GFP-SAP102 and GFP-actin was described previously (Rzadzinska et al., 2004
; Sans et al., 2005
). GFP-PSD-95 and GFP-C3, 5S-PSD-95 were gifts from Dr. David S. Bredt (UCSF, San Francisco, CA); DsRed (pDsRed-Monomer-C1) was a gift from Dr. Zu-Hang Sheng (NIH, Bethesda, MD). GFP (pEGFP-N3) was purchased from Clontech, Mountain View, CA. GFP-SAP102ΔSH3GK, GFP-SAP102ΔSH3 and GFP-SAP102ΔGK were generated by mutating Y483 to a stopcodon (TAT to TAG), deleting the regions between 526A and 585V, and mutating E589 to a stopcodon (GAG to TAG), respectively. GFP-SAP102PDZ1FH encompassed the mutation 161 F →H, GFP-SAP102PDZ2FH had 256 F→H, and GFP-SAP102PDZ3FH had 416 F→H. Each of the three mutants abolishes the PDZ binding between a PDZ domain and the C-terminal carboxylate group of group1 PDZ ligands (Doyle et al., 1996
; Regalado et al., 2006
A mouse monoclonal antibody to PSD-95 was purchased from ABR (Rockford, IL; clone 7E3-1B8) and was used at 1:100 for immunofluorescence. A rabbit polyclonal antibody to SAP102 (JH62514) was a gift from Johannes Hell and was used at 1:3000 for immunofluorescence. The PSD-95 antibody that was used for immunogold staining was from BD Bioscience (San Jose, CA). All of these antibodies were characterized and compared previously (Sans et al., 2000
; Davies et al., 2001
All animal procedures were done in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH publication 85-23) under NIDCD protocol #1167-07. Hippocampal neurons were cultured on coverlips from E18 fetal rat hippocampus (Sans et al., 2001
). The neurons were transfected on 17 days in vitro (DIV) using CalPhos Mammalian Transfection Kit (Clontech, Palo Alto, CA) (Sans et al., 2003
). Three to four days later, neurons were fixed with a 1:1 mixture of methanol and acetone at −20°C for 4 minutes. Then on ice, neurons were washed with PBS, permeablilized in 0.25% Triton-X-100 for 5 minutes, blocked in PBS containing 10% normal goat serum (NGS) for 1 hour, incubated with primary antibody in PBS containing 3% NGS and 0.1% Triton-X-100 (N-T-PBS) for 1 hour, washed with PBS, incubated with FITC- or CY3- conjugated secondary antibody in N-T-PBS for 30 minutes, and then washed again with PBS. Finally, the coverslips were mounted using ProLong Gold Antifade reagent (Invitrogen, Carlsbad, CA), and imaged using an UltraVIEW ERS confocal Microscope (PerkinElmer, Fremont, CA). When labeled by SAP102 or PSD-95 antibody, the average immunofluorescence intensity of GFP-SAP102 or GFP-PSD-95 transfected neurons was estimated to be 2 times as bright as that of the non-transfected neighboring neurons.
Postembedding immunogold labeling was based on established methods (Petralia and Wenthold, 1999
; Petralia et al., 2003
; Petralia et al., 2005
; Yi et al., 2007
). Briefly, rats were perfused with 4% paraformaldehyde plus 0.5% glutaraldehyde, and sections were cryoprotected and frozen in a Leica EM CPC (Vienna, Austria), and further processed and embedded in Lowicryl HM-20 resin using a Leica AFS freeze-substitution instrument. Thin sections were incubated in 0.1% sodium borohydride+50 mM glycine/Tris-buffered saline + 0.1% Triton X-100 (TBST), followed by 10% NGS in TBST, and primary antibody in 1% NGS/TBST overnight, and then immunogold labeling in 1% NGS in TBST plus 0.5% polyethylene glycol (20,000 MW). Finally, sections were stained with uranyl acetate and lead citrate. For double labeling, the 2 primary antibodies were applied together and so were the 2 secondary antibodies. Corresponding controls, lacking the primary antibody, showed only rare gold labeling. Images were stored in their original formats and final images for figures were prepared in Adobe Photoshop: levels and brightness/contrast of images were minimally adjusted, evenly over the entire micrograph.
Hippocampal neurons were cultured on poly-L-lysine coated glass-bottom dishes (MatTek, Ashland, MA) at E18, and then transfected on 16~18 DIV. Neurons were used for FRAP experiment 4 days after transfection. The culture medium was exchanged with pre-warmed Tyrode Solution before experiments. Tyrode Solution contained (in mM) NaCl 145, KCl 5, HEPES 10, Glucose 10, Glycine 0.005, CaCl2 2.6, and MgCl2 1.3 (pH adjusted to 7.4 with NaOH). Temperature was kept at 37°C using a Zeiss TempModule system. Depending on the experiments, 10 μM Jasplakinolide (EMD, La Jolla, CA), 100 μM DL-APV and 20 μM NBQX-disodium salt hydrate (Tocris, Ellisville, MO) were added into Tyrode Solution 30–60 minutes before FRAP recording and during FRAP recording. Control coverslips were incubated with prewarmed Tyrode Solution or 1% DMSO instead of the drugs.
All images were captured using a Zeiss LSM510 confocal microscope with a 100x objective, using 5x optical zoom and a 256×256 pixel resolution. The pinhole was set to 1000 and the 488 laser output was 70%. Spines of interest were bleached 10 times at 100% laser transmission. These conditions are sufficient to bleach the fluorescence of a spine to background level in a fixed preparation. During our FRAP measurements, we used speed 9 which took 0.5 second to finish a scan. A series of images was captured before and immediately after photobleaching with 3% laser transmission. Five images were captured before bleaching; their average fluorescence intensity was set to 100%. For most experiments, images were captured every 3 sec for the first minute after bleaching, and then at 2, 3, 4, 5, 10, 15 and 20 minutes.
ImageJ software (NIH, Bethesda, MD) was used for aligning the images and measuring fluorescence intensity of the region of interest in time-lapse photography. For each movie, the mean intensity of an untransfected area was measured as background and was subtracted from the original intensity. Graphpad Prism software (Graphpad software, La Jolla, CA) was used for curve fitting.
We divided the total fluorescence of spine proteins into the immobile fraction (fi
) and the mobile fraction (fm
). The mobile fraction was divided into a slow mobile fraction (fs
) and a rapid mobile fraction (fr
)(Star et al., 2002
The immobile fraction is:
The mobile fraction of the spines is described as:
represents the fluorescence of region of interest after full recovery from bleaching, Fb
represents the background fluorescence intensity, and F0
is the mean fluorescence before bleaching.
The slow mobile fraction is reflected by FRAP during a 20 minute period, curve fit by one-phase exponential equations:
The rapid mobile fractions were detected immediately after photobleaching. According to equation (1)
they could be given by the equation:
The half recovery time (τ1/2) of the rapid fraction was calculated by Graphpad Prime software, using the total binding model.
The half recovery time of a freely diffusing protein in spines is calculated by comparing the target protein to pEGFP. pEGFP, which is presumable to be freely diffusing in the cytoplasm, had a τ1/2
=0.9 seconds in our system. τ1/2
is related to the cube root of the protein size (Star et al., 2002
). Therefore, free diffusion of GFP-SAP102 and GFP-PSD-95 both would have a τ1/2
= 1.4 seconds, since they had similar molecular weights of 113 and 109 kD. τ1/2
of the free diffusional GFP-SAP102ΔSH3GK (80 kD) would be 1.2 second in our system.
The immunochemistry data were analyzed using MetaMorph software (Universal Imaging Corporation, West Chester, PA). For spine/dendrite ratio analysis, mushroom spines of secondary dendrites and adjacent dendrite shaft regions were defined under the DsRed channel. Then under the GFP channel, the regions of interest were reloaded. The mean intensity of GFP tagged protein in spine regions and dendrite regions was measured. In each neuron, a minimum number of 10 pairs of spines and adjacent dendrite regions from at least 2 dendrites were used for determining a mean spine/dendrite ratio. In total, 5–15 pyramidal neurons from 2–3 parallel transfections were analyzed. Statistical significance between two data sets was calculated using a Student’s t-test.
The perpendicular distance from the center of immunogold particle to the postsynaptic membrane was measured using ImageJ software. The distribution of the gold particles was analyzed by IGOR pro software (WaveMetrics, Portland, OR). Gold particles were included that were completely or partially over the synapse. The gold particles between pre- and post- synaptic membrane were given a negative number. Presynaptic particles were not included.