Dynamic Regulation of AMPAR Phosphorylation In Vivo Following Acute Behavioral Stress

Abstract

The tuning of glutamatergic transmission is an essential mechanism for neuronal communication. α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) are ionotropic glutamate receptors that mediate fast synaptic transmission. The phosphorylation states of specific serine residues on the GluA1 and GluA2 AMPAR subunits are considered critical post-translational modifications that regulate AMPAR activity and subcellular trafficking. While behavioral stress, via stress hormones, exerts specific alterations on such glutamatergic processes, there have been conflicting data concerning the influence of stress on AMPAR phosphorylation in different brain regions, and the post-stress signaling mechanisms mediating these processes are not well delineated. Here, we examined the dynamics of phosphorylation at three AMPAR serine residues (ser831-GluA1, ser845-GluA1, and ser880-GluA2) in four brain regions [amygdala, medial prefrontal cortex (mPFC), dorsal hippocampus, and ventral hippocampus] of the rat during the hour following behavioral stress. We also tested the impact of post-stress corticosteroid receptor blockade on AMPAR phosphorylation. Both GluA1 subunit residues exhibited elevated phosphorylation after stress, yet post-stress administration of corticosteroid receptor antagonists curtailed these effects only at ser831-GluA1. In contrast, ser880-GluA2 displayed a time-dependent tendency for early decreased phosphorylation (that was selectively augmented by mifepristone treatment in the amygdala and mPFC of stressed animals) followed by increased phosphorylation later on. These findings show that the in vivo regulation of AMPAR phosphorylation after stress is a dynamic and subunit-specific process, and they provide support for the hypothesis that corticosteroid receptors have an ongoing role in the regulation of ser831-GluA1 phosphorylation during the post-stress interval.

Keywords: AMPA; Amygdala; Phosphorylation; Prefrontal cortex; Spironolactone; Stress.

Fig. 1

Exposure to acute EP stress increased plasma corticosterone levels. Corticosterone data sampled from trunk blood 10 and 60 min after the end of stress. Data were collapsed across the Drug factor. Inset Spironolactone exposure was associated with elevated corticosterone levels in the stress conditions at 10 min. Error bars show standard error of the means (SEMs). The number sign indicates a significant difference compared to the non-stressed control group. Asterisk indicates a significant t test compared to the vehicle-treated group. AMY amygdala, DH dorsal hippocampus, mPFC medial prefrontal cortex, VH ventral hippocampus

Fig. 2

Exposure to acute EP stress had differential effects on GluA1 and GluA2 phosphorylation. a Overall, the ser831 and ser845 GluA1 residues show increased phosphorylation compared to non-stressed control after a 30-min exposure to an elevated platform. Data were collapsed across brain region and time factors. The number sign indicates a significant difference compared to the non-stressed control group. Error bars represent the SEMs. b Trend lines showing mean phosphorylation levels for ser831-GluA1, ser845-GluA1, and ser880-GluA2 at 10 and 60 min post-stress. Data from stressed groups shown with black solid lines; data from non-stressed groups shown with gray dashed lines. AMY amygdala, DH dorsal hippocampus, mPFC medial prefrontal cortex, VH ventral hippocampus

Fig. 3

Post-stress corticosteroid blockade decreased ser831-GluA1 phosphorylation. a Ser831-GluA1 phosphorylation data showing group means with the data split by the stress and drug factors. Data were collapsed across time and brain region factors and were normalized to the vehicle-treated non-stressed controls. b Ser831-GluA1 phosphorylation data showing group means split by brain region. Data were collapsed across the drug, time, and brain region factors. c Ser845-GluA1 phosphorylation data showing group means with the data split by the stress and drug factors. Data were collapsed across time and brain region factors and were normalized to the vehicle-treated non-stressed control. d Ser845-GluA1 phosphorylation data showing group means split by brain region. Data were collapsed across the drug, time, and brain region factors. The number sign indicates a significant difference compared to the vehicle-treated non-stressed control group. Asterisk indicates a significant difference compared to the vehicle-treated stressed control group. ⁺indicates a significant group difference based on brain region. Error bars show SEMs. AMY amygdala, DH dorsal hippocampus, mPFC medial prefrontal cortex, mif mifepristone, spi spironolactone, veh vehicle, VH ventral hippocampus

Fig. 4

Post-stress mifepristone treatment temporarily augmented ser880-GluA2 phosphorylation in the amygdala and mPFC of stressed animals. Ser880-GluA2 phosphorylation data at 10 min post-stress showing group means with the data split by the stress, brain region, and drug factors. Asterisk indicates a significant difference compared to the vehicle-treated stressed control group. Error bars show SEMs. AMY amygdala, DH dorsal hippocampus, mPFC medial prefrontal cortex, mif mifepristone, spi spironolactone, veh vehicle, VH ventral hippocampus