PKA has a critical role in synaptic delivery of GluR1- and GluR4-containing AMPARs during initial stages of acquisition of in vitro classical conditioning

Abstract

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.

FIG. 1.

Inhibition of cyclic AMP-dependent protein kinase (PKA) by Rp-adenosine-3′,5′-cyclic monophosphorothioate (Rp-cAMPs) suppresses acquisition and expression of classical conditioning. A: physiological records of abducens nerve recordings taken from an experiment in which the compound Rp-cAMPs blocked expression of conditioned responses (CRs). The traces show an abducens nerve CR (arrow) followed by the unconditioned response (UR) recorded in the 2nd pairing session prior to drug application (Normal). Record obtained during application of Rp-cAMPs in the 4th pairing session in which the expression of CRs was blocked but the UR was unaffected (Rp-cAMPs). Washout of drug in the 6th session resulted in reexpression of CRs (arrow, Wash). The conditioned stimulus (CS) and unconditioned stimulus (US) are indicated at the bottom. B: acquisition curves (means ± SD) of the percentage of abducens CRs following Rp-cAMPs treatment at the beginning of conditioning to test for acquisition (left) or after CRs had been obtained to test for CR expression (right). Application of Rp-cAMPs resulted in no acquisition and attenuated expression of CRs. *Indicates significant difference from session 2. P values are given in the text throughout.

FIG. 2.

The Ca²⁺-calmodulin–dependent protein kinase II (CaMKII) antagonist autocamtide-2–related inhibitory peptide (AIP) attenuates both the acquisition and expression of conditioning. A: physiological records of abducens nerve recordings taken from an experiment in which AIP reversibly blocked CR expression. B: acquisition curves of the percentage of abducens CRs show that AIP significantly attenuated both acquisition (left) and expression (right) of CRs. *Indicates significant difference from session 2.

FIG. 3.

Western blots demonstrating specificity of the antibodies used in the present study. Antibodies were tested on naive brain tissue from turtle (T) or rat (R). Bands appeared at the expected molecular weights in both species. See text for details.

FIG. 4.

Characterization of the onset of protein phosphorylation during early conditioning as determined by phosphospecific antibodies and Western blotting. Blots for phospho- and total protein are shown, whereas quantitative data of the ratio of phospho- to total protein is plotted. Significant levels of phosphorylation for PKA (A), CaMKII (B), Ca²⁺-calmodulin–dependent protein kinase IV (CaMKIV, C), and cAMP-response-element-binding protein (CREB, D) occur within 15 min of application of the conditioning stimuli. Ps1, pseudoconditioning for one session; C₁₅, conditioning for 15 min; C1, conditioning for one pairing session; C2, conditioning for 2 sessions; C5, conditioning for 5 sessions. *Indicates significant differences from Ps1.

FIG. 5.

Selectivity of PKA and CaMKII antagonists and effects on p-CREB. A: bath application of Rp-cAMPs during 2 sessions of paired stimulation significantly reduced the conditioning-related increase in PKA phosphorylation to pseudoconditioned levels, whereas AIP had no significant effect. B: application of AIP for 2 sessions suppressed phosphorylation of CaMKII, whereas Rp-cAMPs failed to have a significant effect. C: application of Rp-cAMPs for 2 pairing sessions significantly attenuated phosphorylation of CREB compared with conditioning. Coapplication of Rp-cAMPs with the general CaMKII antagonist KN-62 for 2 sessions completely inhibited CREB phosphorylation to levels similar to pseudoconditioning. *Indicates significant differences from Ps2; # indicates significant differences from C2.

FIG. 6.

Evidence that PKA is downstream from and activates extracellular signal-regulated kinase (ERK). A: bath application of Rp-cAMPs for 2 pairing sessions inhibited the conditioning-related activation of ERK to pseudoconditioned levels. Application of AIP for 2 sessions was ineffective in inhibiting ERK. B: application of the PKA activator Sp-cAMPs alone for the equivalent time period of 2 pairing sessions (with no electrical stimulation) induced phosphorylation of ERK to levels similar to those obtained after conditioning. C: application of the selective MEK–ERK antagonist PD98059 for 2 sessions failed to affect the conditioning-induced activation of PKA, suggesting that ERK does not feed back onto PKA signaling pathways. *Indicates significant differences from Ps2.

FIG. 7.

The N-methyl-d,l-aspartate receptor (NMDAR) antagonist d,l-2-amino-5-phosphonopentanoic acid (d,l-AP5) does not affect phosphorylation of either PKA or CaMKII in early conditioning. A: application of d,l-AP5 during conditioning for 15 min (C₁₅) or one pairing session (C1) failed to attenuate phosphorylated levels of PKA, which were similar to values obtained after conditioning in normal saline. B: application of d,l-AP5 also did not affect phosphorylation of CaMKII in early conditioning. *Indicates significant differences from Ps1.

FIG. 8.

Synaptic incorporation of glutamate receptor 1 (GluR1)- and GluR4-containing α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) is dependent on PKA, whereas GluR4 incorporation also requires CaMKII. A: confocal images of selected abducens motor neurons from the different experimental groups show punctate staining for synaptophysin (Syn) and colocalization of GluR1 (GluR1 + Syn) and GluR4 AMPAR subunits (GluR4 + Syn) with synaptophysin. Colocalization of AMPARs (red) with Syn (green) is defined by overlapping (yellow) or adjacent puncta and are indicated by the arrowheads. B: quantitative analysis of Syn, GluR1 + Syn, and GluR4 + Syn punctate staining for the different experimental groups is plotted. Preparations conditioned for 2 pairing sessions (C2) showed significantly greater punctate staining for Syn, GluR1 + Syn, and GluR4 + Syn compared with the pseudoconditioned group (Ps2). Treatment with the PKA inhibitor Rp-cAMPs for 2 sessions (Rp2) significantly reduced the conditioning-related increase in synaptophysin and the colocalization of GluR1 and GluR4 with synaptophysin. Application of the CaMKII blocker AIP for 2 sessions (AIP2) did not affect elevated levels of Syn or GluR1 + Syn, but significantly attenuated GluR4 + Syn. Application of the PKA activator Sp-cAMPs (Sp2) increased punctate staining for synaptophysin and induced synaptic incorporation of GluR1 and GluR4 subunits. Coapplication of Sp-cAMPs with d,l-AP5 (Sp2 + d,l-AP5) for 2 sessions had no effect on GluR1 synaptic insertion but significantly attenuated GluR4. *Indicates significant differences from Ps2. Scale bar = 2 μm.

FIG. 9.

GluR1 AMPAR subunits are phosphorylated in early stages of conditioning. Western blots using phosphorylation site-selective antibodies indicate that GluR1 subunits are phophorylated at both Ser845 and Ser831. Quantitative analysis shows that the average levels of phosphorylation are significant after one pairing session (C1) or after 25 min of paired stimulation. *Indicates significant differences from Ps1.

FIG. 10.

Two-stage model of the acquisition phase of in vitro classical conditioning. The first stage of acquisition occurs during the 1st pairing session but prior to when CRs are recorded (top line) and involves synaptic insertion of GluR1-containing AMPARs to activate silent synapses. This process does not involve protein synthesis of GluR1 but the translocation of existing receptors. Shortly after the onset of paired CS–US stimulation PKA, CaMKII, and CaMKIV are phosphorylated. Activation of CREB is depicted downstream of PKA and the CaMKs because inhibitors of these block phosphorylation of CREB. Western analysis further shows that brain-derived neurotrophic factor (BDNF), a known target for CREB, is synthesized after phosphorylation of PKA and the CaMKs (at 25 min) and that ERK is activated shortly after that, which leads to synaptic incorporation of GluR1-containing AMPARs. Data also suggest that GluR1 subunits are directly phosphorylated at Ser845 and Ser831, PKA, and CaMKII sites, which could provide an additional or parallel route for GluR1 modification and trafficking. The 2nd stage of acquisition occurs during the 2nd pairing session when CRs are recorded (bottom line) and involves NMDAR-dependent synthesis and synaptic insertion of GluR4-containing AMPARs that are thought to replace GluR1. The synaptic incorporation of GluR4 subunits is associated with the acquisition of CRs. This stage requires protein kinase C (PKC) and is also ERK dependent.