Primary hippocampal cultures and transfection
All animal experiments were approved by the Institutional Animal Care and Use Committee of the Karolinska Institutet. Primary hippocampal cultures were prepared from E18 Sprague Dawley embryos of either sex as previously described [10] with the following modification: twice a week, half the media was changed to Neurobasal (Invitrogen) with 2% B27 (Invitrogen), 1% penicillin/streptomycin and 0.5 mM L-glutamine (Sigma-Aldrich). After 20–22 days in culture, cells were transfected using Lipofectamine 2000 (Invitrogen). 24–48 hours after transfection cells were collected for experiments.
Constructs: Norbin-GFP, mGluR5-mCherry and mCherry [2], PSD-95-mCherry and PSD-95-BFP [10, 11], Homer-DsRed (kindly provided by Dr. Daniel Choquet) and LifeAct-mCherry [12] (Addgene, plasmid 40908).
Immunocytochemistry
Cells were rinsed briefly in Krebs Ringer Buffer, consisting of, in mM, 110 NaCl, 4 KCl, 1 NaH2PO4 · H2O, 25 NaHCO3, 1.5 CaCl2 · H2O, 1.2 MgCl2 · 6H2O, 10 D-glucose and 20 Hepes, and fixated with 4% paraformaldehyde (Sigma-Aldrich) in PBS for 10 minutes at room temperature. Cells were rinsed with PBS followed by 2 minutes of permeabilization using 0.2% Triton X-100 (Sigma-Aldrich) in PBS. Blocking was done with 10% normal goat serum (NGS, Jackson ImmunoResearch Laboratory Inc.) for 1 hour, followed by incubation with primary antibodies diluted in PBS with 5% NGS for 1 hour at room temperature. After repeated rinsing, cells were incubated with secondary antibodies in PBS with 5% NGS for 1 hour at room temperature. Cells were rinsed repeatedly and mounted in Prolong Gold antifade reagent (Invitrogen).
Antibodies
Primary antibodies: anti-Norbin rabbit polyclonal antibody which specificity has been tested in Norbin knockout mice [2] (kind gift from Prof. Paul Greengard), anti-PSD-95 mouse monoclonal, (1:500, Abcam), anti-PSD-95 rabbit polyclonal (1:500, Abcam) and anti-actin mouse monoclonal (1:3000, BD Transduction Laboratories). Secondary antibodies: Abberior STAR 440SX goat anti-mouse IgG (1:50, Abberior), Abberior STAR 512SX goat anti-rabbit IgG (1:50, Abberior), Alexa Fluor 488 goat anti-rabbit (1:500, Invitrogen) and Alexa Fluor 568 goat anti-mouse (1:500, Invitrogen).
Confocal imaging
Imaging and photobleaching was done on a Leica TCS SP5 CW microscope with a 63x/1.4 NA oil objective (Leica). 488 nm and 561 nm excitation wavelengths were used and detection was done at 495–555 nm and 570–650 nm in LAS AF software (Leica). For simultaneous FRAP measurements 128 × 128 pixel images were acquired of spines or 2.5 μm dendritic segments with a pinhole of 2 airy units as follows (image acquisition interval): 5 frames baseline (0.5 s), 5 frames bleach (0.21 s), 5 frames post-bleach (0.21 s) and 10 frames post-bleach (5 s). Cells were kept in KREBS solution at 37°C. Analysis was done in a custom written Matlab (The MathWorks) script. The mobile pool was calculated as the mean of the last two measured intensity values. The half time of recovery was derived from a linear fit between the two measured intensity values closest to 50% the mobile pool.
For intensity comparisons between spines and dendrites in Norbin-GFP and mCherry co-transfected neurons, z-stacks of 512 × 512 pixel images were acquired, with a step size of 0.38 μm and a pinhole of 1 airy unit. Line regions over spine heads and adjacent dendritic segments were selected in maximum projections of the z-stacks and the mean intensities were calculated using ImageJ (http://rsbweb.nih.gov/ij/).
3D structured illumination microscopy (3D-SIM)
Transfected cells were fixated in 4% paraformaldehyde and mounted in Prolong Gold. 3D-SIM imaging was performed using a Plan-apochromat 63X/1.4 NA oil objective on an ELYRA PS.1 (Carl Zeiss) microscope. Excitation wavelengths were 488 nm and 561 nm, with detection at 495–575 nm and 570–650 nm respectively. In the case of triple labeling an additional wavelength of 405 nm was used with detection at 420–480 nm. 1002 × 1004 pixel images, averaging over 4 frames were acquired in 3 rotations. Final images were reconstructed using ZEN 2011 software (Carl Zeiss). The ELYRA PS.1 system was calibrated using fluorescent beads (40 nm), yielding a lateral resolution of <100 nm and an axial resolution of ~275 nm.
STED
EasySTED [13] was extended to two colors for this work. In brief, excitation light from synchronized 470 and 510 nm pulsed diodes (LDH-P-C-470B/-510B, Picoquant) was combined with depletion light from a 592 nm continuous wave laser (MPBC), coupled via an optical fiber (P1-488 PM-FC-2, Thorlabs) to a beam scanner (YANUS, Till Photonics) into the microscope stand (DMI6000CS, Leica Microsystems) equipped with a 100x/1.4 Oil objective lens (Leica). Underneath the objective lens, a segmented wave plate (Abberior GmbH) selectively re-shaped the 592 nm depletion beam into a bright ring with a central intensity zero. Emission was separated by a 550/49 nm beam splitter (AHF) and guided to an avalanche photo diode (SPCM-AQRH-14-FC, Perkin-Elmer) via a multi-mode fiber (Thorlabs, ~1.5 Airy units). A notch filter for 594 nm (AHF) suppressed residual STED light. Excitation light pulses were alternating between 470 and 510 nm, providing excitation for the two channels in sequence. The signal from the detector was for each excitation pulse separated into two channels by time gated detection in (electronics by MPI for Biophysical Chemistry, Göttingen). To further increase resolution and reduce background, the ~5 ns wide detection windows were delayed ~1 ns with respect to the respective excitation pulses [14]. Resolution was assessed by fitting a Gaussian function to line profiles of spots in the Norbin channel, with ~40 nm full width at half maximum (FWHM) (Figure 1C,D). We used a PCI-6259 board (National Instruments) and the software Imspector (MPI) to drive the scanner and for data acquisition.
Co-immunoprecipitation and PSD fractions
Co-immunoprecipitation experiments were performed on hippocampi lysate from Sprague Dawley rats as previously described [15] using antibodies for Norbin, actin and PSD-95. PSD fractions were prepared as previously described [16]. Briefly, brain homogenate was fractionated by differential centrifugation and the PSD fraction was obtained through centrifugation in a sucrose gradient. The proteins were separated on a bis-tris gradient gel, transferred to a membrane and immunoblotted against PSD-95 and Norbin.
Statistical analysis
Non-parametric Wilcoxon rank sum test was used to test the significance of intensity differences between Norbin and mCherry in dendrites and spines. For analyzing colocalization using Pearson’s correlation coefficient (PCC), we used custom software written in Matlab. Individual spines were selected and cropped, normalized and thresholded using Otsu’s method. The PCC for the correlation between the two channels was then computed. FRAP and PCC data was analyzed using non-parametric Kruskal-Wallis test with Bonferroni’s correction for multiple comparisons.