Quantitative analysis of multivesicular bodies (MVBs) in the hypoglossal nerve: evidence that neurotrophic factors do not use MVBs for retrograde axonal transport

Abstract

Multivesicular bodies (MVBs) are defined by multiple internal vesicles enclosed within an outer, limiting membrane. MVBs have previously been quantified in neuronal cell bodies and in dendrites, but their frequencies and significance in axons are controversial. Despite lack of conclusive evidence, it is widely believed that MVBs are the primary organelle that carries neurotrophic factors in axons. Reliable information about axonal MVBs under physiological and pathological conditions is needed for a realistic assessment of their functional roles in neurons. We provide a quantitative ultrastructural analysis of MVBs in the normal postnatal rat hypoglossal nerve and under a variety of experimental conditions. MVBs were about 50 times less frequent in axons than in neuronal cell bodies or dendrites. Five distinct types of MVBs were distinguished in axons, based on MVB size, electron density, and size of internal vesicles. Although target manipulations did not significantly change MVBs in axons, dystrophic conditions such as delayed fixation substantially increased the number of axonal MVBs. Radiolabeled brain- and glial-cell derived neurotrophic factors (BDNF and GDNF) injected into the tongue did not accumulate during retrograde axonal transport in MVBs, as determined by quantitative ultrastructural autoradiography, and confirmed by analysis of quantum dot-labeled BDNF. We conclude that for axonal transport, neurotrophic factors utilize small vesicles or endosomes that can be inconspicuous at transmission electron microscopic resolution, rather than MVBs. Previous reports of axonal MVBs may be based, in part, on artificial generation of such organelles in axons due to dystrophic conditions.

Fig. 1A-C. Schematic of the hypoglossal nerve and representative images at low magnification

A. Hypoglossal nerve showing the target (tongue), myelinated hypoglossal nerve with area used for analysis, and hindbrain. All sampling and analysis was done within a 2 mm segment of the nerve proximal to the hindbrain. In some cases, the target was injected with buffer or neurotrophic factor (NTF). B. Example of a longitudinal section through the hypoglossal nerve showing a myelinated (M) axon shaft. Organelles (arrowheads) are visible in the axoplasm at this magnification. All organelles of interest were subsequently examined at higher magnification (examples shown in Figs. 2, 5). C. Example of a cross-section through the hypoglossal nerve. Some of the organelles that are potential MVBs are marked with arrowheads. Organelles of interest were examined at higher magnification. Scale bar for B, C (shown in C) = 2 μm.

Fig. 2A-H. Images showing five types of MVBs and MVB-like organelles in hypoglossal axons

A. Type-1: Two MVB-like organelles (arrowheads) containing small-sized internal vesicles scattered throughout the matrix. The left MVB-like organelle appears to be located in close vicinity of a microtubule. B. Type-2: Small MVB-like organelle with small-to-medium-sized internal vesicles, some of them adjacent to the outer membrane (arrowhead). C. Type-3: Classic, large MVB with large, distinct internal vesicles. D. Type-4: Large MVB with large internal vesicles that are less distinct (arrowhead), with internal vesicles showing increased electron density. E. Type-5: Large MVB-like organelle with a single, external membrane that lacks distinct demarcations in some areas (arrowhead). The internal vesicles appear partially fused, and the MVB begins to resemble a transitional state between an MVB and a late endosome. F-H. Serial sections through a type 1 MVB. The small MVB in panel G is visible in only one section (70-80 nm), proving that it is spherical rather than tubular. Scale bar for A-E = 250 nm (shown in E). Scale bar for F-H = 250 nm (shown in G).

Fig. 3A-C. Quantification of MVB size and fractional area (FA) of five MVB types in hypoglossal neurons

A. In axons, most MVBs are small, < 0.05 μm², while most MVBs in the soma are 3-4 times larger, 0.1-0.2 μm². B. In normal axons, small MVBs (types 1 and 2), classic (types 3 and 4), and late-endosomal type MVBs (type 5) make up a similar fraction (about one third) of the total fractional area (FA) of MVBs. C. With delayed fixation, the FA of small MVBs, types 1 and 2, decreases, while the FA of large MVBs, especially the late endosomal type 5, increases.

Fig. 4A-D. Morphological features of MVBs at the node of Ranvier and in the axon shaft of the hypoglossal nerve

A. Representative section showing the axon at the node of Ranvier. The node is characterized by a lack of myelin and significantly restricted area of axoplasm (AP) in the “bottleneck” region (between arrows). B. High magnification of the axon shaft, characterized by dense myelin layers. Organelles (arrowheads) in the axon shaft are less numerous than in the paranode/node region. C. High magnification of the node of Ranvier. Note two MVBs (arrowheads), type 3 and type 4, in near symmetric positions, as well as numerous other organelles. D. High magnification of multivesicular-like organelle in an axon shaft apparently caught during membrane invagination (arrowhead) and formation of an internal vesicle. Scale bars: A = 2 μm, B and C =1 μm, D = 250 nm.

Fig. 5A-D. Acid phosphatase activity is rare in axonal MVBs

Immediately perfused and fixed hypoglossal nerves were processed for acid phosphatase activity (A-D). A. Acid phosphatase-positive large MVBs were found in axons, but they were rare. B. Small MVBs, types 1and 2, did not show acid phosphatase activity. C. MVB type 4 lacks acid phosphatase activity. D. Lysosome in Schwann cell, very close to myelin, is acid phosphatase positive (positive control). Scale bars (same for A and B) = 250 nm.

Fig. 6A-D. Delayed fixation significantly increases total fractional area of MVBs and total number of MVBs/axon

A. Cross-section of an axon with delayed fixation is shown at high magnification. Section shows node of Ranvier, as defined by lack of myelin and Schwann cell cytoplasm between myelin layers (asterisk). Organelles that accumulated within bottlenecked axoplasm (AP) include an autophagosome (arrowhead), mitochondria (Mi), classic MVB (large arrow) and MVB-like organelle (small arrow). B. Fractional area of mitochondria and autophagosomes was significantly increased in the node of Ranvier in axons that were treated with delayed fixation. C. Fractional area (FA) of MVBs (all types combined) was significantly increased with delayed fixation (DF) when compared to perfused, immediately fixed axons (Normal fixation, NF). D. Estimated number of MVBs (all types combined) per axon was significantly increased when axons were treated with delayed fixation (DF), indicating that new MVBs have formed. Error bars = SEM; *p<0.05; **p<0.025; ***p<0.005; NF: Normal (immediate) fixation; DF: delayed fixation; Scale bar for A = 500 nm.

Fig. 7A-E. Retrogradely transported neurotrophic factors are not associated with MVBs in hypoglossal axons

I¹²⁵-GDNF and I¹²⁵-BDNF were visualized by autoradiography of thin sections coated with a monolayer emulsion (A-D). A. Silver grain (SG) representing I¹²⁵-GDNF, in axoplasm is not associated with any organelle. B. SG representing I¹²⁵-GDNF in axoplasm next to an undefined structure (arrowhead). C. SG, representing I¹²⁵-BDNF, is not associated with MVB type 1 (arrowhead). D. Dense SG in axoplasm is near, but not associated with MVB-like organelles (arrowheads). Quantum dot-conjugated BDNF (QD-BDNF) was evident by intense electron density of QDs in thin sections (E-F). E. Example of QD-BDNF in the node of a heavily-labeled axon. Many QDs appear to be associated with microtubules (arrows). At higher magnification (inset) it is apparent that the uniformly sized QD-BDNF is composed of multiple QDs (white arrows), possibly within a small endosomal organelle. Scale bar for inset = 50 nm. F. QD-conjugated BDNF (arrow) is located in close vicinity, but not within a “miniature” (type 1) MVB (arrowhead). Scale bars (same for panels A-D) = 200 nm.