Synaptic Organization of the Human Temporal Lobe Neocortex as Revealed by High-Resolution Transmission, Focused Ion Beam Scanning, and Electron Microscopic Tomography

Abstract

Modern electron microscopy (EM) such as fine-scale transmission EM, focused ion beam scanning EM, and EM tomography have enormously improved our knowledge about the synaptic organization of the normal, developmental, and pathologically altered brain. In contrast to various animal species, comparably little is known about these structures in the human brain. Non-epileptic neocortical access tissue from epilepsy surgery was used to generate quantitative 3D models of synapses. Beside the overall geometry, the number, size, and shape of active zones and of the three functionally defined pools of synaptic vesicles representing morphological correlates for synaptic transmission and plasticity were quantified. EM tomography further allowed new insights in the morphological organization and size of the functionally defined readily releasable pool. Beside similarities, human synaptic boutons, although comparably small (approximately 5 µm), differed substantially in several structural parameters, such as the shape and size of active zones, which were on average 2 to 3-fold larger than in experimental animals. The total pool of synaptic vesicles exceeded that in experimental animals by approximately 2 to 3-fold, in particular the readily releasable and recycling pool by approximately 2 to 5-fold, although these pools seemed to be layer-specifically organized. Taken together, synaptic boutons in the human temporal lobe neocortex represent unique entities perfectly adapted to the "job" they have to fulfill in the circuitry in which they are embedded. Furthermore, the quantitative 3D models of synaptic boutons are useful to explain and even predict the functional properties of synaptic connections in the human neocortex.

Keywords: EM tomography; active zones; human temporal lobe neocortex; quantitative three-dimensional models of synaptic boutons; synaptic boutons; synaptic vesicles; transmission and focused ion beam scanning EM.

Figure 1

Synaptic organization of the neuropil in the human temporal lobe neocortex (TLN). (A), Low-power electron microscopy (EM) micrograph of the neuropil of L2 in the gyrus temporalis medialis of the TLN processed for TEM analysis. Here, several synaptic complexes, composed of a dendritic shaft or spine and a synaptic bouton, are highlighted in transparent yellow (synaptic boutons, or SBs) and transparent blue (dendritic shafts or spines). Note the high density of axo-spinous synaptic complexes. (B), Same layer, gyrus, and color code as shown in (A), but here, the tissue sample was processed for focused ion beam scanning EM (FIB-SEM) analysis. Note the different structural appearance of the neuropil between the TEM (A) and FIB-SEM (B) processed tissue samples. Scale bar in (A,B), 1 µm.

Figure 2

Structural characteristics of SBs and their target structures in the human TLN as revealed by FIB-SEM. (A), Large dendrite (de, transparent blue) with three terminating SBs (sp1–sp3, transparent yellow of different shape and size in L6 of the gyrus temporalis medialis. In two SBs (sb1, sb2), a clear AZ is visible, whereas sb3 is lacking an AZ. (B), Small caliber dendrite (de) receiving synaptic input from three SBs, one of which invaginating the dendrite in L5 of the gyrus temporalis medialis. (C), Elongated spine (sp) emerging with a long spine neck from a dendrite (de) in L3 of the gyrus temporalis medialis with two opposing SBs (sb1, sb2) terminating on the spine head. AZs are marked by arrowheads. Same color code as in A. (D), Large mushroom spine (sp) with a short spine neck originating from a dendritic segment (de) in L2 of the gyrus temporalis inferior receiving input from a relatively large end terminal SB with synaptic vesicles (SVs) distributed over the entire terminal. In all images, AZs are marked by arrowheads, and the same color code is used as in (A). Scale bar in (A–D), 0.5 µm.

Figure 3

Structural characteristics of SBs in the human TLN as revealed by FIB-SEM. (A), Large SB (sb) invaginating a dendritic spine (sp) in L5 of the gyrus temporalis medialis. The AZ is marked by arrowheads. Note the cluster of unmyelinated axons (asterisks) close to the end terminal bouton. (B), Large synaptic bouton (sb) terminating on a large spine head (sph) of a short-necked spine (spn) in L6 of the gyrus temporalis. (C), Dendrite (de) with two SBs (sb1, sb2); sb1 with a large tangled structure (highlighted in transparent yellow) within the interior of the bouton invaginating the dendrite, whereas sp2 shows a “normal” appearance. The AZ in sb2 is marked by arrowheads. (D), Small caliber astrocytic processes (asterisks) identifiable by their darker appearance in the surrounding neuropil in L6 of the gyrus temporalis inferior. Infrequently, these astrocytic processes receive synaptic input by an SB (sb) identifiable by the establishment of a prominent AZ (arrowheads). Scale bar in (A–D), 0.5 µm showed layer-specific differences. It has been shown recently that the size of the readily releasable (RRP) dynamically regulates multivesicular release in mice [30], which also seemed to be the case at SBs in the human TLN.

Figure 4

Structural characteristics of SBs and their target structures in the human TLN as revealed by TEM. (A), Large putative inhibitory (sb, transparent yellow) terminating on a large dendrite (de, transparent blue) in L2 of the gyrus temporalis inferior with a large macular, non-perforated AZ (arrowheads) containing thousands of SVs. (B), Typical example of an axonal segment, giving rise to an end terminal SB (sb) innervating two adjacent small dendritic spines with relatively small AZs (arrowheads) in L3 of the gyrus temporalis inferior. Note the appearance of SVs also in the axon. Same color code as in A. (C), End terminal SB (sb) synapsing on a large stubby spine (sp) with a large spine head emerging directly from a dendrite (de) in L6 of the gyrus temporalis medialis. Note the two AZs (arrowheads) and the large spine apparatus (asterisk) in the spine head. Same color code as in (A). (D), A small dendritic segment (de) giving rise to an elongated spine with a small spine neck (spn) leading to a large spine head (sph). Two SBs (sb1, sb2) terminate directly on the spine head (sb1) and on the dendrite (sb2). AZs are marked by arrowheads. Same color code as in (A). Scale bar in (A–D), 0.5 µm.

Figure 5

Structural characteristics of SBs in the human TLN as revealed by TEM. (A), EM micrograph of SBs (sb, transparent yellow) terminating on a dendrite (de, transparent blue) with an adjacent “astrocytic bouton” (astb, transparent yellow) terminating on a small dendritic spine (transparent blue) in L3 of the gyrus temporalis medialis. Note the dark appearance typical for astrocytes and the content of vesicles containing gliotransmitter. AZs are marked by arrowheads. (B), two spines (sp) with a relatively large spine head and short spine necks one containing a prominent spine apparatus (framed area) in L6 of the gyrus temporalis medialis. The right spine with a large macular, non-perforated AZ (arrowheads) receiving input from an end terminal SB, whereas on the left spine display, no obvious contact is visible. (C), Large SBs (sb1) invaginating two spines (sp1, sp2) in L2 of the gyrus temporalis inferior next to another spine (sp3) receiving input from another SB (sb2). AZs are marked by arrowheads. Scale bar in (A–C), 0.5 µm.

Figure 6

EM tomography of synaptic complexes and active zone with “docked” SVs. (A), Single EM micrograph of a tomographic TILT series through a synaptic complex between a spine (sp) and a synaptic bouton (sb) in L6a of the human TLN. The framed area indicates a large spine apparatus and arrowheads indicate the AZ. “Docked” vesicles are highlighted in transparent green. Note the cluster unmyelinated at the synaptic complex. (A1), High magnification of the AZ shown in A. The synaptic bouton (sb) is on top the spine (sp) at the bottom of the images. Here, the “docked” SVs (arrowheads) are clearly visible. Note the omega-shaped SV (black arrowhead) indicating the already occurred release of a quantum of neurotransmitter. (B), Synaptic bouton (sb) establishing a contact (arrowheads) with an astrocytic process (ast) identified by its darker appearance in L6b of the human TLN. (B1), Higher magnification of the AZ shown in B. Here, two SVs are already fused with the presynaptic membrane as marked by arrowheads. Note also the large dense core vesicle (asterisk). (C), Large macular, non-perforated AZ between a spine (sp) and a synaptic bouton (sb) with two ‘docked vesicles’ in L5 of the human TLN. (D), Large non-perforated AZ at a synaptic complex between a dendritic shaft (sh) and a synaptic bouton (sb) with four “docked” SV (arrowheads), two with an omega-shaped appearance. Note the SV (black arrowhead) close, but not fused with the presynaptic density. Scale bar in (A), (B) 0.5 µm and in (A1), (B1), (C), (D) 0.25 µm.

Figure 7

3D-volume reconstructions of SBs and their target structures in the human TLN 3D reconstructed from FIB-SEM and TEM z-stacks. (A), Dendritic segment (blue) in L2 receiving dense synaptic input by seven SBs (yellow, sb1–sb7) reconstructed from a large z-stack using FIB-SEM. In two SBs, the bouton cover was omitted, and in one, it was made transparent to allow the visualization of the total pool of SVs (green dots), the AZ (red), and mitochondria (white). Note the different shape and size of the synaptic terminals and the content of SVs. (A1), (A2), (3D)—reconstruction of the total pool of SVs in the spine (sb5) and shaft (sb7) SB shown in A as marked by asterisks. Note the large difference in the pool of SVs and the lack of mitochondria in the large shaft bouton. (A3), Representative example of the dense astrocytic ensheathment (white contour) of an axo-spinous synaptic complex. The astrocytic coverage isolates the synaptic complex from the neuropil and other synaptic complexes, and fine astrocytic processes can be followed to reach the synaptic cleft. (B–D), Three representative examples of SBs and their target structures 3D reconstructed based on serial ultrathin sections and TEM imaging. All SBs are given in their proportional size to each other. (B), Large end terminal SB in L2. Here, the cover of the SB and that of the postsynaptic spine was made transparent yellow and blue to visualize the pool of SVs (green dots), mitochondria (white), and the three AZs (red) in the presynaptic terminal. (C), SB with a comparably small pool of SVs terminating on a stubby spine in L2. (D), Two SBs (sb1, sb2) terminating on the same mushroom spine in L2. Note the different geometry and size of SBs, the different number, shape, and size of the AZs, the pool of SVs, and the content or lack of mitochondria. Scale bar in (A–D), 0.5 µm.