Methods for Uncovering the Mechanisms of AMPA Receptor Trafficking

Information storage in the brain involves alterations in the strength of communication between neurons. This requires activity-dependent, long-lasting changes in synaptic transmission. Long-term potentiation (LTP) is a long lasting use-dependent increase in the efficiency of excitatory synaptic transmission that has been suggested to underlie certain forms of learning and memory [1]. The induction of LTP requires Ca entry through the N-methyl-D-aspartate receptor (NMDAR). However, the region within the synapse whose regulation results in LTP is still controversial. Some groups suggest a pre-synaptic modification that results in an increase in the amount of glutamate released, whereas others suggest a post-synaptic modification, such as an increase in the number of receptors or a change in receptor properties [2]. Interestingly, the description of the silent synapse, synapses that contain NMDAR only but could acquire α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor (AMPAR) as a result of synaptic activity, support a post-synaptic mechanism [3–5]. Glutamate receptors mediate most excitatory synaptic transmission in the brain. The ionotropic glutamate receptors are clustered with associated downstream signalling molecules that are found in the post-synaptic density (PSD), and the synaptic clustering of these receptors is critical for rapid and efficient synaptic transmission. This complex of receptors with signalling molecules can also undergo dynamic changes. In particular, changing the number of glutamate receptors at the synaptic membrane could constitute a critical mechanism for rapidly altering synaptic strength. Two major classes of ionotropic glutamate receptors exist, the NMDAR and the AMPAR. A third class, the kainate receptors, will not be discussed here as it has been reviewed elsewhere [6]. Both NMDAR and AMPAR are highly concentrated at excitatory synapses linked to the PSD but they interact with different sets of scaffolding proteins. In addition, whereas NMDAR are very stably localized at the PSD, AMPAR cycle rapidly to and from the synaptic membrane. This difference in trafficking behavior of NMDAR versus AMPAR may reflect their use of different mechanisms for anchoring to the PSD. The conversion of a silent synapse to a synapse with AMPAR, a change that creates a functional synapse, has been proposed as one of the main mechanisms for LTP induction. For these reasons, studies of the trafficking of AMPAR and their cycling in and out of the synapse have provided a large step in understanding LTP. Synaptic organization, assembly and trafficking have been studied extensively for other receptors, such as the acetylcholine receptor at the neuromuscular junction [7]. AMPAR trafficking has been one of the most extensively studied properties of the excitatory synapse because of its implication in synaptic plasticity. The complexity of the organization of the brain makes assaying receptor properties without disrupting the system difficult. A complete picture of synaptic regulation comes from combining a wide range of methodological approaches, such as electrophysiology, cell biology, biochemistry and genetics. The development of new methods or the adaptation of methods used in other systems has allowed a better understanding of the trafficking of AMPAR. Mechanisms of AMPAR trafficking have been reviewed elsewhere [3,8,9], and this chapter will present an overview of the different approaches that have been used to study the trafficking of AMPAR and its implication for synaptic plasticity, focusing on the contribution and the importance of each method.