Immunofluorescence studies of extrasynaptic NMDARs in vitro
We first studied the organization of extrasynaptic NMDARs in cultures of hippocampal neurons at the LM level. We compared live surface labeling of native receptors using anti-NR2A and NR2B rabbit antibodies in cultures of hippocampal neurons at 3 WIV, with live surface labeling of transfected neurons at 2-3 WIV using anti-FLAG antibodies to detect flag-NR2A and flag-NR2B. In each case, in parallel immunolabeling was carried out with an antibody raised in guinea pigs against the presynaptic marker, VGLUT, and also antibodies raised in mice and directed against candidate proteins that are thought to associate with NMDA receptors, i.e. anti-PSD-95/93, SAP102, pan-cadherin, N-cadherin, and catenin antibodies.
For the native NMDA receptors, VGLUT immunolabeling indicated that ~ half of the puncta for both NR2A and NR2B were localized extrasynaptically (,). PSD-95/93 and SAP102 antibodies all showed substantial overlap (~ one third) with the extrasynaptic puncta of NR2A and NR2B labeling with no significant differences seen between NR2A and NR2B (). However, while both PSD-95/93 and SAP102 labeling indicated that these proteins are found outside synapses, the relative intensity of synaptic PSD-95/93 was greater than that observed at extrasynaptic sites whereas there was less difference in intensity between synaptic and extrasynaptic puncta of SAP102 (). This suggests that PSD-95/93 is more concentrated at synapses while SAP102 is more widespread.
Figure 1 Triple immunofluorescence labeling of hippocampal cultures using live surface labeling of NR2A or NR2B (555; red), followed by labeling for the presynaptic marker VGLUT (647; blue), and a third protein (488; green) including PSD-95/93, SAP102 (Neuromab), (more ...)
Figure 2 High magnifications of parts of micrographs (additionally processed for brightness and contrast) from triple immunofluorescence labeling of hippocampal cultures with 1) live surface labeling of NR2A (red; 555 for D,E; 647 [STED] for G,H) or NR2B (red-555; (more ...)
Antibodies against cadherin and catenin showed an interesting pattern of light to moderate labeling in the dendrites and dense labeling in glial processes, which could be seen to enwrap the dendrites. Dendritic labeling, as for PSD-95 and SAP102, showed substantial overlap (~ one third) with the extrasynaptic puncta of native NR2A and NR2B labeling with no significant differences seen between NR2A and NR2B.
We also compared the patterns of co-distribution of scaffolding and adhesion proteins with extrasynaptic native NR2A and NR2B in the proximal dendrites with those found at a distance from the cell body. Proximal dendrites that are near the cell body tend to be thick (<105 μm from the soma; average 1.9 μm in diameter) whereas more distal ones are generally thinner (>105 μm from the soma; average 0.9 μm in diameter). This comparison was important because using LM for visualization, it is difficult to distinguish between proteins that are attached directly to the cell surface from those that are situated at various levels below the plasma membrane in the cell cytoplasm. This difficulty is greater for thicker dendrites even when using confocal sections. The portion of extrasynaptic NR2B that overlapped with PSD-95/93 was significantly higher () in thicker versus thinner dendrites. No other significant differences between thick, proximal versus thin, distal dendrites were evident. This was in contrast to proteins colocalizing with synaptic
NR2s. For thin, distal dendrites, it was more common for SAP102 or pan-cadherin to colocalize with NR2A than with NR2B (Fig. S1
). In comparisons of NR2A-containing synapses on thick, proximal versus thin, distal dendrites, colocalization of NR2A with PSD-95/93 was more common in thick, proximal dendrites, and colocalization of NR2A with SAP102 was more common in thin, distal dendrites (Fig. S1
). On thick, proximal dendrites, colocalization of NR2B with PSD-95/93 was more common compared to co-distribution in distal dendrites (Fig. S1
). Other associations were not significant (Fig. S1
Punctal size was always significantly larger for synaptic compared to extrasynaptic NR2A and NR2B puncta (). We did not see any significant difference in punctal size between native and transfected NMDARs, although we did find that transfected NMDAR puncta were larger for both synaptic and extrasynaptic receptors at 2 WIV compared to 3 WIV (data not shown). Native NMDARs at 2 WIV were not studied in detail. Other aspects of transfected NMDAR distribution were not examined in detail with LM in this study, although overall patterns of labeling were similar to native.
We also examined some triple-labeled sections in greater detail to investigate possible morphological evidence for the association of native extrasynaptic NMDARs with PSD-95/93, SAP102, cadherin and catenin. Typically, native NR2A- or NR2B-labeled synaptic puncta were large and showed substantial overlap with the puncta produced by VGLUT (). However, in some cases, smaller puncta were resolved. These could form a partial ring around a VGLUT punctum. This suggests that these represent perisynaptic locations or possibly, small groups of receptors near the inside border of the synaptic active zone. Similarly, examples were found of partial rings of small puncta for NR2A or NR2B where there was no overlap with a VGLUT punctum, i.e., extrasynaptic NMDARs. Note in the example in how these extrasynaptic red puncta surround a punctum of PSD-95/93. This supports the idea that there is some direct association between NMDAR and PSD-95/93 puncta. These NMDAR labeled puncta were at the minimum size that can be resolved with LM. They are probably real structures since they were seen with 4-frame averaging and fusion of the confocal stack. However, they would be better characterized if resolution was improved. Thus, a preliminary study using the Leica STED microscope was carried out. This gave superresolution for one color in the x and y axes. We used the superresolution for live surface labeling of NR2A receptors co-labeled with VGLUT and SAP102 (the latter 2 were at standard confocal resolution only). Now, numerous fine puncta as small as about 100 nm, including some that may be perisynaptic around presynaptic terminals were observed (; compare with perisynaptic SAP102 immunogold labeling in Fig. S2F
Thus, the in vitro LM investigation revealed that extrasynaptic NMDARs show some close associations with the MAGUKs, PSD-95 and SAP102, as well as with the adhesion proteins, cadherin and catenin.
EM immunoperoxidase studies of live surface labeling for NMDARs in vitro
The LM in vitro studies were extended to the EM level to identify the specific structures associated with surface extrasynaptic NMDARs. In order to study the definitive distribution of extrasynaptic surface NMDARs in cultures of hippocampal pyramidal neurons, we developed a method for correlating the LM and EM localizations of immunoperoxidase labeling using live, surface labeling of native or transfected NR2A and NR2B-containing receptors (, ). This involved firstly at the LM level, the identification and photographing of labeled neurons () followed by sectioning of the labeled neuron for EM studies. Visualization was via the regular procedure and with silver/gold toning (see Methods).
Figure 3 LM/EM correlation of immunoperoxidase/DAB live surface labeling of a hippocampus culture 2 WIV, transfected with myc-NR2B and labeled with myc antibody. Extrasynaptic sites of labeling are indicated by arrowheads, p=presynaptic terminal, asterisk=postsynaptic (more ...)
Figure 4 Examples of EM immunoperoxidase/DAB live surface labeling of a hippocampus culture 3 WIV, labeled with NR2A (A,B) or NR2B (D-F) antibody or control lacking the primary antibody (C; same experiment as for NR2A (A,B)). Extrasynaptic sites of labeling are (more ...)
Generally, the distribution of the immunoperoxidase/DAB reaction product was seen in patches on the extrasynaptic surface of dendrites and spines. When a patch was at a point of contact with another process, the reaction product was associated with the membranes of both the process and the dendrite or spine surface. This is expected since the chromagen reaction is associated with labeling of extracellular epitopes. Thus it is localized within the narrow cleft between processes. The neuron in shows a hippocampal pyramidal neuron that was transfected with the myc-NR2B clone and live surface labeled with anti-myc antibodies at 2 WIV. Typically, the main dendrites of a large neuron in culture were covered with bundles of thinner processes that mostly run parallel to the length of the dendrite. These processes included axons, dendrites and glial processes. The most common were axons. At the EM level, these axons could be identified definitively by following them along their length to areas with large numbers of synaptic vesicles (). Other presumptive axons were identified by their straight, non-tapering profile and the fact that they are filled with microtubules (, ). Distinctive examples of labeling between presumptive axons and dendrites are shown in ; examples with the soma are shown in , and at spine synapses in . In all these examples, as described for the LM studies, some labeling extended into the perisynaptic region. Synapse identification was not always definitive and it may be that some apparent associations represent early synaptic contacts. In some cases, endosomes were seen just subjacent to the surface labeling (e.g. ). Analysis of 65 extrasynaptic labeled regions (as noted above, each of these regions would be equivalent to a punctum identified with LM) on the dendrites and cell body of this neuron (taken from several serial sections) showed that more than half of the extrasynaptic labeled regions were adjacent to axons or presumptive axon profiles. Most other profiles could not be identified. These extrasynaptic labeled regions ranged in length from 51 - 768 nm (mean = 251 ± 18 nm; ). Synaptic or possible synaptic labeled regions ranged from 128 - 384 nm (mean = 221 ± 32 nm). In an analysis of 35 labeled regions along a single dendrite profile in a 2 WIV culture transfected with the flag-NR2A clone and live surface labeled with anti-flag antibodies, ~25 % of the extrasynaptic regions were between the dendrite and adjacent axons. Approximately 50% of the regions were between at least 1 axon and another process in the bundle surrounding the dendrite (data not shown). For this single dendrite profile, the extrasynaptic, labeled surface regions ranged from 50-560 nm (mean = 227 ± 19 nm; ).
Width of labeled region of extrasynaptic NMDARs using immunogold localization of NR1 in CA1 stratum radiatum of hippocampus, or DAB/immunoperoxidase of hippocampal neuronal cultures
For labeling of native NMDARs in cultures, 2 studies were completed (). For these, we did not concentrate on a single labeled neuron for the LM and EM correlation as for the exogenous NMDARs since the native receptor labeling was widespread in cultured neurons. Anti-NR2A () and anti-NR2B antibodies () usually produced distinctive labeling at extrasynaptic sites. These included the side of spines () and sites along the dendritic shaft (). The extrasynaptic labeled regions ranged from 51 - 538 nm in length (mean = 198 ± 12 nm; N = 50) and 51 - 602 nm (mean = 186 nm ± 11; N = 69) for NR2A and NR2B, respectively. These mean lengths are larger than those found with immunogold labeling (; see also, in brain sections, below). This is probably due to the greater sensitivity of immunoperoxidase labeling (Petralia, 1997
; Petralia and Wenthold, 1998
; further references in the supplemental text
) as well as some lateral diffusion of the DAB reaction product. No significant differences were found for the two measurements of extrasynaptic puncta between exogenous flag-NR2A and myc-NR2B and between the NR2A and NR2B native receptors. There was however a significant difference between the combined totals of exogenous (flag-NR2A+myc-NR2B; mean = 243 ± 13 nm; N = 100) versus native (NR2A+NR2B; mean = 191 ± 8 nm; N=119) extrasynaptic NR2 regions (p = 0.00127). By comparison, identified synaptic labeled regions for native receptors ranged from 115 to 369 nm (mean = 184 ± 20 nm; N=13) and 128 to 333 nm (mean = 223 ± 13 nm; N=23) for NR2A and NR2B, respectively. Thus, the average size of synaptic and extrasynaptic sites were similar. The greater range of the extrasynaptic labeling may reflect the lack of restriction of an extrasynaptic region to a limiting area such as that defined by a PSD.
In summary, in vitro EM analysis revealed that extrasynaptic NMDARs are on sides of spines and along dendrite shafts. The vast majority of these sites are at contact points with processes adjacent to the neuron the most common of which are axons and axon terminals.
Electron microscope localization of NR1 in brain sections
Distribution of cell surface proteins in neuronal cells in culture is not necessarily representative of their distribution in vivo in the brain itself. We examined therefore the distribution of NMDA receptors in sections from the CA1 stratum radiatum of the hippocampus of 3 P37 (), 2 P10, and 2 P2 () rats using post-embedding immunogold methods with an antibody directed against the NR1 C2 C-terminal exon. NMDA receptor distribution was also determined in 3 P35 rats using pre-embedding immunoperoxidase/DAB methods and the anti-NR1 C2 antibody (). This replicates the methodology that was used for the in vitro labeling
Figure 5 Immunogold labeling with NR1 antibody in the adult hippocampus CA1 stratum radiatum (10 nm gold in A-D and 5 nm gold in E-J). Extrasynaptic or perisynaptic sites of labeling are indicated by arrowheads; p=presynaptic terminal, asterisk=PSD, d=dendrite (more ...)
Figure 7 Immunogold labeling with NR1 antibody in the P2 hippocampus CA1 stratum radiatum (5 nm gold). Extrasynaptic or perisynaptic sites of labeling are indicated by arrowheads; p=presynaptic terminal, asterisk=PSD, and d=dendrite shaft. NR1-labeled extrasynaptic (more ...)
Figure 6 EM immunoperoxidase/DAB labeling with NR1 antibody in the adult hippocampus CA1 stratum radiatum; those in E-H were processed further with silver/gold toning. Extrasynaptic or perisynaptic sites of labeling are indicated by arrowheads; p=presynaptic terminal, (more ...)
Postembedding immunogold labeling for NR1 in the adult hippocampus showed abundant labeling with 5, 10, or 15 nm gold secondary antibodies in the PSDs with less labeling at extrasynaptic sites. Extrasynaptic immunogold labeling in dendrites () and spines () was usually adjacent to other cell processes. Often, these could be identified as axons/axon terminals () or presumptive glia (; defined in Methods; also see the supplemental text
concerning the glial marker, GFAP). In most cases, there was no obvious density present under the membrane in these areas. Occasionally, a faint density was evident when compared to adjacent PSDs ().
The size and distribution of extrasynaptic immunogold labeling for NR1 was quantified in this adult tissue (; ). A total of 146 extrasynaptic sites of immunogold labeling (5 nm gold particles; 263 particles) on dendrites (68 sites) and postsynaptic spines (78 sites) in the CA1 stratum radiatum of 2 P37 rats were analyzed. Of the sites on spines, 35/78 were within 100 nm of the PSD (i.e., they were perisynaptic). Nearly all of the extrasynaptic sites were in close contact with an adjacent process. Using the criteria to define axon/axon terminal-like or glia-like described earlier, for extrasynaptic/perisynaptic contacts on postsynaptic spines, 55% and 23% were adjacent to axon/axon terminal-like and glia-like processes, respectively compared to 47% and 31% for contacts on dendrites (). The remaining associated processes included postsynaptic spines, dendrites or were not identified. For those sites that contained more than 1 gold particle (i.e., N = 77 sites with 2-6 particles), the gold particles were found along an average length of 24.6 nm of the cell membrane (range: 5.3 - 92.7 nm; ). Thus, all of the patches seen suggest that the extrasynaptic sites are smaller than synapses, which when identified by morphology in the CA1 stratum radiatum are about 195 nm in length (Tao-Cheng et al., 2007
Gold labeling for NR1 in a random sample of synapses from the CA1 stratum radiatum was also studied. It was noted that the spread of gold labeling in a synapse covered only a portion of the width of the synapse. The density of labeling was 0.99 ± 0.08 gold particles per postsynaptic membrane profile (N = 281 synapse profiles from two P37 rats with no significant difference between animals). This value is similar to that reported previously for NR1 in synapses (Petralia et al., 1999
). These synapse profiles also had 0.33 gold particles per extrasynaptic membrane (0.22 ± 0.04 for perisynaptic plus 0.11 ± 0.03 for the remainder of the extrasynaptic membrane). A sample of mediumsized dendritic profiles in the same sample area (N = 26 profiles from 2 animals, average dimensions 1.5 × 4.8 μm) had up to 6 gold particles on the extrasynaptic membrane (mean = 2.08 ± 0.37 gold particles per profile). While immunogold labeling is not a good measure of the absolute quantity of receptors due to its insensitivity (Petralia et al., 1999
), this suggests that a portion of NMDARs is found outside the postsynaptic membrane on spines and dendrite.
For the immunoperoxidase labeling experiments, the distribution of NR1 was examined first with LM. This showed a wide distribution of labeling throughout the brain as described previously using a polyclonal antibody made to the identical region of NR1 (data not shown; Petralia et al., 1994b
). At the EM level, the DAB reaction product was localized to subregions of the dendrites and spines (). For NR1 labeling of postsynaptic spines, many spines showed only light labeling in the PSD but dense labeling in the membrane on the side of the spine (; the examples shown are from 3 animals), indicating that NR1 is localized to the perisynaptic/extrasynaptic areas. Examples of dense labeling in the PSD are not shown (except for moderately dense labeling in ), but have been published previously for NR1 and NR2 antibodies using immunoperoxidase/DAB (Petralia et al., 1994a
). These papers also included some examples of perisynaptic or extrasynaptic labeling on spines, although they were not described in the text (see also Aoki et al., 1994
). Note particularly in , the nearly identical shape and position of the labeled area from 2 different animals using 2 different methods. Similar patches of labeling could be found in discrete areas of the dendrite surface membrane (; examples shown are from 3 animals). The typical widths of these patches of labeling on dendrites and spines were ~ 100 - 200 nm (); the smallest were ~30 nm. In most cases, labeled patches of membrane were contacted directly by an adjacent process. Most contacts appeared to be with axon terminals () or glial processes (; as described for immunogold above). In dendrites, some surface membrane patches of labeling were associated with labeling in adjacent tubulovesicular structures within the dendrite (; examples shown are from 2 animals). These may be endosomal structures (Washbourne et al., 2004
; Wang et al., 2008
) or other organelles (such as modified components of the endoplasmic reticulum, Golgi, or trans
-Golgi network) involved in trafficking of NMDARs in dendrites (Sytnyk et al., 2004
; Jeyifous et al., 2009
We also performed semi-quantification of labeling from sections from two animals, using the silver/gold toning immunoperoxidase method. About thirteen percent of labeled spine synapses (N = 62) showed silver/gold labeling throughout the spine, while ~37% showed preferential labeling of the extrasynaptic surface and ~50% showed preferential labeling of the extrasynaptic surface plus the postsynaptic membrane. This indicates that at least one third of labeled spine synapses have extrasynaptic labeling that is not a simple diffusion artifact and thus it probably represents extrasynaptic NMDARs. This is consistent with our immunogold study described above. Also in this material, 79% of labeled dendrite profiles (N =42) showed some labeling on the dendrite surface. All the labeled dendrites showed labeling in endosomes and presumptive endosomal structures.
In contrast to the adult, regions of extrasynaptic NR1 labeling often had associated densities in the hippocampus at P2 (). Similar densities have been described previously, labeled with NR1 (polyclonal), NR2A/B, PSD-95, SAP102 and SynGAP antibodies (Sans et al., 2000
; Petralia et al., 2003
). Also, similar structures have been described using stronger fixative regimes (Fiala et al., 1998
). Many of these regions of extrasynaptic NR1 labeling that have associated densities are not contacted directly by another process, particularly those that have fairly dense densities (). These are called “bare densities” and they are possibly remnants of former synapses (Sans et al., 2000
). Other regions of extrasynaptic NR1 labeling that have associated densities have light densities and these may represent new contacts with axonal growth cones that will develop into synapses (). Surface labeling with the NR1 antibody was associated with clathrin-coated pits and associated endocytic structures at P2 () as noted previously using another NR1 and NR2A/B antibodies (Petralia et al., 2003
). Other labeled regions of extrasynaptic NR1 were similar to those described in the adult, but overall processes were more difficult to identify due to their immature state, and less of the surface of processes was in contact with other processes at this age. Thus a detailed classification of these contacts was not practical for this study (note: we also carried out some labeling at an intermediate age between P2 and adult, i.e. P10 using immunogold; see supplemental text
Thus, localization of extrasynaptic NMDARs in vivo resembles that seen in vitro with close associations of these sites with adjacent processes, particularly axons and axon terminals, but also with glia and other processes such as dendrites or postsynaptic spines. This distribution was corroborated by two methods, immunogold for greater specificity of localization and immunoperoxidase for greater sensitivity. The study also revealed that early postnatal ages showed evidence of distinct dense material at many of the extrasynaptic NMDAR sites compared to adults.
EM studies of proteins associated with NMDARs
This study so far has revealed the specific localizations of extrasynaptic NMDARs bothin vitro and in vivo. In this section, we extend our earlier in vitro LM studies to investigate the association of extrasynaptic NMDARs with the scaffolds PSD-95/93, SAP102 and the adhesion proteins, pan-cadherin, N-cadherin, and catenin at the EM level. Double-labeling immunogold and single labeling immunoperoxidase methods were both used.
Catenin showed distinctive labeling patterns consistent with its known distribution (). Immunogold double labeling revealed that catenin was co-distributed with extrasynaptic NMDA receptors (; n = 2 animals). Similar distributions were found in cultured hippocampal neurons at 3 WIV (). Labeling patterns appeared to be similar using anti-N-cadherin and anti-pan-cadherin antibodies, although the labeling was not as strong or definitive as observed with antibodies directed against catenin (for synapses see Petralia et al., 2005
). This localization represents the association of NMDARs near cadherin/catenin bridges that are used for adhesion at contact points.
Figure 8 Immunogold double labeling with NR1 antibody (5 nm) and β-catenin (15 nm), in the adult hippocampus (A-F) or in hippocampal culture (G,H; 3 WIV). Extrasynaptic or perisynaptic sites of labeling are indicated by arrowheads; p=presynaptic terminal, (more ...)
The anti-PSD-95 antibody that works well for EM immunogold labeling results in a distribution of immunoreactivity that is highly localized to PSDs in the adult hippocampus (Sans et al., 2000
; Yi et al., 2007
). Immunoreactivity is also prevalent in distinctive densities, such as in “bare densities” in the P2 hippocampus and in extrasynaptic attachment plaques found in glomeruli in the adult cerebellum (Petralia et al., 2002
). Consequently, here little anti-PSD-95 immunoreactivity was found on the extrasynaptic regions of neurons in vivo
or in vitro
(which show little or no density as noted above), although occasional examples were seen (Fig. S2A-C
). The SAP102 antibody that works well for EM immunogold labeling is an antibody raised in rabbits; thus double labeling with 2 rabbit antibodies is problematic. However, some double labeling for SAP102 and NR1 subunits was carried out using an NR1 mouse monoclonal antibody (Fig. S2D, E
). Double labeling for SAP102 and PSD-95 also was carried out (Fig. S2F
). These studies revealed some distinctive extrasynaptic sites of single labeling for SAP102 (Fig. S2D,F
) that were similar to extrasynaptic sites seen for NR1 labeling. In addition, we did preembedding immunoperoxidase/DAB of the ABR PSD-95/93 or the Neuromab SAP102 (that were used for immunofluorescence) in brain sections of 2 adult rats (Fig. S3
). At the LM level, neuronal labeling for either antibody was distinctive in the hippocampus and many other regions of the brain. Control sections were unlabeled (data not shown). In the pyramidal neurons of the hippocampus, labeling in apical dendrites was moderate for PSD-95/93 and dense for SAP102. Both antibodies also appeared to label the mossy fibers of the stratum lucidum. With EM, labeling in the hippocampus (CA1stratum radiatum; CA3 stratum lucidum) for PSD-95/93 was particularly abundant in postsynaptic spines (Fig. S3A
). Labeling was also seen in dendrites and in some axon terminals with some labeling at extrasynaptic sites (Fig. S3B
). For SAP102, compared to PSD-95/93, labeling density and frequency were higher in dendrites and lower in spines (Fig. S3C-E
). SAP102 dendrite labeling was often concentrated in patches associated with transport vesicles where the labeling frequently extended to the adjacent dendrite cell membrane. It was also seen at distinct extrasynaptic sites (Fig. S3E
). Similarly to PSD-95/93, labeling was found in some axon terminals. Overall, the immunoperoxidase/DAB data were consistent with immunofluorescence data that showed that SAP102 labeling is more prevalent in dendrites than PSD-95/93 labeling.
Thus, in this section, we showed that sites with extrasynaptic NMDARs can be associated with adhesion proteins. This is probably relevant especially since these sites are usually at contact points with adjacent processes. MAGUKs such as SAP102 and PSD-95 can associate with extrasynaptic NMDARs. This probably corresponds to the colocalizations of extrasynaptic NMDARs and MAGUKs shown with in vitro LM studies described above.