Gonadal steroid 17β-estradiol (E2) exerts rapid, non-genomic effects on neurons and strictly regulates learning and memory through altering glutamatergic neurotransmission and synaptic plasticity. However, its non-genomic effects on AMPARs are not well understood. Here, we analyzed the rapid effect of E2 on AMPARs using single-molecule tracking and super-resolution imaging techniques. We found that E2 rapidly decreased the surface movement of AMPAR via membrane G protein-coupled estrogen receptor 1 (GPER1) in neurites in a dose-dependent manner. The cortical actin network played a pivotal role in the GPER1 mediated effects of E2 on the surface mobility of AMPAR. E2 also decreased the surface movement of AMPAR both in synaptic and extrasynaptic regions on neurites and increased the synaptic dwell time of AMPARs. Our results provide evidence for understanding E2 action on neuronal plasticity and glutamatergic neurotransmission at the molecular level.
The gonadal steroid 17β-estradiol (E2) rapidly alters glutamatergic neurotransmission, but its direct effect on the AMPA receptor (AMPAR) remains unknown. Live-cell single-molecule imaging experiments revealed that E2 rapidly and dose-dependently alters the surface movement of AMPAR via membrane estrogen receptors with distinct effects on somas and neurites. The effect of E2 on the surface mobility of AMPAR depends on the integrity of the cortical actin network.
Main textThe gonadal steroid 17β-estradiol (E2) plays a role in a wide range of biological actions from fertility to neuroprotection (1,2). The cellular effects of E2 have been proposed to be mediated by slow transcriptional action through classic nuclear receptors (ERα and ERβ). In addition to its classic genomic effects, E2 rapidly alters the function of receptors and the activity of second messengers through membrane estrogen receptors such as the typical membraneassociated ERα and ERβ as well as G protein-coupled estrogen receptor 1 (GPER1) (3). E2 promptly regulates glutamatergic neurotransmission and synaptic plasticity. For instance, E2 decreases AMPA miniature excitatory postsynaptic current frequency within minutes (4).Surface trafficking of glutamate receptors such as AMPA receptors (AMPARs) plays critical roles in excitatory neurotransmission (5) and synaptic plasticity (6). However, whether E2 affects AMPAR surface trafficking is unknown. To examine the effect of E2 on the surface trafficking of the glutamate receptor, we applied E2 to live neurons (differentiated from PC12 cells) ( Supplementary Fig. 1a, b, c). We imaged single endogenous GluR2-AMPA, which is the most abundant AMPAR subunit in neurons (7), and mGluR1, a metabotropic glutamate receptor involved in rapid membrane action of E2 (8), using ATTO-labeled antibodies and livecell total internal reflection fluorescence (TIRF) microscopy ( Fig. 1a; Supplementary Movies 1-4).GluR2-AMPAR and mGluR1 molecules exhibited Brownian and confined motion (Fig. 1a, b). The diffusion coefficients of GluR2-AMPAR (DAMPAR) and mGluR1 (DmGluR1) were significantly greater on neurites than on somas (Fig. 1c, d), indicating that the surface movements of glutamate receptors are faster on neurites.Application of 100 pM E2 decreased DAMPAR on somas within 5 minutes; however, it did not affect DAMPAR on neurites (Fig. 2a). The effect of E2 was selective for GluR2-AMPAR, since E2 did not change DmGluR1 (Fig. 2b; Supplementary Fig. 2b). In contrast, 100 nM E2 4 decreased DAMPAR on neurites, while DAMPAR on somas remained unchanged (Fig. 2c).Previously, Potier and colleagues demonstrated that 10 nM E2 decreased the diffusion dynamics of NMDA receptor (GluN2B-NMDAR) molecules in hippocampal neurons (9). Our findings indicate that the rapid effect of E2 on the surface trafficking of GluR2-AMPAR is compartment-specific and dose-dependent.
StatisticsThe D values are expressed as the mean ± SEM (in the time series), the mean ± SEM as a percentage of the vehicle value and as the median ± 25-75% (see figure legends)....
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