Corticosterone acutely increases GluN2B-NMDAR, but not GluN2A-NMDAR, surface and synaptic content

To investigate the acute effect of corticosterone on the distribution of NMDAR subtypes embedded into the plasma membrane of live hippocampal neurons, we over-expressed the GluN2A or GluN2B subunit fused to a Super Ecliptic pHluorin (SEP) at its extracellular N-terminus (GluN2A and GluN2B-SEP) to image preferentially (not exclusively, see ref. 22) the surface receptor pool23. The fluorescence intensity of clustered GluN2A- or GluN2B-NMDAR, which has been shown to co-localize with synaptic markers24, was measured over time before and after exposure to 50 nM corticosterone (Fig. 1A). GluN2A-NMDAR clusters were stable over the time within the 30 min period following corticosterone exposure (Fig. 1A–C). In contrast, the fluorescence of GluN2B-NMDAR clusters rapidly and significantly increased already 5 min after corticosterone exposure (Fig. 1A–C). This increase was stable over 30 min as it remained to its highest value during this period (Fig. 1B). Thus, corticosterone acutely increases the clustering of surface GluN2B-NMDAR. We directly measured the surface level of GluN2A- and GluN2B-NMDAR content in glutamate synapse by transfecting neurons with GluN2A and GluN2B subunits containing different extracellular tags and the postsynaptic protein Homer 1c-DsRed (Fig. 1D). After live immunocytochemical staining of the surface GluN2 subunits, corticosterone exposure increased the staining of synaptic GluN2B-NMDAR, significantly decreasing the GluN2A/2B ratio within glutamate synapses (Fig. 1E,F). Collectively, these data indicate that a single acute corticosterone exposure rapidly alters the GluN2A/2B synaptic ratio through a specific alteration of the clustering of GluN2B-NMDAR.

Figure 1 Corticosterone alters surface GluN2B-NMDAR clustering. (A) Dendritic fragments of GluN2A- and GluN2B-SEP expressing neurons before and after exposure to corticosterone (100 nM). Scale bar = 5 µm, scale bar inset = 1 µm. (B) Example of fluorescence intensity of GluN2A- (n = 12 dendritic fields, N = 5 neurons) and GluN2B-SEP (n = 15 dendritic fields, N = 5 neurons) clusters over time. (C) Comparison of GluN2A- and GluN2B-SEP cluster fluorescence intensity before and after exposure to corticosterone. ***p < 0.001, paired Student t-test. (D) Live immunostaining of GluN2A and GluN2B subunits in Homer 1c-DsRed expressing neurons. Scale bar = 10 µm, scale bar inset = 5 µm. (E) Immunostaining of surface GluN2A and GluN2B subunits before and after exposure to corticosterone. Scale bar = 5 µm. (F) Comparison of the fluorescence intensity of GluN2A (n = 12 dendritic fields, N = 5 neurons) and GluN2B (n = 11 dendritic fields, N = 5 neurons) subunit clusters (expressed as ratio) -SEP before and after exposure to corticosterone (50 nM, 20 min). *p < 0.05, Student t-test. Full size image

Corticosterone increases NMDAR-mediated miniature EPSC and sensitivity to a GluN2B antagonist

To examine the functional effect of corticosterone on the synaptic NMDAR-mediated current, corticosterone (100 nM) was applied to hippocampal cultured neurons and NMDAR-mediated miniature EPSCs (NMDAR mEPSCs) were recorded in neurons (Fig. 2A) (see Methods for details). After 20 min, the frequency of NMDAR mEPSC was unaltered (Fig. 2B). However, corticosterone significantly increased the peak amplitude and charge (area under the curve) of NMDAR mEPSCs when compared to vehicle-treated cells (Fig. 2B). The decay times of NMDAR mEPSCs tended to increase, although not significantly, after corticosterone exposure (Fig. 2B). Thus, corticosterone acutely increased the NMDAR-mediated current in spontaneously active glutamate synapses, with a putative recruitment of NMDAR with slower decay kinetics. To specifically test whether GluN2B-NMDAR are recruited following corticosterone exposure, Ro 25–6981 (potent and selective activity-dependent blocker of GluN2B-NMDAR) was bath-applied and the corticosterone-induced changes in NMDAR mEPSCs were measured (Fig. S1). In the presence of Ro 25–6981 corticosterone administration did not alter NMDAR-mEPSCs (Fig. 2G–J), indicating that corticosterone rapidly promotes GluN2B-NMDAR mediated current. Noteworthy, the access resistance was not different between vehicle (23.6 ± 1.7 MΩ) and corticosterone (24.1 ± 2.1 MΩ) treated cells or between Ro-25 (21.3 ± 2.3 MΩ) and Ro-25+corticosterone (20.7 ± 1.6 MΩ) treated cells.

Figure 2 Corticosterone increases amplitude and charged area of NMDAR-mediated mEPSCs. (A) Representative traces of NMDAR mEPSCs after vehicle (<0.1% ethanol) and corticosterone (100 nM), in the presence or absence of Ro25–6981. Scale bar: horizontal 200 ms, vertical 10 pA. (B) Comparison of the inter-event interval, amplitude, decay time and area of NMDAR mEPSCs. mEPSCs were recorded in buffer without (vehicle, n = 14 cells) and with corticosterone (n = 19 cells, red bars). In a separate series of experiments, NMDAR mEPSCs were recorded in the presence of Ro 25–6981 (3 μM, n = 9 cells) or Ro 25–6981 (3 μM) plus corticosterone (100 nM) (n = 7 neurons). *p < 0.05, unpaired Student t-test. Full size image

Corticosterone rapidly alters the surface dynamics of NMDAR

One possible mechanism underlying the corticosterone-induced increase in GluN2B-NMDAR current is an alteration of the receptor trafficking. Indeed, corticosterone acutely modulates the surface dynamics of another glutamate receptor, i.e. the AMPA type25, 26. Furthermore, the synaptic pool of NMDAR depends on the receptor trafficking27. Once at the plasma membrane, NMDAR diffuse within the plasma membrane, a process that continuously provides receptor to synaptic areas28, 29. To test whether corticosterone acutely modulates surface NMDAR in live hippocampal neurons, we first tracked endogenous surface GluN1 subunit (obligatory subunit of NMDAR) using a single nanoparticle detection approach30, 31 (Fig. 3A). In the buffer (PBS) condition, most GluN1-NMDAR exhibited a lower trafficking within glutamate synapse areas when compared to the extrasynaptic compartment (Fig. 3B), likely due to the anchoring by synaptic scaffold proteins32, 33. Strikingly, corticosterone (50 nM, 20 min) reduces the surface dynamics of GluN1-NMDAR in the synaptic (Fig. 3B). In presence of corticosterone, GluN1-NMDAR synaptic dwell-time (time spent within the postsynaptic area) was significantly increased and the diffusion coefficients of synaptic GluN1-NMDAR consistently decreased (Fig. 3C,D). The diffusion down-regulation was mostly explained by the up-shift of the immobile fraction, indicating a higher fraction of immobile NMDAR. Together, these data indicate thus that corticosterone rapidly reduces the surface dynamics of NMDAR within glutamate synapses, favoring their active retention and anchoring.

Figure 3 Corticosterone decreases the surface dynamics of GluN1-NMDAR in hippocampal neurons exposed to corticosterone. (A) Schematic representation of antibody against GluN1 subunit and single QD complex used to label and track surface NMDAR. (B) Representative trajectories of single GluN1-NMDAR in control (buffer, blue) and corticosterone (100 nM, 20 min; red). Note that the traces represent different receptors. The black arrows point toward spines in which glutamatergic synapses were identified. Lower panels, enlarged trajectories located within the postsynaptic densities (gray areas). Starting and ending time of the single trajectories are indicated as for instance time 0 (t 0s ). (C) Comparison of the synaptic dwell-time (expressed in seconds) of surface GluN1-NMDAR in buffer (n = 55 trajectories) or corticosterone (n = 62 trajectories) condition. ***p < 0.001, Student t-test. (D) Comparison of the cumulative distributions of GluN1-NMDAR instantaneous diffusion coefficients in buffer (n = 172 trajectories) and corticosterone (n = 190 trajectories) conditions. ***p < 0.001, Kolmogorov-Smirnov test. Full size image

Corticosterone specifically alters GluN2B-NMDAR surface dynamics and synaptic stabilization through a MR-dependent mechanism

As shown above, corticosterone alters the surface distribution of GluN2B-NMDAR and the surface dynamics of GluN1-NMDAR (Figs 1 and 3). One may suggest that the corticosterone-induced GluN2B-NMDAR redistribution relies on a change in GluN2B-NMDAR surface dynamics. To test this possibility, we tracked single GluN2B-NMDAR at the surface of live hippocampal neurons. Consistently, corticosterone exposure (50 nM, 20 min) significantly decreased GluN2B-NMDAR surface diffusion whereas no change was observed for GluN2A-NMDAR diffusion (Fig. 4A,B).

Figure 4 Corticosterone specifically decreases the surface dynamics of GluN2B-NMDAR in hippocampal neurons exposed to corticosterone. (A) Representative trajectories of single GluN2A- and GluN2B-NMDAR in hippocampal neurons (DIC images) exposed to either buffer or corticosterone (100 nM, 20 min). Scale bar, 5 µm; scale bar inset, 1 µm. (B) Comparison of the cumulative distributions of GluN2A- (buffer, n = 112 trajectories; corticosterone, n = 143 trajectories) and GluN2B-NMDAR (buffer, n = 189 trajectories; corticosterone, n = 191 trajectories) instantaneous diffusion coefficients in buffer and corticosterone conditions. ***p < 0.001, Kolmogorov-Smirnov test. Full size image

To dissect the upstream event that lead to this rapid redistribution of surface GluN2B-NMDAR after corticosterone exposure, we investigated whether a MR agonist (aldosterone, 10 nM, 20 min) was able to produce similar effects. Indeed, it was previously demonstrated in hippocampal neurons that corticosterone rapidly alters the surface trafficking of AMPA receptor through a MR-like pathway25, 26. Aldosterone exposure produced a rapid and stable increase in the fluorescence of GluN2B-NMDAR surface clusters (Fig. 5A–C), similar to the time-course and magnitude of the effect observed after corticosterone (Fig. 1A). This suggests that corticosterone effect on GluN2B-NMDAR surface distribution is mediated by a MR-like signaling pathway. Surface GluN2B-NMDAR were then tracked using single QD imaging in absence or presence of aldosterone. A rapid and significant reduction of GluN2B-NMDAR surface diffusion, similar to the effect produced by corticosterone, was observed with aldosterone (Fig. 5D,E). To test whether these effects were specific to the MR signaling, neurons were exposed to a GR agonist, RU28362 (50 nM). In contrast to aldosterone, RU28362 did not alter GluN2B-NMDAR surface dynamics both 5 and 20 min after GR activation (Fig. 5D,E). Because corticosterone can rapidly cross the plasma membrane and act intracellularly on signaling cascade, we exposed neurons to corticosterone coupled to bovine serum albumin (BSA) (50 nM, 20 min), which is a membrane non-permeate active analog of corticosterone. Strikingly, corticosterone-BSA rapidly decreased the surface diffusion of GluN2B-NMDAR with a significant and maximal effect already observed 5 min after exposure (Fig. 5D,E). Collectively, these data demonstrate that the GluN2B-NMDAR surface dynamics is rapidly and strongly affected by corticosterone through a membrane MR-like signaling pathway.

Figure 5 Aldosterone, a MR activator, alters the surface distribution and dynamics of GluN2B-NMDAR. (A) Dendritic fragments of GluN2B-SEP expressing neurons before and after exposure to aldosterone (10 nM). Scale bar = 5 µm, scale bar inset = 1 µm. (B) Example of fluorescence intensity of GluN2B-SEP clusters over time, before and after aldosterone application. (C) Comparison of GluN2B-SEP cluster fluorescence intensity before and after exposure to aldosterone (10 nM, 25 min exposure; n = 10 dendritic fields, N = 5 neurons). ***p < 0.001, paired Student t-test. (D) Representative trajectories of single GluN2B-NMDAR in hippocampal neurons (DIC images) exposed to either buffer solution, aldosterone (10 nM, 20 min), corticosterone BSA (50 nM, 20 min), or RU28392 (GR agonist, 50 nM, 20 min). Scale bar, 5 µm. (E) Comparison of the cumulative distributions of GluN2B-NMDAR instantaneous diffusion coefficients between aldosterone (buffer, n = 214 trajectories; aldosterone, n = 225 trajectories), corticosterone BSA (buffer, n = 539 trajectories; 5min corticosterone BSA, n = 855 trajectories; 20min corticosterone BSA, n = 864 trajectories), or RU28392 (buffer, n = 229 trajectories; 5min RU28392, n = 175 trajectories; 20min RU28392, n = 119 trajectories). ***p < 0.001, Kolmogorov-Smirnov test. Full size image

Finally, the effect of corticosterone on the GluN2B-NMDAR synaptic content could originate from a higher retention of the receptors within the synapse. Indeed, we previously showed that changes in the synaptic surface dynamics of surface GluN2-NMDAR alter their synaptic content15, 33,34,35. Few minutes after corticosterone exposure, GluN2B-NMDAR synaptic diffusion was significantly reduced, consistent with a higher anchoring of the receptors in the synaptic area (Fig. 6A,B). This effect could be mimicked by the application of aldosterone (10 nM), consistent with the implication of a MR-like signalling in this synaptic retention of GluN2B-NMDAR (Fig. 6A). Noteworthy, the effect of corticosterone and aldosterone on synaptic GluN2B-NMDAR was higher than the ones observed for extrasynaptic GluN2B-NMDAR (e.g. corticosterone effect on synaptic receptor, −95%, and extrasynaptic receptor, −64%) synaptic effect of corticosterone (Figs 4, 5 and 6). Although the mechanism is still undefined, the stabilization of GluN2B-NMDAR by MR-related kinase signalling is likely36. Together, these data unravel that corticosterone rapidly activates MR, leading to an efficient postsynaptic anchoring of GluN2B-NMDAR.

Figure 6 Corticosterone and aldosterone decrease GluN2B-NMDAR surface dynamics within synapses. (A) Representative trajectories of surface GluN2B-NMDAR in hippocampal synapses exposed to either buffer solution, aldosterone (10 nM, 20 min), or corticosterone (100 nM, 20 min). Scale bar, 200 nm. (B) Comparison of the synaptic GluN2B-NMDAR instantaneous diffusion coefficients between buffer and corticosterone (buffer, n = 141 trajectories; corticosterone, n = 138 trajectories), and buffer and aldosterone (buffer, n = 291 trajectories; aldosterone, n = 402 trajectories). Data are expressed as median diffusion coefficient ±25–75% IQR. *p < 0.05, two-tailed Mann-Whitney test. Full size image

Corticosterone-induced AMPAR synaptic potentiation requires NMDAR surface redistribution

The fast remodeling of synaptic GluN2B-NMDAR triggered by corticosterone might theoretically impact the synaptic long-term adaptations observed after stress hormone exposure. Among these, it is well-documented that corticosterone induces long-term potentiation (LTP) of AMPAR in hippocampal synapses, which occlude classical NMDAR-dependent theta-burst and tetanus-induced LTP37. At the cellular level, this effect is mediated by an increased trafficking of AMPAR toward and within the plasma membrane25, 26. Since corticosterone rapidly primes glutamate synapse for potentiation38 and since a decrease in the GluN2A/GluN2B ratio favors LTP11, we directly tested the intriguing possibility that the surface redistribution of GluN2B-NMDAR observed after corticosterone play an instrumental role in triggering corticosterone-induced AMPAR synaptic potentiation. For this, live hippocampal neurons were incubated with corticosterone for 20 min and the synaptic retention of single GluA1-AMPAR (GluA1-QD) was estimated 90 min later in conditions in which NMDAR were either free to diffuse or immobilize by x-linking34 (Fig. 7A). Consistent with previous report38, corticosterone increased the relative content of synaptic GluA1-AMPAR when measured 1.5 h after exposure (Fig. 7A,B). Strikingly, reducing the surface diffusion of NMDAR by x-link fully prevented the corticosterone-induced AMPAR synaptic potentiation (Fig. 7B,C). Together, these data provide thus the first evidence that the well-documented effect of corticosterone on synaptic potentiation requires, among the first steps, a rapid redistribution of surface NMDAR.