Endosomal compartments serve multiple hippocampal dendritic spines from a widespread rather than a local store of recycling membrane

Abstract

Endosomes are essential to dendritic and synaptic function in sorting membrane proteins for degradation or recycling, yet little is known about their locations near synapses. Here, serial electron microscopy was used to ascertain the morphology and distribution of all membranous intracellular compartments in distal dendrites of hippocampal CA1 pyramidal neurons in juvenile and adult rats. First, the continuous network of smooth endoplasmic reticulum (SER) was traced throughout dendritic segments and their spines. SER occupied the cortex of the dendritic shaft and extended into 14% of spines. Several types of non-SER compartments were then identified, including clathrin-coated vesicles and pits, large uncoated vesicles, tubular compartments, multivesicular bodies (MVBs), and MVB-tubule complexes. The uptake of extracellular gold particles indicated that these compartments were endosomal in origin. Small, round vesicles and pits that did not contain gold were also identified. The tubular compartments exhibited clathrin-coated tips consistent with the genesis of these small, presumably exosomal vesicles. Approximately 70% of the non-SER compartments were located within or at the base of dendritic spines. Overall, only 29% of dendritic spines had endosomal compartments, whereas 20% contained small vesicles. Small vesicles did not colocalize in spines with endosomes or SER. Three-dimensional reconstructions revealed that up to 20 spines shared a recycling pool of plasmalemmal proteins rather than maintaining independent stores at each spine.

Fig. 1.

Smooth endoplasmic reticulum forms a network in the dendrite. Top, SER in a single section appears as thin cisternae with wavy membranes (arrows) surrounding a mitochondrion (∗). A mushroom spine (m) and a thin spine (t) originate from the dendrite on this section. Bottom, Three-dimensional reconstruction of the SER demonstrates that the cisternae form a network with larger flat compartments (arrowheads) connected by thin extensions (arrows). Most of the network lies in the periphery of the dendrite, surrounding the mitochondria (not shown in reconstruction). Seventeen spines originate from this segment of dendrite. SER is found in only three spines (∗), two of which are large mushroom spines with spine apparatuses. Scale bar, 1.0 μm.

Fig. 2.

A coated pit (arrow) in a spine head. The synapse (not visible on this section) lies on the opposite side of the spine head. SER (arrowheads) is visible in the spine neck as a flattened sheet and in the dendrite as a thin compartment. Inset, A coated vesicle (arrow) and a coated pit in a dendrite shaft. Scale bar, 0.5 μm.

Fig. 3.

Tubular compartments in dendrites and spines.a, SER (arrowhead) extends into the neck of a thin spine. A non-SER tubular compartment (arrow) at the origin of the spine exhibits a uniform diameter compared with SER. b, A non-SER tubule (arrow) in a spine neck. c, A coated bud (∗) at the tip of a tubule. Note the darker interior of the tubular compartments (arrows) as compared with SER (arrowheads). Scale bar, 0.5 μm.

Fig. 4.

Small vesicles in dendrites. a, A large vesicle (arrowhead) next to a small vesicle (arrow) in a spine head. Note the distinctive difference in size. b, A small vesicle (arrow) in a spine head with a tubular compartment that has a coated tip (arrowhead). c, d, Adjacent serial sections showing a tubular compartment (c, arrowhead) with a small vesicle (d, arrow) of the same diameter near its tip. e, A small vesicle-sized omega figure (arrow) in the plasmalemma at the base of a thin spine (s). A cross-sectioned tubular endosome (arrowhead) is also at the spine origin, near a thinner cross-section of SER (∗). Inset, A small vesicle adjacent to the plasmalemma in a spine head. Scale bar, 0.5 μm.

Fig. 5.

Multivesicular body/tubule complexes in dendrites.a, An MVB with attached tubules (arrows). Compare the flattened appearance of SER (arrowhead) with the more cylindrical shape of an unattached tubule (open arrow) located nearby. b, A tubule (arrow) is in continuity with an MVB only partially visible on this section. SER is located nearby (arrowhead). Scale bar (shown in b fora and b): 0.25 μm. c, Reconstruction of the MVB–tubule complex of band associated compartments. The tubule (arrow) visible in the electron micrograph is continuous with a round MVB (arrowhead) that has a second tubular extension. Several additional tubular compartments are located adjacent to this structure. An isolated MVB (∗) nearby does not have any tubular extensions. Also reconstructed are several large, ellipsoidal vesicles (gray) and two spherical small vesicles (dark gray). Scale bar, 0.5 μm.

Fig. 6.

Amorphous vesicular compartments found in dendrites and spines. a, A clump of amorphous vesicles (arrow), with unattached SER nearby (arrowhead). b, An amorphous vesicle (arrow) adjacent to the plasma membrane in a spine head. Amorphous vesicles were in the head or neck of 5% of the spines examined in the study. Scale bar, 0.5 μm.

Fig. 7.

Gold particles conjugated with BSA were taken in from the extracellular space into endosomal compartments.a, Gold is seen in a coated vesicle (arrow) but not in SER (arrowhead) or a small vesicle (∗) in a mushroom spine. Scale bar, 0.5 μm.b, Gold is seen in the lumen of an MVB in a dendrite. The MVB exhibits an invagination (arrow) that may indicate the formation of an internal vesicle. c, Gold is concentrated in the base of a tubular extension (arrow) from an MVB. d, Gold particles were found in coated pits (arrow) and in tubular compartments with coated tips (arrowhead). e, An amorphous vesicle clump with gold visible in three of the vesicles (arrows). f, Serial section analysis of dendrites detected gold in endocytotic vesicles (cp, coated pits; cv, coated vesicles; lv, large vesicles) and non-SER tubular compartments. Gold was concentrated in MVB–tubule sorting complexes (cplx) and mostly absent from small vesicles (sv). No gold was found in any of the 1031 profiles of SER in the dendrites.

Fig. 8.

The frequency of each type of non-SER compartment (cp, coated pits; cv, coated vesicles;lv, large vesicles; te, tubular endosomes; cplx, MVB–tubule complexes;sv, small vesicles; av, amorphous vesicles; avc, amorphous vesicular clumps) in dendrites. The location of the compartment was determined to be in spines (1: in a spine head or neck), at the base of the spine (2), or in the shaft of the dendrite (3), as shown in the inset.

Fig. 9.

Three-dimensional reconstructions of representative dendritic segments from each age. Endocytotic vesicles (coated pits, coated vesicles, and large vesicles) are shown inyellow. Tubular endosomes, MVBs, and MVB–tubule sorting complexes are red. These structures are infrequent along the length of the dendrites relative to the frequency of spines. Small vesicles (blue) are often located in spines but only occasionally colocalize with endosomes. Amorphous vesicles are shown indark brown. Scale bar, 1 μm.

Fig. 10.

Distribution of endosomal volume. More endosomal compartments are found in spines than at spine origins; however, the bulk of endosomal volume is found in MVBs and sorting complexes, which are localized primarily at spine origins.

Fig. 11.

Venn diagrams showing the percentage of spines that contained SER, an endosomal compartment (coated pit, coated vesicle, large vesicle, tubular endosome, or MVB–tubule complex), or small vesicles at each age. The overlap areas show the percentages of spines with more than one type of compartment. The percentages under the labels give the overall proportion of spines containing a particular type of compartment. Thus, 19.8% of adult spines contained SER, whereas 1% of adult spines contained SER, endosomes, and small vesicles.

Fig. 12.

Diagram of the putative itinerary of endosomal compartments in distal dendrites. Clathrin-coated pits invaginate to form coated vesicles that become large vesicles after the loss of the coat. Large vesicles merge into tubular endosomes and MVB–tubular complexes. Sorted, soma-bound material leaves the dendrite via isolated MVBs. The coated tips of tubular endosomes give rise to small, round vesicles that return to fuse with the plasmalemma. The source and target regions of the plasmalemma for these components are likely to be disparate.