Quantitative 3D Ultrastructure of Thalamocortical Synapses from the "Lemniscal" Ventral Posteromedial Nucleus in Mouse Barrel Cortex

Abstract

Thalamocortical synapses from "lemniscal" neurons of the dorsomedial portion of the rodent ventral posteromedial nucleus (VPMdm) are able to induce with remarkable efficacy, despite their relative low numbers, the firing of primary somatosensory cortex (S1) layer 4 (L4) neurons. To which extent this high efficacy depends on structural synaptic features remains unclear. Using both serial transmission (TEM) and focused ion beam milling scanning electron microscopy (FIB/SEM), we 3D-reconstructed and quantitatively analyzed anterogradely labeled VPMdm axons in L4 of adult mouse S1. All VPMdm synapses are asymmetric. Virtually all are established by axonal boutons, 53% of which contact multiple (2-4) elements (overall synapse/bouton ratio = 1.6). Most boutons are large (mean 0.47 μm3), and contain 1-3 mitochondria. Vesicle pools and postsynaptic density (PSD) surface areas are large compared to others in rodent cortex. Most PSDs are complex. Most synapses (83%) are established on dendritic spine heads. Furthermore, 15% of the postsynaptic spines receive a second, symmetric synapse. In addition, 13% of the spine heads have a large protrusion inserted into a membrane pouch of the VPMdm bouton. The unusual combination of structural features in VPMdm synapses is likely to contribute significantly to the high efficacy, strength, and plasticity of these thalamocortical synapses.

Figure 1.

Selective labeling of VPMdm axons in the mouse S1 cortex. (A) A representative example of a stereotaxically guided iontophoretic BDA injection in VPMdm. The thionin-counterstained coronal section shows the center of the BDA microdeposit (white asterisk). Abbreviations: VPMdm, dorsomedial part of the ventral posteromedial thalamic nucleus; VPL, ventral posterolateral thalamic nucleus; Po, Posterior thalamic nucleus. Coronal level caudal to Bregma (AP; in mm) is indicated on the lower right corner (dorsal to the top, lateral to the right). (B) BDA-labeled VPMdm axons arborizing profusely in barrels (D6–D8 as indicated by the dashed circles) in S1. Non-branched ascending axon segments in layer 5 are indicated by black arrows. (C) Flat map reconstruction from the serial coronal sections of the barrel field zone covered by the BDA-labeled VPMdm axons in this experiment (dashed line). Anteroposterior coronal levels (“B” Bregma, −1 mm, −2 mm) are indicated on top of the map. A standard stereotaxically adjusted map of mouse L4 barrel domains (gray shaded areas) is included, for reference. Samples were taken from barrels D7–8 and E7 (asterisks). (D) High-magnification view of labeled axons at the core of a barrel. Note the frequent axonal varicosities of various sizes, marked by arrowheads. (E) Bar histogram showing the distribution in size of light microscopically identified varicosities/boutons. (F) Bar histogram of the distribution of intervaricosity/bouton distance. Scale bars A,B = 250 μm; D = 5 μm.

Figure 2.

3D serial TEM and FIB/SEM of identified VPMdm boutons in L4 of the S1BF. (A) Durcupan embedded 50μm-thick section containing BDA-labeled VPMdm axons in barrels D6–D8 of S1BF. This section is adjacent to that shown in Figure 1B. The trapezoid contour indicates the borders of a sample excised for subsequent serial ultrathin sectioning and TEM imaging. (B) Sample containing BDA-labeled axons, after block-mounting and trimming. Note the large blood vessel in A and B (arrow) which was used as a landmark. (C) Low power electron micrograph of an individual ultrathin section. For reference, an arrow indicates the same blood vessel in panels A–C. The bleached circle (arrowhead) is the region illustrated in D. (D) Three aligned electron micrographs from consecutive ultrathin sections showing a labeled VPMdm bouton. (E) 3D reconstruction of a VPMdm synaptic bouton (blue) shown in D establishing synaptic contact with 3 different postsynaptic spines (yellow). Note the large mitochondrion (pale blue) associated with the pool of synaptic vesicles (green). One of the PSDs (red) is also visible. (F, G) Durcupan embedded 50μm-thick section containing BDA-labeled VPMdm axons in barrels D8–D7 of S1BF. This tissue section is adjacent to that shown in Supplementary Figure 1D. The green frame indicates the borders of a sample (panel G) excised for subsequent FIB/SEM imaging. The band between the 2 white dashed lines shows the approximate borders of layer 4. (H) Secondary electron image obtained with scanning EM from the block face shown in G. (I) Secondary electron image showing several trenches milled with the focused ion beam to allow SEM imaging of brain tissue under the block face. Stacks of serial images were acquired from all trenches except the one indicated with a red dot. For precise localization, the shape of these trenches is overlaid on the images in panels F–H. (J) 3D FIB/SEM reconstruction of a labeled axonal segment giving rise to a synaptic bouton (blue) with 2 PSDs (red) on a 3-axis background view of the neuropil. A spine postsynaptic to the bouton and its parent dendrite (yellow) is also 3D-reconstructed. Scale bars: A, F–H = 250 μm; C = 5 μm; D, J = 0.5 μm; E = 0.25 μm; I = 100 μm.

Figure 3.

Serial TEM micrographs of BDA-labeled VPMdm thalamocortical boutons (bt) establishing synaptic contacts with dendritic shafts (de). (A, B) VPMdm synaptic boutons establishing synaptic contacts with dendritic shafts. (C) Two synaptic boutons terminate in close proximity on the same dendritic segment. (D) Individual synaptic bouton innervating both a dendritic shaft and spine (sp). Note that the PSDs (arrowheads) are of different shape and size, but always non-perforated. In all figures, PSDs are marked by arrowheads. Scale bars A,B = 0.25 μm; C,D = 0.5 μm.

Figure 4.

Serial TEM VPMdm boutons (bt) establishing synaptic contact with dendritic spines (sp). (A) A labeled VPMdm bouton synapsing on a dendritic spine head. A spine apparatus is indicated by an asterisk. (B) Two dendritic spines innervated by the same VPMdm bouton. (C) Two spine heads completely enwrapped by the bouton (D) VPMdm bouton establishing 2 synaptic contacts with the same dendritic spine (1 non-perforated and 1 perforated). Inset: higher-magnification of one of the PSDs (frame area in D). Some synaptic vesicles are indicated by white arrows. In all figures PSDs are marked by arrowheads. Scale bars A–D = 0.25 μm.

Figure 5.

Serial TEM 3D-volume rendering of VPMdm boutons. (A1–3) Three consecutive ultrathin sections of a BDA-labeled VPMdm bouton. Large numbers of presynaptic vesicles (given in green) are distributed throughout the terminal. The PSD is highlighted in red and marked by arrowheads. (B–F) Examples of 3D-volume reconstructions of individual boutons. In each panel, the outline of the bouton (blue) and PSDs (red) are represented at the top. The bouton’s mitochondria (white) and pool of synaptic vesicles (green) are shown at the bottom. Note the different shape and size of the PSDs. Scale bars A–F = 0.5 μm.

Figure 6.

Serial TEM 3D-volume rendering of VPMdm boutons (blue) establishing synaptic contacts (red) onto dendritic shafts and spines (yellow). (A) A bouton with a non-perforated PSD terminating on a dendritic shaft. (B) A bouton establishing a synaptic contact with a dendritic spine with a ring-like perforation of its PSD. (C) A bouton with 3 PSDs contacting a dendritic shaft (1) and 2 separated spines (2 and 3). (D) A bouton with a horseshoe-like perforated (1) and a ring-like perforated PSD (2) terminating on 2 individual dendritic spines. (E) A bouton innervating 4 dendritic spines. Note the different shape and size of the PSDs. (F) En passant axon with 2 synaptic boutons contacting 2 dendritic spines (dashed line indicates the borders of the boutons). (G) Three labeled boutons (1–3) in close proximity to each other establishing 1 (1–2) and 4 (3) synaptic contacts with dendritic spines. Note the different size of the 3 boutons. All PSDs are represented in red. Scale bar A–G = 0.5 μm.

Figure 7.

VPMdm axonal segments 3D-reconstructed from FIB/SEM image stacks. (A) 3D reconstructions of fifteen axonal segments. Axonal membrane is represented in transparent blue to visualize mitochondria (white). PSDs in spines are colored in red. PSDs in dendrite shafts are colored in green and pointed by an open green arrow. White arrows indicate the location of a protrusion into the VPM bouton from the postsynaptic spine (see Fig. 9). Open white arrows indicate PSDs located in non-varicose axon domains containing mitochondria. Note that the latter lack nearby mitochondria. (B) Samples form a series of FIB/SEM images showing the synapse marked with the letter “B” in panel A. Images shown are spaced 75 nm. The dark BDA precipitate is clearly visible within this non-varicose axonal segment. The arrowhead points to a PSD on a dendritic spine. Scale bars A = 2 μm; B = 0.5 μm.

Figure 8.

Spines of different shape and size postsynaptic to identified VPMdm boutons 3D-reconstructed from FIB/SEM image stacks. About 30 spines with varying shapes were reconstructed. Typical “filopodial” (1), “mushroom” (2), “branched” (3), and “stubby” (4) shapes can be observed. The spine membrane (yellow) is made transparent to allow visualization of the PSDs (red). In addition, PSDs are shown on top of each spine, rotated and at double magnification, to better visualize their shape. PSDs in gray and indicated with an asterisk are those located on the same spine but not corresponding to a contact with a labeled VPM axons. Non-synaptic zones of the spine intruded inside VPMdm boutons are indicated by a blue–green shade on some spines. In those spines with a visible spine apparatus, a 3D reconstruction of this organelle (in blue) is shown on the left of the corresponding spine. Scale bar = 0.5 μm.

Figure 9.

Spine protrusions embedded into VPMdm boutons. (A1–5) Serial TEM images of a synaptic bouton (transparent blue) containing a large spine protrusion (transparent yellow) deeply inserted into it. Mitochondria are shaded in purple, and the PSD in brown. (B) 3D reconstruction of the same synapse. Images in panels A1–5 correspond, respectively, to levels 1–5 in this reconstruction, and are colored likewise. The synaptic vesicles (green) are also shown in the 3D reconstruction. (C,D) Two additional examples of spines with large spine protrusions invading VPMdm boutons reconstructed from FIB/SEM samples. Same color code as in panel B. Scale bars = 0.25 μm.

Figure 10.

Correlations between different structural parameters of the VPMdm boutons (I). Serial TEM and FIB/SEM data are pooled together. (A) Dot plot correlation analysis between bouton surface vs. volume. Note the high correlation as indicated by the linear regression and R². (B) Between bouton volume vs. mitochondrial volume. (C) Bouton surface area vs. PSDs contour surface area. Note that both parameters are not correlated, as indicated by the R². (D) Bar histogram of the distribution of PSD contour surface area for dendritic spines and shafts. There were no statistically significant differences between the 2 distributions (P = 0.876, Kolmogorov–Smirnov test).

Figure 11.

Correlation of structural parameters of VPMdm boutons (II). Panels A–B and F show Serial TEM and FIB/SEM data pooled together. Panels C–E display only TEM data. In the charts, dots plot correlation: (A) between bouton and spine head volume. (B) between PSD surface vs. spine head surface; (C) between the total pool of synaptic vesicles vs. bouton volume; (D) between total pool of synaptic vesicles vs. PSD contour surface area; and (E) between total pool of vesicles and spine head volume. (F) Bar histogram showing the distribution of spine head volumes in spines postsynaptic to VPMdm axons (62 serial TEM and 30 FIB/SEM).

Figure 12.

Graphic summary of structural features of VPMdm synaptic boutons and their innervation pattern on target structures. Serial TEM and FIB/SEM data are pooled together. (A) Synaptic boutons lacking or containing mitochondria. (B) Predominant innervation pattern of VPMdm synaptic boutons. (C) Unusual synaptic specializations observed in spines postsynaptic to VPMdm boutons. From top to bottom, a conventional apposition between a spine and a synaptic bouton covered only by the PSD at the pre-and postsynaptic apposition zone; a spine with its head partially invading the bouton, the PSD is part of the pre- and postsynaptic apposition zone; and a spine with a large and deep protrusion into the bouton. Note that the bouton perimeter remains constant whereas the spine protrusion expands inside it. See also Figure 9B,D.