Blong is delivered to synapses by spontaneous synaptic activity and following the induction of LTP. Inhibition of the former form of GluR-Blong transport results in 35% decrease of AMPA receptor-mediated response at ∼P14 CA3 to CA1 synapses, indicating that delivery of endogenous GluR-Blong-containing receptors by spontaneous synaptic activity is required to maintain approximately one-third of the steady-state AMPA receptor synaptic content. At the same time, synaptic delivery of GluR-Blong-containing receptors following the induction of LTP is responsible for approximately 50% of the stably established potentiation at the ∼P14 CA3 to CA1 synapses. GluR-Blong-containing receptors, via their C-terminal-dependent transport mechanisms, thus clearly contribute to AMPA receptor functions in the juvenile hippocampus.
Experimental Procedures
Acknowledgements We thank Andrea Migala for slice cultures, Andrei Rozov for electrophysiology help, Ulla Amtmann for in situ hybridization, Sabine Gruenewald and Carmen Grosskurth for cell culture, Horst Grosskurth for sequencing, Julia Kuhl for graphics, Sondra Schlesinger for Sindbis(nsp2S726) plasmid and helpful discussions, and Rolf Sprengel and Troy Margrie for critical reading of the manuscript and helpful discussions. This study was supported in part by DFG grant Os 191/1-1 to P.O., the NIH and the Alle Davis and Maxine Harrison Endowment to R.M., the Alzheimer's association and the Fraxa research foundation to J.J.Z. J.J.Z. is a Naples Investigator of the NARSAD foundation and an Alfred P. Sloan Fellow.
Introduction AMPA receptors are the primary mediators of fast excitatory synaptic transmission in the mammalian brain. Modifications of AMPA receptors have been found in several models of synaptic plasticity and neuropathology, including long-term potentiation (LTP) 1, 2, 17, 18, 21, learning and memory 4, 31, excitotoxicity 5, 19, and epileptic seizures 12, 23. Direct modification of AMPA receptors has received much attention as a possible means of modifying synaptic responses, but the mechanisms regulating AMPA/GluR receptor populations are only beginning to be explored. Several reports have indicated that LTP and LTD inducibility change during the postnatal Deferiprone sale in the hippocampus, as well as in various telencephalic structures 3, 10, 30, 33. The number of silent synapses, i.e., synapses expressing functional NMDA but not AMPA receptors 14, 16, has also been shown to be much greater at early postnatal ages than in adults [6]. Induction of LTP at silent synapses has been proposed to be due to the calcium-dependent transition from an absence to a presence of functional AMPA receptors. Several possible mechanisms could be underlying this transition: (1) latent receptors in synaptic plasma membrane could be modified from a quiescent to an active state, (2) occult receptors in synaptic plasma membrane could be rapidly exposed, (3) functional AMPA receptors could be added to synaptic plasma membrane via fusion of intracellular receptor-laden vesicles to synaptic plasma membrane, or (4) extrasynaptic receptors could rapidly migrate and aggregate into synaptic sites. Developmental changes in AMPA receptor ligand binding have been reported in both membrane fractions of rat telencephalon [8], and autoradiograms 13, 26. In particular, quantitative autoradiography with low concentrations of -AMPA revealed an increase in binding above adult levels during the postnatal developmental period in rat hippocampus [26]. Moreover, the changes were primarily due to alterations in ligand binding in the pyramidal and granule cell layers. We have also reported that binding performed with high concentrations of unlabeled AMPA (500 nM) does not indicate a postnatal increase above adult levels, suggesting that the total number of AMPA binding sites does not exhibit an enhanced expression over adult levels. Saturation kinetics revealed the existence of two binding sites with different affinities and maximal numbers for -AMPA 9, 11, 20, 28, and it has been shown that the ratio of high to low affinity sites was changing with development 8, 25. Furthermore, studies on the characteristics of ligand binding in different subcellular fractions have indicated a preferential localization of high-affinity sites in non-synaptic subcellular loci [25]. We subsequently showed using Western blots labeled with antibodies against AMPA receptor subunits that at least a portion of the intracellular population of AMPA/GluR receptors represent nascent, intracellular receptors en route to target sites [27]. These sites can be visualized over the pyramidal and granule cell layers of the hippocampus in autoradiography using low concentrations of -AMPA. Therefore, it was of interest to analyze the changes in GluR subunits in various subcellular fractions during postnatal development, and to compare them with postnatal changes in AMPA binding properties.