The Subcellular Organization of Cortactin in Hippocampus

Light microscopy

Cortactin immunostaining was seen in gray matter throughout the brain, sparing white matter. Staining was prominent in several areas rich in dendritic spines, including the cerebellar cortex, olfactory bulb, striatum, cerebral neocortex, and hippocampal formation (Fig. 2A). Within the hippocampal formation, staining was dense in the neuropil of Ammon's horn and the molecular layer of the dentate gyrus (Fig. 2B). For this study, we focused our attention on CA1, the spiny pyramidal neurons of which have been especially well studied. Immunoreaction was present in CA1 pyramidal neurons, labeling their cell bodies and apical dendrites but sparing the nucleus; somatic staining was organized into small patches (Fig. 2C). No obvious glial staining was observed. Proximal dendrites contained abundant immunoperoxidase reaction product, often associated with filamentous structures (Fig. 2C,D). High-magnification views revealed punctate staining in the stratum radiatum (Fig. 2E), suggesting that cortactin may be concentrated in dendritic spines and/or terminals.

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Figure 2.

Immunoperoxidase staining for cortactin. A, Low-magnification view of a parasagittal section of adult rat brain. Staining is especially prominent in the cerebellar cortex and hippocampus and is also strong in other brain regions rich in dendritic spines. B, Micrograph showing the hippocampal formation. Staining is conspicuous in the molecular layer of the dentate gyrus (DG) and in the neuropil of CA1 (dashed lines mark borders of the main regions within the hippocampal formation). C, D, CA1 of hippocampus, showing stained somata and apical dendrites of pyramidal neurons. The detail in C reveals patchy staining in the somatodendritic compartment, surrounding immunonegative nuclei. E, High-magnification view of the stratum radiatum reveals intensely labeled dendrites; puncta in the neuropil may correspond to dendritic spines. Scale bars: A, 5 mm; B, 500 μm; C, E, 25 μm; D, 50 μm.

To elucidate the organization of cortactin in the neuropil, we performed high-resolution confocal microscopy on material double labeled for cortactin and presynaptic markers. Cortactin in the stratum radiatum was punctate; these puncta were frequently adjacent to synaptophysin-immunopositive puncta. Cortactin puncta often showed small regions of overlap with synaptophysin puncta (Fig. 3A, arrows), but seldom was extensive overlap detected, suggesting that cortactin concentrates in postsynaptic structures. The same pattern was also seen after double labeling with cortactin and VGLUT1, a marker for glutamatergic terminals (Fig. 3B) (Fremeau et al., 2001; Kaneko and Fujiyama, 2002).

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Figure 3.

Subcellular distribution of cortactin in stratum radiatum of CA1. A, Confocal microscopy shows cortactin (red) and the presynaptic marker synaptophysin (green). The apposition of puncta immunopositive for the two markers (arrows in inset) suggests that cortactin may be postsynaptic. B, Double immunofluorescence with cortactin (red) and VGLUT1 (green). The pattern of punctate apposition suggests that cortactin is associated with glutamatergic synapses. C, Cortactin immunostaining was performed on material treated with the lipophilic dye DiO (green), which stains the plasma membrane of scattered pyramidal cells. Inset, Cortactin puncta (c₁) can be seen within DiO-stained (c₂) spines (merge; c₃, arrowheads). D, Double fluorescence with cortactin (red) and phalloidin (green). Results suggest that cortactin localizes profiles also rich in actin. Inset, Arrowheads pointing to both cortactin-positive (d₁) and phalloidin-positive (d₂) sspines. Scale bars: A-C, 5 μm; D, 20 μm; insets, 5 μm.

These results, along with previous data from cultured neurons, suggested that cortactin in the hippocampus might concentrate in dendritic spines. To clarify the issue, we used the lipophilic dye DiO to label the dendritic plasma membrane. This experiment confirmed that cortactin concentrates in dendritic spines in the adult brain (Fig. 3C, arrowheads). Because cortactin has an F-actin-binding domain, we wondered whether it colocalized with actin in specific subcellular compartments. We therefore performed double labeling with phalloidin and found massive dendritic colocalization, especially in spines (Fig. 3D) (cf. Capani et al., 2001a,b).

These LM data show that cortactin is expressed at high levels in spiny neurons and suggest that it concentrates along with actin in dendritic spines.

Electron microscopy

To gain a better understanding of its subcellular organization, we performed immmunoelectron microscopy for cortactin. In cell bodies of CA1 pyramidal neurons, the electron-dense immunoperoxidase reaction product was mostly associated with rough endoplasmatic reticulum (Fig. 4A). Most dendritic shafts contained patches of reaction product, but staining was markedly denser in spines (Fig. 4B,C, asterisks). Reaction product often filled spines, although in some cases it was attenuated or absent from the region just beneath the PSD (Fig. 4 D,E). Reaction product was not detected in axons and axon terminals. Thus, immunoperoxidase electron microscopy confirms our LM observation that cortactin concentrates in spines.

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Figure 4.

Electron micrographs showing immunoperoxidase labeling of cortactin in CA1.A, Reaction product is visible in the cell bodies of two pyramidal neurons, associated with the endoplasmatic reticulum but sparing the Golgi complex. n, Nucleus. B-E, Reaction product accumulates in spines (asterisks) and dendrites (d) but not in presynaptic terminals. Arrowheads in D and E point to the region of immunostained spines, just beneath the PSD, that lacks immunostaining. Scale bars: A, 1 μm; B, 0.5 μm; C-E, 0.25 μm.

The above results suggest that cortactin in spines may lie away from the synaptic junction. However, these data do not allow critical examination of the question, because immunoperoxidase product may migrate away from the enzymatic site, often filling the profile and making it difficult to define the precise location of the antigen. To clarify this issue, we performed a set of experiments with immunogold labeling. We repeatedly tried postembedding techniques, but with little success; presumably the relevant epitope is destroyed or occluded by the preliminary histological processing. We therefore performed preembedding immunogold labeling, followed by silver intensification, to obtain more precise spatial localization of cortactin.

Numerous gold/silver particles coding for cortactin were seen in dendritic shafts of CA1 pyramidal neurons (Fig. 5A). Most of these particles were associated with microtubules, presumably representing a transport pool trafficking between spines and the cell body. Some dendritic labeling was not associated with microtubules; we speculate that this pool is coupled to actin filaments in the dendritic shaft, as hinted by phalloidin/cortactin double immunfluorescent labeling (Fig. 3D). In a number of cases, cortactin (presumably in association with actin filaments) concentrated in the general vicinity of asymmetric synaptic contacts onto dendritic shafts of putative interneurons (Fig. 5B).

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Figure 5.

Preembedding immunogold labeling for cortactin in stratum radiatum of CA1. A, B, Electron micrographs show immunopositive dendrites (d). Most of the silver-enchanced gold particles visible in the apical dendrite in A are associated with microtubules. Gold particles in the dendrite of a possible interneuron (B) concentrate close to asymmetric synaptic appositions. C, Low-magnification view of synaptic neuropil. Arrows point to three immunolabeled spines; d marks an immunopositive dendritic shaft. D, E, High-magnification views showing immunopositive spines. Gold particles extend deep into the spine core (star in D indicates axon terminal with a gold particle). Scale bars: A-C, 0.5 μm; D, E, 0.2 μm.

Immunofluorescence and EM immunoperoxidase indicate that axons and axon terminals do not contain cortactin; however, after immunogold processing, occasional gold particles were seen in axon terminals (Fig. 5D, star). To assess whether these particles might represent unspecific labeling, we performed quantitative analysis of the density of immunogold labeling in different subcellular compartments. Nonspecific background labeling was calculated by measuring particle densities over the nuclei of pyramidal cells, because these were consistently immunonegative with immunofluorescence (see Materials and Methods). Labeling over axon terminals was not significantly above this background (p > 0.3) (Table 1). In contrast, dendritic shafts (8 times above background) and spines (15 times above background) contained significantly higher concentrations of gold particles than did nuclei (p < 0.001).

Labeling was especially prominent in the spines of pyramidal neurons. Gold particles in spines usually spared the immediate region of the PSD (Fig. 5C, arrows). Within spines, gold particles were often associated with electron-dense filamentous material (Fig. 5 D,E), suggesting an interaction between cortactin and the cytoskeleton. In a number of cases, gold particles were seen in association with coated pits and vesicles in spines and dendritic shafts, although these profiles were uncommon.

Because cortactin has been found to increase spine size in cultured neurons, we wondered whether larger spines in the intact brain contain higher concentrations of cortactin. However, examining 236 randomly selected spine profiles, we found a weak negative correlation between labeling density and profile area (r = -0.11). Random stochastic noise inherent in sampling from small profiles is likely to lead to underestimates of correlation. To reduce this noise, we examined correlations after grouping the data into 5, 10, or 20 bins of profile area and found correlations ranging from -0.43 to -0.50. Because work on cultured neurons suggests that cortactin may be important for spine development, we speculated that cortactin density might be positively correlated with spine size during postnatal development. However, a similar weakly negative relationship was also seen in immature animals: in a sample of 102 profiles from P21 hippocampus, r = -0.04, and in a sample of 106 profiles from P14, r = -0.18; again, the magnitude of the correlation, but not its sign, increased with binning.

Distribution of cortactin within spines

To establish the organization of cortactin within spines, we measured the distances of gold particles from the outer leaflet of the postsynaptic membrane, as described previously (Valtschanoff and Weinberg, 2001). Consistent with hints from the DAB material (Fig. 4D,E), cortactin labeling concentrated away from the PSD, in the cytoplasmic core of the spine, with a peak labeling density ∼100-150 nm away from the synaptic membrane (Fig. 6A, ○). This result must be treated cautiously: preembedding techniques may fail to detect antigen within the dense protein matrix of the PSD (Lorincz et al., 2002). This possibility concerned us because some biochemical studies (Husi et al., 2000; Peng et al., 2004) have reported cortactin in the PSD, although others failed to detect cortactin (Walikonis et al., 2000; Yoshimura et al., 2004).

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Figure 6.

Axodendritic distribution of cortactin, α-actinin, and synaptophysin in axospinous synaptic profiles. A, Graph shows the axodendritic distribution of gold particles coding for cortactin (○) and α-actinin (▴) in the spine cytoplasm. The x-axis values represent the distance from the center of the gold particle to the synaptic membrane, along the axodendritic axis (negative values represent gold particles in the synaptic cleft or presynaptic terminal; see inset in C). The y-axis values represent the percentage of the total number of particles counted in each bin. Cortactin lies significantly farther from the PSD than α-actinin. B, Distances of gold particles coding for cortactin from the spine plasma membrane; cortactin concentrates in the spine core. Only particles lying within the spine were considered; the graph was truncated at 150 nm to permit inclusion of data from small spines. Labeling density was weighted to compensate for the reduction in perimeter, for particles lying deep in the cytoplasm. C, Graph shows that gold particles coding for synaptophysin (▵) are restricted to the presynaptic terminal. The dashed line represents the postsynaptic membrane, which corresponds to 0 in A and C (see inset for explanation). Random photos were taken of spines and terminals in the stratum radiatum of CA1 exhibiting a clear synapse and containing at least one gold particle. Data are from 65 profiles for α-actinin, 55 for cortactin, and 55 for synaptophysin. The bin width was 10 nm for A and B and 20 nm for C. Data were smoothed with a three-point weighted running average.

To control for the possibility that methodological limitations might have prevented detection of cortactin in the PSD, we used the same preembedding technique to detect α-actinin, an actin-binding protein previously demonstrated with postembedding techniques to concentrate within the PSD (Wyszynski et al., 1998). We found that particles coding for α-actinin concentrated 50-70 nm from the PSD, significantly closer than cortactin to the synaptic plasma membrane (p < 0.001) (Fig. 6A, ▴). Examining these data further, we found that 9% of the particles coding for α-actinin (17 of 188) lay ≤30 nm from the postsynaptic membrane (a region clearly within the PSD), whereas none of the cortactin particles (0 of 122) lay in this region. It is thus unlikely that methodological limitations prevented detection of cortactin within the PSD. However, ∼10% of the gold particles were <60 nm from the synaptic membrane, close enough to permit a direct interaction with binding partners within the PSD, especially proteins like Shank that lie in its inner layer (Valtschanoff and Weinberg, 2001). As an additional control, we examined the distribution of synaptophysin (Fig. 6C, ▵), confirming that it was exclusively presynaptic, lying mainly >100 nm from the synaptic membrane. Thus, our data indicate that the major pool of cortactin lies within the spine core and suggests that the modest fraction associated with the synapse lies at the cytoplasmic fringe of the PSD.

It still remains possible from the above data that cortactin accumulates close to the plasma membrane but away from the PSD. To test this possibility, we measured the distance of gold particles coding for cortactin to the spine plasma membrane (Fig. 6B). The virtually complete absence of cortactin within 30 nm of the plasma membrane, in contrast to its accumulation ∼100-150 nm away, confirmed that cortactin concentrates in the spine core.

Proteins that regulate actin remodeling

The Arp2/3 complex, a key regulator of actin polymerization, is a likely component of spine remodeling (Machesky and Insall, 1998; Svitkina and Borisy, 1999; Meyer and Feldman, 2002). Arp2/3 functions efficiently only when activated; its best-known activator is the Wiskott-Aldrich syndrome protein (WASP) (Mullins, 2000), but cortactin can also activate Arp2/3-mediated actin assembly (Uruno et al., 2001; Weaver et al., 2001). However, cortactin has a special branch-stabilizing activity that WASP lacks (Uruno et al., 2003). Thus, cortactin is likely to have different cellular functions than the WASP protein family.

Complete stabilization of the actin network would prevent the spine from functioning as a plastic receptive unit (Kasai et al., 2003). Cortactin in model systems provides only partial stabilization, thus permitting continued flexibility or growth (Uruno et al., 2003); we suggest that cortactin plays an analogous role in dendritic spines. Furthermore, cortactin moves from the spine into the dendritic shaft after activation of NMDA receptors (Hering and Sheng, 2003), a stimulus previously found to stabilize spine morphology (Star et al., 2002). Intriguingly, profilin, another regulator of actin polymerization (Finkel et al., 1994), moves into the spine head after NMDA receptor activation (Ackermann and Matus, 2003). Because profilin acts to stabilize spine morphology, we suggest that the NMDA receptor-mediated exchange of profilin for cortactin may represent an activity-dependent switch regulating actin stability.

Spine morphology and cortactin

Recent experiments in cultured neurons (Hering and Sheng, 2003) suggest a role for cortactin in spine development. Cortactin can bind to Shank, a scaffold protein previously shown to increase spine size. However, this association is apparently not the primary mediator of the cortactin effect, because Shank overexpression increased spine head thickness, whereas cortactin overexpression increased spine length; furthermore, cortactin mutants unable to bind to Shank also increase spine length (Hering and Sheng, 2003). Our findings extend this work by showing that cortactin concentrates in spines in the mature brain. Unexpectedly, our data suggest that cortactin is more concentrated in small spines, at variance with results from cultured neurons (Hering and Sheng, 2003). Cortactin might play functionally different roles in developing and mature neurons, but we also saw a negative correlation in spines from the immature hippocampus. Overexpression experiments may not reflect the physiological function of cortactin in vivo; alternatively, one could speculate that small spines containing high concentrations of cortactin have been “marked” for subsequent enlargement. Although purely observational, our results are consistent with Kasai's model (Kasai et al., 2003; Matsuzaki et al., 2004) in that small spines, presumably available for plasticity-dependent remodeling, contain more cortactin than large, presumably stable spines.

Distribution of cortactin within spines

Our data show that cortactin concentrates at a considerable distance (∼100-150 nm) from the synapse, suggesting that it interacts with actin in the spine core, away from the PSD. This apparent exclusion was not only from the immediate vicinity of the postsynaptic membrane but also at nonsynaptic membranes. That this is unlikely to reflect methodological limitations is supported by our results with α-actinin; moreover, using the same technique, we can detect other proteins that concentrate close to the membrane and avoid the spine core (Racz et al., 2004). We propose that the spine core represents a previously unrecognized domain playing a special role in cytoskeletal organization and/or spine remodeling.

The present results do not exclude that cortactin may bind to Shank in the PSD. We detected a small amount of cortactin at the cytoplasmic fringe of the PSD, and previous work from our laboratory found Shank to concentrate in the cytoplasmic side of the PSD (Valtschanoff and Weinberg, 2001). Together with previous work (Naisbitt et al., 1999; Husi et al., 2000; Peng et al., 2004), the present data suggest that although a small amount of cortactin lies close to the PSD, it is only weakly associated with it. Thus, unlike the Shank-PSD-95-GKAP (guanylate kinase-associated protein) complex, cortactin is unlikely to be an integral component of the PSD. Our data are consistent with the presence of two distinct pools of cortactin within the spine, a small synaptic pool and a larger core pool, only indirectly coupled to synaptic activity. We speculate that these two pools have distinct functions: the core pool is situated to mediate changes in spine shape, whereas the synaptic pool might help to modify the composition or shape of the PSD in response to specific patterns of activity (Desmond and Levy, 1986; Geinisman et al., 2000; Malinow and Malenka, 2002; Marrone and Petit, 2002; Harris et al., 2003).

Cortactin in dendrites

We found that cortactin is also concentrated in dendritic shafts, although to a lesser degree than in spines. This may represent a transport pool of cortactin en route from the cell body to spines. However, cortactin has also been implicated in the morphogenesis of dendritic trees: δ-catenin can bind to cortactin (Weed et al., 2000); by activating Arp2/3-induced actin polymerization, this catenin-cortactin complex induces process elongation, at least in cultured neurons. In contrast, when δ-catenin (which lies at the plasma membrane) is tyrosine phosphorylated, the dendritic tree becomes highly branched, apparently because the phosphorylation prevents the catenin-cortactin association (Martinez et al., 2003). Thus, our data raise the possibility that cortactin may also play a structural role in mature dendrites.