Electron tomographic structure and protein composition of isolated rat cerebellar, hippocampal and cortical postsynaptic densities

Abstract

Electron tomography and immunogold labeling were used to analyze similarities and differences in the morphology and protein composition of postsynaptic densities (PSDs) isolated from adult rat cerebella, hippocampi, and cortices. There were similarities in physical dimensions and gross morphology between cortical, hippocampal and most cerebellar PSDs, although the morphology among cerebellar PSDs could be categorized into three distinct groups. The majority of cerebellar PSDs were composed of dense regions of protein, similar to cortical and hippocampal PSDs, while others were either composed of granular or lattice-like protein regions. Significant differences were found in protein composition and organization across PSDs from the different brain regions. The signaling protein, βCaMKII, was found to be a major component of each PSD type and was more abundant than αCaMKII in both hippocampal and cerebellar PSDs. The scaffold molecule PSD-95, a major component of cortical PSDs, was found absent in a fraction of cerebellar PSDs and when present was clustered in its distribution. In contrast, immunogold labeling for the proteasome was significantly more abundant in cerebellar and hippocampal PSDs than cortical PSDs. Together, these results indicate that PSDs exhibit remarkable diversity in their composition and morphology, presumably as a reflection of the unique functional demands placed on different synapses.

Keywords: electron cryotomography; electron tomography; immunogold labeling; postsynaptic density; receptors; scaffold proteins.

Figure 1

Isolation of PSDs from Cerebella, Hippocampi and Cortices. (A) Western blots illustrating loss of synaptic vesicle protein SV2 and enrichment of PSD scaffold PSD-95 in synaptic junction (Syn. J) and PSD fractions in comparison to brain homogenate (Hom.) and synaptosome (Syn.) fractions. Eight micrograms of total protein were loaded in each lane. (B) Electron micrographs of negatively stained isolated PSDs from cerebella (Cereb.), hippocampi (Hippo.) and cortices (Cortical). Scale bar = 100 nm.

Figure 2

Histograms of PSD Surface Area. Histograms depicting surface area distributions of PSDs isolated from cerebella, hippocampi and cortices. The peak for all PSD types was approximately 0.2 ξm². N = 120.

Figure 3

Negative Stain Tomographic Reconstructions of PSDs Isolated from Cortices and Hippocampi. All images are 10 nm cross-sections through final tomographic reconstruction. PSDs isolated from cortices and hippocampi were composed of large dense protein regions (black horizontal arrows) with small areas lacking protein (black vertical arrows). Asterisks highlight detergent resistant membranes attached to the PSDs. Gold was used as a fiducial marker to align image series during reconstruction. Scale bar = 100 nm.

Figure 4

Negative Stain Tomographic Reconstructions of PSDs Isolated from Cerebella. All images are 10 nm cross-sections through final tomographic reconstruction. The majority of PSDs isolated from cerebella were: composed of large dense protein regions with small areas lacking protein (Dense Cereb.); more globular in texture (Granular Cereb.); or latticelike in appearance (Lacy Cereb.). Black horizontal arrows indicate areas of dense protein, vertical arrows indicate areas without protein density and asterisks highlight detergent resistant membranes attached to the PSDs. Gold used as a fiducial marker to align images during reconstruction can be seen in some of the images. Scale bar = 100 nm.

Figure 5

Negative Stain Tomographic Reconstructions of PSDs Isolated from Cortices, Hippocampi, and Cerebella. All images are 10 nm cross-sections through final tomographic reconstructions. The left panels show examples of cortical (A), hippocampal (B), and cerebellar PSDs (C-D). Black horizontal arrows indicate areas of dense protein, vertical arrows indicate areas without protein density and asterisks highlight detergent resistant membranes attached to the PSDs. Enlarged regions of each image on the left are shown in the right panels that illustrate the finer structural features of PSDs from cortices (A), hippocampi (B), and cerebella (C-E). An example of a dense cerebellar PSD (C), granular cerebellar PSD (D) and a lacy cerebellar PSD (E) are shown. All PSDs were composed of filamentous and globular proteins, indicated with white horizontal and vertical arrows, respectively, in the right panels. Gold used as a fiducial marker to align images is occasionally visible. Scale bar = 100 nm.

Figure 6

Cryotomographic Reconstructions of PSDs Isolated from Cortices and Hippocampi. All images are 10 nm cross-sections through final tomographic reconstructions. PSDs isolated from cortices and hippocampi were composed of large dense protein regions with small areas lacking protein. Black horizontal arrows indicate areas of dense protein, vertical arrows indicate areas without protein density and asterisks highlight detergent resistant membranes attached to the PSDs. Gold used as a fiducial marker to align images is occasionally visible. Scale bar = 100 nm.

Figure 7

Cryotomographic Reconstructions of PSDs Isolated from Cerebella. All images are 10 nm cross-sections through final tomographic reconstructions. The majority of PSDs isolated from cerebella where composed of large dense protein regions with small areas lacking protein (Dense Cereb.), although some PSDs were more globular in texture (Granular Cereb.), and others were latticelike in appearance (Lacy Cereb.). Black horizontal arrows indicate areas of dense protein, vertical arrows indicate areas without protein density and asterisks highlight detergent resistant membranes attached to the PSDs. Gold used as a fiducial marker to align images is occasionally visible. Scale bar = 100 nm.

Figure 8

Protein Occupancy of PSDs Isolated from Cerebella, Hippocampi and Cortices. Whisker plot showing individual and mean protein-to-volume ratios, defined as the total number of voxels containing protein divided by the total number of voxels contained within the PSD. Values from each of twelve cryo-preserved PSDs from each brain region are shown as black diamonds. Within the whisker plots, the black squares represent the mean, the line represents the median, the pyramids represent the interquartile range and the error bars show the 95% to 5% range of the data.

Figure 9

Comparison of Protein Profiles of Isolated PSDs. Deep purple stained SDS-gel of separated proteins from PSDs isolated from cortices, hippocampi, and cerebella. The major bands in each lane are indicated with asterisks and the position of molecular weight standards (in kDa) are indicated to the left of the gel. Three micrograms of total protein were loaded in each lane.

Figure 10

Ripley’s K Function Analysis of a Cerebellar PSD Immunogold Labeled for PSD-95. (A) Negative stain electron micrograph of a PSD from cerebella immunogold labeled for PSD-95. (B) Coordinate model for the PSD in A. Connected red points define the boundary, while the gold is represented as black points. (C) H-function for the example PSD shown in A and B. Simulated complete spatial randomness (black solid line) was normalized to zero and the Ripley’s K function of the experimental data is shown as a red line. Non-random distribution was determined by whether the red line crossed either the maximum and minimum envelopes (dashed lines) as defined by 1000 simulations of complete spatial randomness.