Diverse synaptic terminals on rat stapedius motoneurons

Abstract

Stapedius motoneurons (SMN) mediate the contraction of the stapedius muscle, which protects the inner ear from injury and reduces the masking effects of background noise. A variety of inputs to SMNs are known to exist, but their terminal ultrastructure has not been investigated. We characterized the synaptic terminals on retrogradely labeled SMNs found just ventromedial to the facial motor nucleus. About 80% of the terminals contained round synaptic vesicles. One type (Sm Rnd) had small, round vesicles filling the terminal with occasional dense core vesicles and formed an asymmetric synapse. Sm Rnd terminals were small with lengths of apposition to the SMN less than 3 microm. Partial reconstructions from serial sections demonstrated that these terminals formed up to three synapses per terminal. Another terminal type (Lg Rnd) had large, round vesicles and asymmetric synapses. Most Lg Rnd terminals were small but some were extensive, e.g., abutting the SMN for up to 10 microm. One of these terminals formed at least seven synapses. Another terminal type (Pleo) had pleomorphic vesicles and symmetric active zones that, in some cases, were invaginated by spines from the SMN. A fourth uncommon terminal type (Het Rnd) had round vesicles of heterogeneous sizes and asymmetric synapses. A fifth rare terminal type (Cist) had large, round vesicles and an accompanying subsurface cistern in the SMN. These were generally the same kinds of terminals found on other motoneurons, but the high proportion of round vesicle synapses indicate that SMNs receive mostly excitatory inputs.

FIG. 1

HRP-labeled SMN. Center image: low magnification electron micrograph of SMN (neuron R1) with reaction product granules (one indicated) and diffuse darkening of the cytoplasm. Lower left: drawing of the left half of a coronal section of a rat brainstem showing the location of the SMN ventromedial to the left motor nucleus of the facial nerve (VII). The pyramidal tract is outlined at the midline. PVCN posteroventral cochlear nucleus. Upper right: bright-field photomicrograph of the epoxy-embedded labeled SMN in an 80-μm thick Vibratome section. Scale bar = 10 μm (center image), scale bar = 1 mm (lower left), scale bar = 25 μm (upper right).

FIG. 2

Representative electron micrographs showing three major terminal types forming synapses on SMNs. A Sm Rnd synaptic terminal densely packed with small, round vesicles and containing a few dense core vesicles (one indicated with DCV). B Lg Rnd synaptic terminal containing large, round vesicles. C Pleo synaptic terminal with pleomorphic vesicles. A cleft of uniform width and a postsynaptic density in the SMN is seen in each example, designated by arrowheads. The terminals shown in this figure were found on SMN R2. Scale bar = 0.25 μm.

FIG. 3

A–C Five types of synaptic terminals (see Key) on labeled SMNs (cyan). Synapses are marked by arrowheads. A Synaptic terminals with pleomorphic vesicles (Pleo, red), with large, round vesicles (Lg Rnd, orange), or a cistern in the SMN rather than a postsynaptic density (Cist, green). B Synaptic terminal with numerous small, round vesicles (Sm Rnd, yellow) and dense core vesicles (DCV). C Synaptic terminal with round vesicles of heterogeneous sizes (Het Rnd, lavender). D Low magnification electron micrograph showing the distribution of synaptic terminals on the SMN cell body and proximal dendrites. There are 12 Sm Rnd terminals (yellow), 6 Lg Rnd terminals (orange), and 2 Pleo terminals (red) in this section. These synaptic terminals (and other terminals that could not be verified to form synapses) covered 33% of the membrane of the soma and 44% of the membrane of the proximal dendrites. Scale bar = 10 μm. Overall summary of the number of synapses (E) and terminals (F) for the five types: Sm Rnd small, round+DCV; Lg Rnd large, round; Pleo pleomorphic; Het Rnd heterogeneous round; Cist cistern. These data are from the four labeled SMNs from all three rats. A–C are from R2, and D is from R1. Scale bar = 0.5 μm (A–C), scale bar = 10 μm (D).

FIG. 4

Electron micrographs showing examples of Lg Rnd and Pleo synaptic terminals. A Lg Rnd terminal (orange) having the appearance of “beads on a string.” Two parts of this terminal, connected in another section, abut the SMN (cyan). One synapse, convex with respect to the cytoplasm, is indicated (arrowheads). Another synapse was located near the coated pit (indicated on figure) but two sections removed. Note reaction product granules in the SMN. The image is comprised of two digitally merged photomicrographs. B Another Lg Rnd terminal (orange) that abuts the SMN for over 9 μm. The abutment is not wholly contiguous in that small glial processes sporadically intercalate. A synapse with a concave postsynaptic density is visible in this section (arrowheads); three other synapses were formed in other sections. This image is comprised of three digitally merged photomicrographs. C A portion of another Lg Rnd (orange). Note that there are abundant synaptic vesicles although there was no synapse in this part of the terminal. There is a separate axonal swelling (magenta) with a mixture of clear vesicles and large granular dense core vesicles (asterisk). The swelling is in the perifacial neuropil and separated from the SMN by the Lg Rnd terminal. Note that these dense core vesicles are appreciably larger than those in Sm Rnd terminals (yellow, DCV). A Pleo terminal (red) is also visible in the neuropil. D Pleo terminal (red) forming a complex relationship with two finger-like, spinous protrusions of an SMN. One spine is connected to the SMN (shown in this section) and receives a synapse (arrowheads). The other spine was connected to the SMN through the series of sections. Pleo vesicles are clustered at the synapse, although there are not many in the rest of the terminal. Much of the terminal area is taken up by mitochondria. A and C are from R1; B and D are from R2. Scale bar = 1.0 μm (A and B), scale bar = 0.5 μm (C and D).

FIG. 5

View of a reconstruction of a terminal (orange) formed onto an SMN (blue, neuron R1). The reconstruction was generated with 21 serial sections, one of which is shown in Fig. 4A. The terminal forms seven synapses on the SMN (light color, one shown by arrowheads). The origin of the swelling is the myelinated stalk visible at the lower right (my, dark orange color). The gray structure at left is an unlabeled dendrite in neuropil that receives a synapse from the reconstructed terminal. Scale bar = 1 μm.

FIG. 6

Apposition lengths for Sm Rnd, Lg Rnd, and Pleo terminals. Sm Rnd terminals (A) were small with a restricted distribution of apposition lengths ranging from 0.52 to 2.77 μm (mean = 1.65 μm, n = 35). In contrast, Lg Rnd terminals (B) had a broader distribution of apposition lengths ranging from 0.97 to 9.33 μm (mean = 2.77 μm, n = 20). Pleo terminals (C), like Sm Rnd terminals, had short apposition lengths (mean = 1.57 μm, range 0.74 to 2.54 μm, n = 13).

FIG. 7

A High-magnification electron micrograph of a terminal with round vesicles of heterogeneous sizes (Het Rnd, same section as Fig. 3C). A few noticeably large vesicles (arrow) are scattered in the terminal. Arrowheads denote synapse onto SMN R2. Scale bar = 0.5 μm. B Distribution of vesicle areas for the terminal pictured in A. Most vesicles distribute around 12 × 100 nm² but a few larger vesicles distribute around 32 × 100 nm² (arrow).

FIG. 8

Detail of a cistern-type (cistern) synaptic terminal on the soma of neuron R2. This is a different section than Fig. 3A. The bilaminar cistern parallels the abutment of the terminal and the SMN. Note that the cistern is continuous with the endoplasmic reticulum (ER) of the SMN. Large, round vesicles are seen filling the terminal. Scale bar = 0.5 μm.

FIG. 9

A summary of the diverse terminal types on SMNs. The SMNs studied were located ventromedial to the facial motor nucleus. Numbers indicate the relative frequency of occurrence (%).