Conversion of Silent Synapses Into the Active Pool by Selective GluR1-3 and GluR4 AMPAR Trafficking During In Vitro Classical Conditioning

Abstract

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.

Coordinate action of pre- and postsynaptic brain-derived neurotrophic factor is required for AMPAR trafficking and acquisition of in vitro classical conditioning

“…We reported previously that preexisting GluR1 AMPAR subunits are delivered to silent synapses during early stages of conditioning, followed by synthesis and insertion of GluR4 subunits that support CR acquisition (Mokin et al, 2007). In the present study, bath application of BDNF for the equivalent of two pairing sessions induced the same postsynaptic glutamate receptor modifications that was observed after two sessions of conditioning.…”

“…In previous studies we consistently observed that levels of the presynaptic protein synaptophysin were significantly elevated during conditioning Mokin et al, 2007). Western blotting was performed to assess whether BDNF induces a similar enhancement of presynaptic proteins.…”

“…Conditioning in this model has been shown to involve transient delivery of synaptic GluR1-containing AMPARs followed by synaptic insertion of GluR4 subunits (Mokin et al, 2007). Here, using immunostaining and confocal imaging of abducens motor neurons, we examined whether BDNF application results in the synaptic delivery of either GluR1 or GluR4 subunits, or both.…”

“…Furthermore, studies demonstrate that protein kinase C (PKC) activation of ERK MAPK signal transduction pathways regulate GluR4 synaptic insertion during conditioning, whereas ERK signaling, but not PKC, regulates insertion of GluR1 Zheng and Keifer, submitted). Apart from these postsynaptic modifications during conditioning, expression of the presynaptic vesicle-associated protein synaptophysin was also consistently enhanced during the acquisition of conditioning Mokin et al, 2007). Given the evidence implicates a role for BDNF in pre-and postsynaptic modifications in LTP, the present study was carried out to examine BDNFinduced mechanisms of synaptic plasticity during classical conditioning in this in vitro preparation.…”

“…More recently we have shown that conditioning is associated with two waves of synaptic AMPAR insertion in abducens motor neurons. Initially, GluR1-containing AMPARs are trafficked to synapses to unsilence them (Mokin et al, 2007). This is followed by NMDA-dependent synaptic delivery of GluR4-containing AMPARs that is associated with the acquisition of conditioned responses (CRs; Mokin and Keifer, 2004;.…”