BDNF-induced synaptic delivery of AMPAR subunits is differentially dependent on NMDA receptors and requires ERK

Abstract

Previous studies using an in vitro model of eyeblink classical conditioning in turtles suggest that increased numbers of synaptic AMPARs supports the acquisition and expression of conditioned responses (CRs). Brain-derived neurotrophic factor (BDNF) and its associated receptor tyrosine kinase, TrkB, is also required for acquisition of CRs. Bath application of BDNF alone induces synaptic delivery of GluR1- and GluR4-containing AMPARs that is blocked by coapplication of the receptor tyrosine kinase inhibitor K252a. The molecular mechanisms involved in BDNF-induced AMPAR trafficking remain largely unknown. The aim of this study was to determine whether BDNF-induced synaptic AMPAR incorporation utilizes similar cellular mechanisms as AMPAR trafficking that occurs during in vitro classical conditioning. Using pharmacological blockade and confocal imaging, the results show that synaptic delivery of GluR1 subunits during conditioning or BDNF application does not require activity of NMDARs but is mediated by extracellular signal-regulated kinase (ERK). In contrast, synaptic delivery of GluR4-containing AMPARs during both conditioning and BDNF application is NMDAR- as well as ERK-dependent. These findings indicate that BDNF application mimics AMPAR trafficking observed during conditioning by activation of some of the same intracellular signaling pathways and suggest that BDNF is a key signal transduction element in postsynaptic events that mediate conditioning.

Fig. 1

Confocal images of abducens motor neurons showing punctate staining for the presynaptic marker synaptophysin (Syn; green), GluR1 AMPAR subunits (red), or GluR4 AMPAR subunits (red) from each of the conditioned groups examined: pseudoconditioned for two sessions (Ps2), conditioned for two sessions (C2), and conditioned during application of AP-5 for two sessions (AP-5). Colocalization of AMPAR with synaptophysin punctate staining is also shown (GluR1+Syn, GluR4+Syn) and indicated by the arrowheads. Scale bar = 2 μm.

Fig. 2

Confocal images of punctate staining of abducens motor neurons for synaptophysin, GluR1, and GluR4 AMPAR subunits from each of the BDNF-treated groups examined: BDNF application for two sessions (BDNF2), coapplication of BDNF with AP-5 for two sessions (AP-5), and coapplication of BDNF with PD98059 for two sessions (PD). Colocalization of AMPAR with synaptophysin punctate staining is also shown (GluR1+Syn, GluR4+Syn) and indicated by the arrowheads. Scale bar =2 μm.

Fig. 3

Quantitative analysis of punctate staining of individual puncta for GluR1, GluR4, and synaptophysin (Syn), and for colocalized staining for GluR1 + Syn, and GluR4 + Syn from the different treatment groups. * indicates significant differences from Ps2; # indicates significant differences from C2; ‡ indicates significant differences from BDNF2.

Fig. 4

Western blot analysis of protein expression for GluR1 and GluR4 AMPAR subunits and synaptophysin from naive and BDNF-treated groups. Levels of GluR1 were high in naive preparations or after treatment with BDNF and remained high following coapplication of BDNF with AP-5 or PD98059. In contrast, BDNF-induced increases in protein levels for both GluR4 and synaptophysin were reduced after coapplication of BDNF with either AP-5 or PD98059. Actin loading controls are shown in the bottom panel.