Care and handling of animals were in accordance with NIH Animal Care and Use Committee Guidelines. Postnatal day (P) 15-21 Sprague-Dawley rats, injected with 1-1.2% CTB bilaterally through the superior colliculus, were maintained on a 12:12-hr light:dark cycle.
1. Retinal Tissue Fixation
2. Freeze-substitution
NOTE: This freeze-substitution method is modified from an earlier published protocol 19,20. Also, it is crucial that the instruments are very cold (wear gloves); otherwise, the tissue may thaw partially when touched with the instruments. All of these steps are done within the AFS chamber and the instruments are never allowed to move above the rim of the chamber. Similarly, proper cooling of all chemicals used in the AFS is necessary.
3. Postembedding EM Immunogold Immunocytochemistry
NOTE: Postembedding immunocytochemistry is performed as described 23,24,25.
4. Quantification
The results presented here demonstrate strikingly different subsynaptic localization patterns of GluA 2/3 and NMDARs on RGC dendrites in rat retina, as described previously 24,25. 77% of GluA 2/3 immunogold particles in RGC dendritic profiles were located within the PSD (Figure 1A), similar to most central synapses. However, NMDARs were located either synaptically or extrasynaptically. 83% of GluN2A immunogold particles were localized in the PSD (Figure 1C, 2A, 2B), preferentially at OFF synapses, and the gold particles were more concentrated at the center of the PSD (Figure 2C). In contrast, the large majority of particles labeling for GluN2B (96%) were located outside the PSD (Figure 1B, 2A, 2B), distributed primarily along the extrasynaptic plasma membrane, with the peak particle density at 180 nm from the edge of the PSD (Figure 2B). In particular, GluN2B exhibited a preference for ON synapses. In a subset of experiments, simultaneous labeling of GluN2A, PSD-95 and CTB indicated that GluN2A and PSD-95 were colocalized in the PSD of OFF RGC dendrites (Figure 1D), where NR2A was in the dendritic membrane while PSD-95 was beneath the membrane, suggesting that they are anchored at the PSD. A significant difference in gold density between perisynaptic membrane and mitochondrial membranes was observed, suggesting a specific labeling in the former (Figure 2D).
Figure 1. Immunogold Labeling Showing Synaptic and Perisynaptic Localization of Glutamate Receptors at RGC Dendrites Labeled by CTB. (A): double immunogold labeling of GluA2/3 (10 nm gold) and CTB (18 nm gold). Presynaptic ribbons indicated by arrowheads. Small gold particles are clustered (arrow) in the PSD of RGC processes. (B) double immunogold labeling of GluN2B (10 nm gold) and CTB (18 nm gold); small gold particles (arrow) are on the extrasynaptic plasma membrane. (C) double immunogold labeling of GluN2A (10 nm gold) and CTB (18 nm gold). Similar to GluA2/3, small particles are clustered within the PSD. (D) Triple immunogold labeling of GluN2A (5 nm gold), PSD-95 (10 nm gold) and CTB (18 nm gold). GluN2A gold particles (small arrows) and PSD-95 gold particles (large arrows) are co-localized within the PSD on individual CTB-positive RGC dendrites. Inset: higher magnification of the PSD. Scale bar: 0.1 µm (A = C = D, B). These are new micrographs based on data published in Zhang and Diamond 24,25. Please click here to view a larger version of this figure.
Figure 2. Quantitative Comparison of NMDAR Localization in RGC Dendrites. (A) A histogram showing the tangential distribution of immunogold for GluN2A (n = 53 profiles ) and GluN2B (n = 56 profiles ) within and outside the PSD. The perisynaptic region was divided into 60 nm bins. (B) A histogram showing labeling density of immunogold particles for GluN2A (n = 53 profiles) and GluN2B (n = 56 profiles). The perisynaptic region was divided into 180 nm bins from the edge of the PSD. (C) A histogram showing the tangential distribution of the total number of immunogold particles for GluN2A (n = 44 profiles) and GluN2B (n = 3 profiles) at all profiles with labeling within the PSD. (D) Comparison of particle density of immunogold particles for GluN2B (n = 53 and 33 profiles) and GluN2A (n = 9 and 30) in the perisynaptic membranes and mitochondrial membrane, respectively, in the CTB-positive RGC processes. These histograms are modified from histograms published in Zhang and Diamond 24,25. Please click here to view a larger version of this figure.
Paraformaldehyde | EMS | 15710 | |
Glutarldehyde | EMS | 16019 | |
NaH2PO4 | Sigma | S9638 | |
Na2HPO4 | Sigma | 7782-85-6 | |
CaCl2 | Sigma | C-8106 | |
BSA | Sigma | A-7030 | |
Triton X-100 | Sigma | T-8787 | |
NaOH | Sigma | 221465 | |
NaN3 | JT Baker | V015-05 | |
Glycerol | Gibco BRL | 15514-011 | |
Lowicryl HM 20 | Polysciences | 15924-1 | |
Tris-Base | Fisher | BP151-500 | |
Tris | Fisher | 04997-100 | |
Anti-GluN2A | Millipore | AB1555P | Dilution 1/50 |
Anti-GluN2B | Millipore | AB1557P | Dilution 1/30 |
Anti-GluA2/3 | Millipore | AB1506 | Dilution 1/30 |
Anti-PSD-95 | Millipore | MA1–046 | Dilution 1/100 |
Donkey anti-rabbit IgG-10 nm gold particles | EMS | 25704 | Dilution 1/20 |
Donkey anti-mouse IgG-10 nm gold particles | EMS | 25814 | Dilution 1/20 |
Donkey anti-mouse IgG-5 nm gold particles | EMS | 25812 | Dilution 1/20 |
Donkey anti-goat IgG-18 nm gold particles | Jackson ImmunoResearch | 705-215-147 | Dilution 1/20 |
Formvar-Carbon coated nickel-slot grids. | EMS | FCF2010-Ni | |
Uranyl acetate | EMS | 22400-1 | |
Methanol | EMS | 67-56-1 | |
Lead citrate | Leica | ||
Leica EM AFS | Leica | ||
Leica EM CPC | Leica | ||
Ultromicrotome | Leica | ||
JEOL 1200 EM | JEOL | ||
liquid nitrogen | Roberts Oxygen | ||
Propane | Roberts Oxygen | ||
CTB | List Biological Laboratories | 104 | 1-1.2% |
Anti-CTB | List Biological Laboratories | 703 | Dilution 1/4000 |
Retinal ganglion cells (RGCs) receive excitatory glutamatergic input from bipolar cells. Synaptic excitation of RGCs is mediated postsynaptically by NMDA receptors (NMDARs) and AMPA receptors (AMPARs). Physiological data have indicated that glutamate receptors at RGCs are expressed not only in postsynaptic but also in perisynaptic or extrasynaptic membrane compartments. However, precise anatomical locations for glutamate receptors at RGC synapses have not been determined. Although a high-resolution quantitative analysis of glutamate receptors at central synapses is widely employed, this approach has had only limited success in the retina. We developed a postembedding immunogold method for analysis of membrane receptors, making it possible to estimate the number, density and variability of these receptors at retinal ribbon synapses. Here we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of retinal fixation, 2) freeze-substitution, 3) postembedding immunogold electron microscope (EM) immunocytochemistry and, 4) quantitative visualization of glutamate receptors at ribbon synapses.
Retinal ganglion cells (RGCs) receive excitatory glutamatergic input from bipolar cells. Synaptic excitation of RGCs is mediated postsynaptically by NMDA receptors (NMDARs) and AMPA receptors (AMPARs). Physiological data have indicated that glutamate receptors at RGCs are expressed not only in postsynaptic but also in perisynaptic or extrasynaptic membrane compartments. However, precise anatomical locations for glutamate receptors at RGC synapses have not been determined. Although a high-resolution quantitative analysis of glutamate receptors at central synapses is widely employed, this approach has had only limited success in the retina. We developed a postembedding immunogold method for analysis of membrane receptors, making it possible to estimate the number, density and variability of these receptors at retinal ribbon synapses. Here we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of retinal fixation, 2) freeze-substitution, 3) postembedding immunogold electron microscope (EM) immunocytochemistry and, 4) quantitative visualization of glutamate receptors at ribbon synapses.
Retinal ganglion cells (RGCs) receive excitatory glutamatergic input from bipolar cells. Synaptic excitation of RGCs is mediated postsynaptically by NMDA receptors (NMDARs) and AMPA receptors (AMPARs). Physiological data have indicated that glutamate receptors at RGCs are expressed not only in postsynaptic but also in perisynaptic or extrasynaptic membrane compartments. However, precise anatomical locations for glutamate receptors at RGC synapses have not been determined. Although a high-resolution quantitative analysis of glutamate receptors at central synapses is widely employed, this approach has had only limited success in the retina. We developed a postembedding immunogold method for analysis of membrane receptors, making it possible to estimate the number, density and variability of these receptors at retinal ribbon synapses. Here we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of retinal fixation, 2) freeze-substitution, 3) postembedding immunogold electron microscope (EM) immunocytochemistry and, 4) quantitative visualization of glutamate receptors at ribbon synapses.