In the last several years it has become clear that AMPA type glutamate neurotransmitter receptors are rapidly transported into and out of synapses to strengthen or weaken their function. The remarkable dynamics of AMPA receptor (AMPAR) synaptic localization provides a compelling mechanism for understanding the cellular basis of learning and memory, as well as disease states involving cognitive dysfunction. Here, we summarize the evidence for AMPAR trafficking as a mechanism underlying a variety of learned responses derived from both behavioral and cellular studies. Evidence is also reviewed supporting synaptic dysfunction related to impaired AMPAR trafficking as a mechanism underlying learning and memory deficits in Alzheimer’s disease. We conclude that emerging data support the concept of multistage AMPAR trafficking during learning and that a broad approach to include examination of all the AMPAR subunits will provide a more complete view of the mechanisms underlying multiple forms of learning.

The cyclic AMP-dependent protein kinase (PKA) signaling pathway has been shown to be important in mechanisms of synaptic plasticity, although its direct and downstream signaling effects are not well understood. Using an in vitro model of eyeblink classical conditioning, we report that PKA has a critical role in initiating a signaling cascade that results in synaptic delivery of glutamate receptor 1 (GluR1)- and GluR4-containing alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) in abducens motor neurons during conditioning. PKA and the Ca(2+)-calmodulin-dependent protein kinases (CaMKs) II and IV are activated early in conditioning and are required for acquisition and expression of conditioned responses (CRs). cAMP-response-element-binding protein (CREB) is also activated early in conditioning but is blocked by coapplication of inhibitors to PKA and the CaMKs, suggesting that CREB is downstream of those signaling cascades. Moreover, evidence suggests that PKA activates extracellular signal-regulated kinase, which is also required for conditioning. Imaging studies after conditioning further indicate that colocalization of GluR1 AMPAR subunits with the synaptic marker synaptophysin requires PKA, but is insensitive to the N-methyl-d-aspartate receptor (NMDAR) inhibitor d,l-AP5. PKA activation also leads to synaptic localization of GluR4 subunits that, unlike GluR1, is dependent on NMDARs and is mediated by CaMKII. Together with previous studies, our findings support a two-stage model of AMPAR synaptic delivery during acquisition of classical conditioning. The first stage involves synaptic incorporation of GluR1-containing AMPARs that serves to activate silent synapses. This allows a second stage of NMDAR- and protein kinase C-dependent delivery of GluR4 AMPAR subunits that supports the acquisition of CRs.

Mokin M, Zheng Z, Keifer J. Conversion of silent synapses into the active pool by selective GluR1-3 and GluR4 AMPAR trafficking during in vitro classical conditioning. J Neurophysiol 98: 1278 -1286, 2007. First published June 27, 2007 doi:10.1152/jn.00212.2007. The conversion of silent synapses into active sites is hypothesized to be a primary mechanism underlying learning and memory processes. Here we used an in vitro model of classical conditioning from turtles that demonstrates a neural correlate of eyeblink conditioning to examine whether the conversion of silent synapses has a role in this form of associative learning. This was accomplished by direct visualization of AMPA receptor (AMPAR) and N-methyl-D-aspartate receptor (NMDAR) subunits colocalized with synaptophysin (Syn) using immunofluorescence and confocal microscopy. In naive preparations, there was a relatively high level of synapses immunopositive for NR1-Syn alone interpreted to be silent synapses. After early stages of conditioning during acquisition of conditioned responses (CRs), there was a significant increase in the colocalization of GluR1-3 AMPAR subunits at NR1-immunopositive synaptic sites. Later in conditioning, levels of GluR1-3 declined and enhanced colocalization of GluR4-containing AMPAR subunits at synapses was observed. The trafficking of these subunits during conditioning was NMDAR mediated and was accompanied by protein synthesis of GluR4 subunits. Examination of the postsynaptic density fraction confirmed the early and late synaptic insertion of GluR1-3 and GluR4, respectively, during conditioning. These findings suggest that there is differential trafficking of synaptic AMPARs during classical conditioning. Existing GluR1-3 AMPAR subunits are initially delivered to silent synapses early in conditioning to unsilence them followed by synthesis and insertion of GluR4 AMPAR subunits that are required for acquisition and expression of CRs.

Brain-derived neurotrophic factor (BDNF) gene expression critically controls learning and its aberrant regulation is implicated in Alzheimer's disease and a host of neurodevelopmental disorders. The BDNF gene is target of known DNA regulatory mechanisms but details of its activity-dependent regulation are not fully characterized. We performed a comprehensive analysis of the epigenetic regulation of the turtle BDNF gene (tBDNF) during a neural correlate of associative learning using an in vitro model of eye blink classical conditioning. Shortly after conditioning onset, the results from ChIP-qPCR show conditioning-dependent increases in methyl-CpG-binding protein 2 (MeCP2) and repressor basic helix-loop-helix binding protein 2 (BHLHB2) binding to tBDNF promoter II that corresponds with transcriptional repression. In contrast, enhanced binding of ten-eleven translocation protein 1 (Tet1), extracellular signal-regulated kinase 1/2 (ERK1/2), and cAMP response element-binding protein (CREB) to promoter III corresponds with transcriptional activation. These actions are accompanied by rapid modifications in histone methylation and phosphorylation status of RNA polymerase II (RNAP II). Significantly, these remarkably coordinated changes in epigenetic factors for two alternatively regulated tBDNF promoters during conditioning are controlled by Tet1 and ERK1/2. Our findings indicate that Tet1 and ERK1/2 are critical partners that, through complementary functions, control learning-dependent tBDNF promoter accessibility required for rapid transcription and acquisition of classical conditioning.

MECP2 mutations underlying Rett syndrome cause widespread misregulation of gene expression. Functions for MeCP2 other than transcriptional are not well understood. In an ex vivo brain preparation from the pond turtle Trachemys scripta elegans, an intraexonic splicing event in the brain-derived neurotrophic factor (BDNF) gene generates a truncated mRNA transcript in naïve brain that is suppressed upon classical conditioning. MeCP2 and its partners, splicing factor Y-box binding protein 1 (YB-1) and methylcytosine dioxygenase 1 (Tet1), bind to BDNF chromatin in naïve but dissociate during conditioning; the dissociation correlating with decreased DNA methylation. Surprisingly, conditioning results in new occupancy of BDNF chromatin by DNA insulator protein CCCTC-binding factor (CTCF), which is associated with suppression of splicing in conditioning. Knockdown of MeCP2 shows it is instrumental for splicing and inhibits Tet1 and CTCF binding thereby negatively impacting DNA methylation and conditioning-dependent splicing regulation. Thus, mutations in MECP2 can have secondary effects on DNA methylation and alternative splicing.DOI: http://dx.doi.org/10.7554/eLife.25384.001