Synaptic organization of cortico-cortical communication in primates

Abstract

In cortical circuitry, synaptic communication across areas is based on two types of axon terminals, small and large, with modulatory and driving roles, respectively. In contrast, it is not known whether similar synaptic specializations exist for intra-areal projections. Using anterograde tracing and three-dimensional reconstruction by electron microscopy (3D-EM), we asked whether large boutons form synapses in the circuit of somatosensory cortical areas 3b and 1. In contrast to observations in macaque visual cortex, light microscopy showed both small and large boutons not only in inter-areal pathways, but also in long-distance intrinsic connections. 3D-EM showed that correlation of surface and volume provides a powerful tool for classifying cortical endings. Principal component analysis supported this observation and highlighted the significance of the size of mitochondria as a distinguishing feature of bouton type. The larger mitochondrion and higher degree of perforated postsynaptic density associated with large rather than to small boutons support the driver-like function of large boutons. In contrast to bouton size and complexity, the size of the postsynaptic density appeared invariant across the bouton types. Comparative studies in human supported that size is a major distinguishing factor of bouton type in the cerebral cortex. In conclusion, the driver-like function of the large endings could facilitate fast dissemination of tactile information within the intrinsic and inter-areal circuitry of areas 3b and 1.

Keywords: Saimiri sciureus; axon terminal; electron microscopy; human; multivariate analyses; serial section reconstruction; tract tracing.

Figure 1.

Anterograde BDA labeling and distribution of large boutons at the light microscopy level. A, B: Injection sites. Representative light microscopic images of the injection sites (A) taken from the sections labeled by stars in the graphical reconstructions (B) in the 6 cases. The lateral spread of the core and halo regions are shown by black and grey, respectively, across the sections where BDA injections could be identified. White dots indicate the electrode track. Upper panels: area 3b injections, lower panels show are 1 injections. Depth of the uppermost and lowermost sections from the pial surface, with clearly detectable BDA injection site are also indicated. Note the injection of the full cortical thickness in cases P and Mo with area 1 injections. C, D: Small and large boutons identified by light microscopy. Representative light microscopic image showing numerous thin fibers decorated by small boutons (diameter ≤ 1 μm) in a terminal axon arborization patch of area 3b (C). Arrowheads show terminal-like structures with stalk, and arrow indicates varicosity. An example of an axonal branch bearing 2 small varicosities (arrows) are shown in the inset. Example of large boutons (diameter > 1 μm) appearing as varicosities of long-range intra-areal and inter-areal horizontal axonal fibers in area 1 (D). E: Distribution of the large boutons (diameter > 1 μm, each marker corresponds to one large bouton) after aligning the merged series of 6 cases using the injection sites (i.j.) and the border between area 3b (a3b) and area 1 (a1) as fiducial landmarks. Only boutons in areas 3b and 1 are shown. Grey squares show large boutons labeled by area 3b injections and black dots show boutons labeled after area 1 injections. Large markers indicate boutons reconstructed by EM. Injections were made into digit 2 (d2) distal finger pad representation except one case with area 1 injection, where digit 4 (d4) distal finger pad representation was injected. Note that the distance of bouton closest to the injection site was 690 μm. Dotted lines: areal borders, arrows: injection sites, sc: central sulcus. r: rostral, m: medial. Scale bars: 200 μm on A and B, 5 μm on C and D and 1 mm on E. F: Areal distribution of large boutons in areas 3b and 1. Inter-areal ratio represents the inter-areal fraction of the total number observed in the injected and target areas. Squares indicate individual cases and stars show averages.

Figure 2.

Ultrastructural features of BDA-labeled somatosensory cortical endings. A-C: Electron microscopic cross sections of small boutons (diameter ≤ 1 μm light microscopy size) forming synapses with dendritic spines (sp). D-F: Electron microscopic cross sections of large boutons (diameter > 1 μm light microscopy size) forming axospinous (E, F) and axodendritic (D, d: dendrite) synapses. All BDA-labeled boutons establish asymmetric synaptic contact (arrowheads) in the forms of simple (A-C, E) and complex perforated synapses (D, F). Note the lack of mitochondrion (m) in some boutons (A). Note also that boutons can contain more than one mitochondrion (D, F). In A, and also on D and F, note the unstained synaptic vesicles (v) appearing as white dots in the dark NiDAB precipitate, and aggregating near the synaptic contact in the bouton. On C note an invagination (arrow), which lacks synaptic membrane specialization within the bouton. Scale bar: 0.5 μm on A- D, F and 1 μm on E.

Figure 3.

Two examples of serially reconstructed labeled large boutons. A: Light microscopic image of a large axonal varicosity examined ultrastructurally in B-D. B: Boundaries of the reconstructed bouton based on the serial ultrathin sections are shown. Arrows labeled by D₁, D₂ and D₃ identify sections shown in D. C: Three-dimensional structure of the synaptic organization of the labeled bouton (beige) is shown after surface rendering with opaque (C₁) and transparent coloring (C₂). Spines are shown in grey, mitochondrion is blue and synaptic membrane specialization is green. D: A short sample of the electron microscopic series showing two asymmetric synapses (arrowheads) formed by the labeled bouton with dendritic spines (sp1, sp2). Note numerous synaptic vesicles (white dot-like structures) and an invagination (arrow see also on B), which lacks synaptic membrane specialization within the bouton. The bouton also contains a mitochondrion (m). E-G: Another example of the serially reconstructed labeled large bouton. Conventions are the same as B-D. Note asymmetric synaptic membrane specialization on F (arrowheads). H: For comparison a 3D structure of a small bouton is shown with an opaque (H₁) and a transparent beige coating surface (H₂). The bouton contains a mitochondrion (blue) and creates a synapse (green) with a spine (grey). Scale bars: 5 μm on A and 1 μm on B-H.

Figure 4.

Comparing the surface and volume of the BDA-labeled bouton groups. A: Surface, B: Volume and C: Surface/volume ratio of small, and large boutons. To calculate ratios the data was transformed (²√surface and ³√volume) to maintain dimensional coherence. Plots show individual boutons (open circles) and average (horizontal bar). The small solid symbol in the center of the open markers label boutons with well circumscribed PSD. Note an outlier (filled circle) exhibiting very large surface (A) and volume (B). However, this bouton (large transparent circle) exhibited similar surface/volume ratio to the rest of the population (C). D: Correlation of volume and surface resulted in the grouping of small (open triangles) and large (open circles) boutons. The outlier with very large surface (A) and volume (B), is shown by the black circle. Solid symbol in the center of a marker indicate boutons with PSD. E: Note that boutons with and without PSD exhibit similar distributions and high correlation of the surface and volume (PSD: R = 0.90, noPSD: R = 0.98). Note the regrouping of some LM large boutons into the cluster of small boutons. F: Distribution of small boutons lacking (grey triangles) or containing (black triangles) mitochondrion also exhibit high correlation of the surface and volume (R = 0.77). G,H: Histograms showing the surface and volume distribution in the population of small and large boutons. For better visibility giant varicosities were not included. The absence of data at 2.4–2.9 μm² and 0.52–0.62 μm³ revealed the separation of clusters.

Figure 5.

Frequency of basic ultrastructural features of the BDA-labeled boutons grouped on the basis of surface-volume correlation. A: Ratio of boutons forming multiple synaptic contacts via multiple PSDs. B: Ratio of synaptic contacts with perforated PSDs. C: Ratio of mitochondrion containing boutons. The single giant bouton, which established synaptic contact was not included in the comparisons.

Figure 6.

Comparison of ultrastructural features of the small and large boutons. A: Bouton surface, B: Bouton volume, C: Bouton surface per volume. To calculate the ratios data was transformed (²√surface and ³√volume) to maintain dimensional coherence. D: Mitochondrial surface per bouton, E: Mitochondrial volume per bouton, F: Mitochondrial surface per bouton surface, G: Mitochondrial volume per bouton volume, H: PSD surface, I: PSD surface per bouton surface, J: PSD surface per mitochondrial surface, K: Bouton shape factor, L: PSD shape factor. Note, that the shape factor of PSDs did not differ significantly from 1 (one sample t-test, df = 29, p = 0.4 with a mean ± sd of 1.1 ± 0.7), while the shape factor of boutons was significantly smaller than 1 (one sample t-test, df = 29, p = 0.04 with a mean ± sd of 0.9 ± 0.2). Mean is shown by horizontal bar and circles represent individual boutons. Asterisk indicates significant difference for the non-corrected p values. **: 0.005 < p ≤ 0.01. ***: p ≤ 0.005.

Figure 7.

Results of the PCA analyses. A: Scree plot showing the eigenvalues and the percentage of variance explained by the 11 principal components. B: The importance of the variables measured by the modeling power, which is defined as the explained standard deviation. C: Distribution of the loading factors (transformed values showing the contribution of the variable to the PCA model) P1 and P2 for the first and second principal components. The greater a variable is away from the origin, the more influential that variable has. Diagonal positioning in opposite quadrants means negative correlation between the variables. D: Case-wise analysis shows the tendency of grouping the black squares and open circles, which represent small and large boutons, respectively, along the two principal components. Distribution of the scores (distances of transformed values of the variables from the origin along the PCs) of boutons for the first principal component (T1) plotted against the scores for the second principal component (T2). Ellipse outlines ±3sd and indicates that there was no outlier in the dataset. M/B sf: mitochondrial surface/bouton surface, M/B vol: mitochondrial volume/bouton volume, B shf: bouton shape factor, B sf/vol: bouton surface/volume, PSD/M sf: PSD surface/mitochondrial surface, PSD/B sf: PSD surface/bouton surface, PSD shf: PSD shape factor.

Figure 8.

Results of the PCA analyses of boutons from layers 4 and 5 of the human temporal cortex. A: Scree plot, B: Variables importance, C: Distribution of the loading factors and D: Case-wise analysis. Boutons of layer 4 are marked by black rectangles and boutons of layer 5 are shown by open circles. Note the appearance of outliers, which all except one belong to boutons of layer 5. spher: sphericity, circ: circularity. All other conventions are the same as in Figure 7. Note that this analysis was based on 9 variables as opposed to the 12 variables used for analyzing the BDA labeled boutons. Note also on D the outliers, which is consisted of layer 5 boutons (circle with cross) with one exception from layer 4 (cross with the oblique rectangle in the center).

Figure 9.

Correlation of volume and surface of boutons of layers 4 and 5 of the human temporal cortex. L4: layer 4 (rectangles), L5: layer 5 (circles). Note that the highest and lowest values are composed almost exclusively of L5 and L4 boutons, respectively.

Figure 10.

Results of the PCA analyses of BDA labeled boutons using a set of variables matching those used for analyzing the human data. A: Variables importance, B: Case-wise analysis. Black rectangles: small boutons, open circles: large boutons. Conventions are the same as in Figure 7.