Corticosterone alters AMPAR mobility and facilitates bidirectional synaptic plasticity.

Methodology/Principal Findings Using immunocytochemistry and live cell imaging techniques we show that corticosterone selectively increases surface expression of the AMPAR subunit GluR2 in primary hippocampal cultures via a glucocorticoid receptor and protein synthesis dependent mechanism. In agreement, we report that corticosterone also dramatically increases the fraction of surface expressed GluR2 that undergo lateral diffusion. Furthermore, our data indicate that corticosterone facilitates NMDAR-invoked endocytosis of both synaptic and extra-synaptic GluR2 under conditions that weaken synaptic transmission.

Materials and Methods Dispersed hippocampal neuronal cultures and immunocytochemistry

The experiments were carried out with permission of the local Animal Committee of the Erasmus Medical Center and University of Bristol. Primary hippocampal cultures were prepared from embryonic day 18 (E18) rat brains as described [14] . Cells were plated on coverslips coated with poly-D-lysine (30 µg/ml) and laminin (2 µg/ml) at a density of 75,000/well. Hippocampal cultures were grown in Neurobasal medium supplemented with B27, 0.5 mM glutamine, 12.5 µM glutamate and penicillin/streptomycin. At DIV13-20 hippocampal neurons were incubated with GluR1 (Calbiochem (1∶8) and GluR2 (Zymed (1∶80) N-terminal antibodies (10 µg/ml) at 37°C for 15 min [12] . After washing in DMEM medium, the neurons were fixed for 5 min with 4% formaldehyde/4% sucrose in phosphate-buffered saline (PBS). Neurons were then washed three times in PBS for 30 min at room temperature and incubated with secondary antibody conjugated to Alexa488 (1∶400) or Alexa568 (1∶400) in staining buffer without TritonX-100 (0.2% BSA, 0.8 M NaCl, 30 mM phosphate buffer, pH 7.4) overnight at 4°C. Neurons were then washed three times in PBS for 30 min at room temperature and mounted. For total staining cells were fixed for 5 min with 4% formaldehyde/4% sucrose in phosphate-buffered saline (PBS). Next, cells were incubated with GluR1 (1∶5000) and GluR2 C-terminal antibodies (1∶500) [15] in staining-buffer with TritonX-100 overnight at 4°C. Neurons were then washed three times in PBS for 30 min at room temperature and incubated with secondary antibody conjugated to Alexa488 (1∶400) or Alexa568 (1∶400) in GDB with TritonX-100 for 2 h at room temperature and washed three times in PBS for 30 minutes. Confocal images were obtained with sequential acquisition settings at the maximal resolution of the microscope (1024×1024 pixels). Morphometric analysis and quantification were performed using MetaMorph software (Universal Imaging Corporation). For details see supplementary materials and methods .

Image analysis and quantification

Confocal images stained neurons were obtained with sequential acquisition settings at the maximal resolution of the microscope (1024×1024 pixels). Each image was a z-series of 6–10 images each averaged 2 times. The resulting z-stack was ‘flattened’ into a single image using maximum projection. The confocal settings were kept the same for all scans when fluorescence intensity was compared. Morphometric analysis and quantification were performed using MetaMorph software (Universal Imaging Corporation). For the quantification of surface antibody staining, images were acquired with use of a 40× objective with 1.0× electronic zoom and the average intensity of the soma and dendrites was measured in MetaMorph. Acquisition of the images as well as morphometric quantification was performed under “blinded” conditions. Statistical analysis was performed with student's t test assuming a two-tailed and unequal variation. N defined as the number of quantified neurons.

Biotinylation assays

High-density hippocampal cultures were prepared as described previously (13After 14 DIV, neurons were treated with 100 nM CORT for 3 h or treated vehicle only and membrane and total fractions were prepared [16] . Immunoblotting was performed using a rabbit polyclonal antibody to GluR1 (Upstate; 0.6 µg/ml) and mouse monoclonal antibodies to GluR2 (Chemicon; 1 µg/ml), Transferrin receptor (Sigma 1 µg/ml), β-actin (Sigma; 0.5 µg/ml) and α-tubulin (Sigma; 0.5 µg/ml). Quantitative densitometric analysis was performed using NIH Image J.

Electrophysiology

Coverslips were placed in a recording chamber mounted on an upright microscope (Nikon E600FN), continuously perfused with artificial cerebrospinal fluid (aCSF) (32°C, 2–3 ml/sec; containing in (mM): NaCl (120), KCl (3.5), MgSO 4 (1.3), NaH 2 PO 4 (1.25), CaCl 2 (2.5), Glucose (10.0) and NaHCO 3 (25.0), pH 7.4) and kept fully submerged. Whole cell patch clamp recordings were made using an AXOPATCH 200B amplifier (Axon Instruments, USA), with electrodes from borosilicate glass(1.5 mm outer diameter, Hilgerberg, Malsfeld, Germany). The electrodes were pulled on a Sutter (USA) micropipette puller. The pipette solution contained (in mM): 120 Cs methane sulfonate; CsCl (17.5); HEPES (10); BAPTA (5); Mg-ATP (2); Na-GTP (0.5); QX-314 (10); pH 7.4, adjusted with CsOH; pipette resistance was between 3–6 MΩ. Under visual control (40× objective and 10× ocular magnification) the electrode was directed towards a neuron with positive pressure. Once sealed on the cell membrane (resistance above 1 GΩ) the membrane patch under the electrode was ruptured by gentle suction and the cell was kept at a holding potential of −70 mV. The liquid junction potential caused a shift of no more than 10 mV, which was not compensated during mEPSCs recording. Recordings with an uncompensated series resistance of