FIGURE 1.

Synaptic spinules are fundamental components of excitatory synapses in CA1. (A) Single image from the 5 nm isotropic FIB-SEM image volume of CA1 hippocampus. Excitatory asymmetric postsynaptic densities (PSDs) are indicated by a red asterisk. Note the large apical pyramidal cell dendrites that flank the left and right sides of the image that reduce excitatory synapse density throughout this image volume. (B) Cartoon showing quantification of the percentage of excitatory presynaptic spinule-bearing boutons (SBBs), presynaptic boutons without spinules (non-SBBs), perforated postsynaptic densities (Perf PSDs), macular PSDs, SBBs enveloping multiple spinules, SBBs with multiple synapses, and spinules with clathrin-coated tips (CT) versus those without clathrin-coats (no-CT). (C) Representative example of an SBB (purple) with CT spinules from its postsynaptic spine (PSs; pink) partner. Upper and middle panels are adjacent FIB-SEM images showing the invagination and envelopment of one of three PSs spinules into this SBB. Bottom panel shows the 3D reconstruction of this PSs projecting three spinules into the SBB. Note that this SBB forms a second synapse (PSD in red) with a second spine (not shown). (D) Upper and middle panels show adjacent FIB-SEM images of an adjacent bouton (AdjAx, green) projecting a no-CT spinule into an SBB (purple) that has a synapse with a postsynaptic spine (uncolored PSD, top left of SBB). Bottom panel shows the 3D reconstruction of this AdjAx with its large no-CT spinule enveloped by the SBB, and the SBB’s PSD (red) with the postsynaptic spine (not shown). Note the second small CT AdjAx spinule projecting into the very bottom of this SBB. (E) Upper and middle panels show two FIB-SEM images of an adjacent spine (no PSD with this SBB; AdjS; orange) projecting (top panel) a large no-CT spinule into an SBB that becomes fully enveloped (lower panel). Bottom panel shows the 3D reconstruction of this AdjS, SBB, and SBB’s PSD (red). Note the spinule’s small (∼30 nm) invagination site (top and bottom panels) that expands into a large, enveloped structure that extends through the majority of this SBB’s length (middle and bottom panels). (F) Upper and middle panels show two successive FIB-SEM images of an adjacent dendrite (AdjD, yellow) projecting a no-CT spinule into an SBB (purple), with the spinule fully enveloped by the SBB (middle panel). Bottom panels show the 3D reconstruction of the surface of this dendrite and its non-synaptic spinule encapsulated by this SBB with a synapse (red) onto a dendritic spine from a separate dendrite (not shown). (G) 3D reconstruction of a segment of a dendrite and its spines, a majority of which project spinules into SBBs. All spinules from an individual spine are pseudocolored with the same color. Note that individual spines may project both CT and no-CT spinules (e.g., red and cyan spinules). (G₁,G₂) Single FIBSEM sections showing large no-CT spinules within SBBs (purple) emanating from distinct spines (arrows), and the 3D reconstruction of these SBBs (at right) displaying the enveloped spinule and PSDs (red). (G₃) FIB-SEM image and 3D reconstruction showing a small CT spinule (cyan) enveloped by an SBB (purple), displaying an annulus-shaped perforated PSD (red). (G₄) FIB-SEM image and 3D reconstruction showing an SBB enveloping a spine head spinule (peach) and displaying two macular PSDs (red). (G₅) 3D reconstruction (arrow origin) and two successive FIB-SEM images showing an inhibitory presynaptic bouton (blue) with a synapse (symmetric PSD at left of bouton) onto a large pyramidal cell apical dendrite, and two enveloped spinules (pink and yellow) from nearby synaptic spines. (H) 3D reconstruction of four excitatory axons (orange, purple, magenta, and green), and one inhibitory axon (blue), projecting AdjAx spinules into each other, with visible PSDs (red). Arrows point to FIB-SEM images of the same SBB and AdjAx spinule relationships. (H₁) Two successive FIB-SEM images showing an adjacent axon projecting a large no-CT spinule (orange) containing presynaptic vesicles into an SBB (purple). (H₂) Two successive FIB-SEM images showing an adjacent presynaptic bouton (magenta) projecting a large no-CT spinule into an SBB (purple). Note that the individual synapses that both boutons have with postsynaptic spines are visible as dark PSDs at top and bottom of image at left. (H₃) FIB-SEM image (left) and 3D reconstruction (right) showing the reciprocal relationship to panel (H₂), with SBB from magenta axon enveloping an AdjAx spinule from the purple axon’s presynaptic bouton and PSDs shown in red. (H₄) Two successive FIB-SEM images showing an AdjAx (magenta) with a spinule into an inhibitory presynaptic bouton (blue). (H₅) Two successive FIB-SEM images showing another AdjAx (green) with a spinule into this same inhibitory presynaptic bouton (blue) with a synapse (symmetric PSD) onto a dendritic shaft (top FIB-SEM image and red PSD opposite green spinule in 3D reconstruction). Scale bars (A) 1 μm, (C–H) 0.5 μm; Scale cubes (C–H) 0.5 μm per side.