Jacob Davidowitz

Expression of a novel combination of fast and slow troponin T isoforms in rabbit extraocular muscles

SummaryThe properties of extraocular muscles (EOMs) are quite different from those of the trunk and limb. Here we show that there is a novel pattern of troponin T (TnT) expression in EOMs which most likely contributes to the fine control of ocular movement and may reflect their innervation by cranial motoneurons. Three regions of the muscle were analysed to distinguish the TnT isoforms present in the fast singly-innervated fibres from those in the multiply-innervated fibres. More than 95% of the TnT in the singly-innervated fibres is TnT3f, which exhibits the most graded response to changes in calcium concentration during activation (Schachatet al., J. molec. Biol.198, 551–4). In multiply-innervated fibres, which exhibit tonic contractures, the slow troponin T TnT2s is expressed. While neither TnT3f nor TnT2s is unique to EOM, this pattern is unusual in two respects: first, both TnT3f and TnT2s are minor components of the trunk and limb musculature, and second, most muscles express several fast and both slow TnT species. Although EOM occupies a highly specialized physiological niche, its unusual physiology is not reflected in the presence of new TnT isoforms but in the expression of a different ratio of the known species of TnT.

Light and electron microscopic serial analysis of mouse extraocular muscle: Morphology, innervation and topographical organization of component fiber populations

Mouse superior rectus extraocular muscle was examined in serial section by light and electron microscopy. By such analysis, it was possible to discriminate single versus multiple innervation, characteristics of internal cell morphology, and topographical distribution of the respective fiber populations within the muscle. Singly innervated (SIF) and multiply innervated fibers (MIF) were observed, both in an orbital surface layer and in the underlying global region of the muscle. Five morphologically distinct fiber types (three SIF and two MIF) were discriminable in terms of fiber diameter, mitochondrial richness, development of the sarcoplasmic reticulum, and myofibrillar size. Many fibers both SIF and MIF, terminated variously along the length of the muscle. The diameter of orbital MIF typically varied from one end of the fiber to the other by a factor of about three; the global MIF were of essentially constant diameter. The junctional complexity varied among the respective types of SIF. The MIF of both the global and orbital regions exhibited comparable ranges of complexity in their neuromuscular junctions.

Notes on Technic: “Clearing” Steel Knife Epon Sections in a Polystyrene Film Sandwich

Serial sectioning epoxy embedments by steel knife permits rapid light microscope survey of large tissue volumes, and preselection of areas of interest for electron microscopy. Acetate film (Hollander 1970) and Turtox plastic slides (West 1972) have been suggested as substrates upon which the sections may be “cleared” with an added layer of cured epoxy. In our experience, these substrates are excessively adherent to Epon, and “cleared” sections thinner than 40-50 μm cannot be released from them reliably. The following method is suitable for processing Epon sections 10 or more microns thick.

Myotonic muscle in mouse. A light and electron microscopic study in serial sections.

Abstract The EDL muscle of the Bar Harbor Strain C57BL 6Jdy 2j mouse was studied in serial section by light and electron microscopy. Phase contrast microscopy of 15 μm epon sections revealed fiber abnormalities such as central nucleation, atypical fiber diameter and regions of focal fiber necrosis; fiber splitting detected in such sections was a prominent feature of the myotonic muscle. Ultrathin resectioning of selected 15 μm sections showed additional alterations such as dilation of the sarcoplasmic reticulum, mitochondrial conglomerations and disruptions, sarcolemmal infolding and breakdown, and myofibrillar degeneration. The most notable neuropathic changes included distortion in shape and decrease in numbers of synaptic vesicles as well as axonal retraction, reduplication of the basement membrane, and the presence of dense bodies between axon and junctional sarcolemma. These data indicate the involvement of both muscle fiber and motor end plate in mouse myotonic dystrophy.

Cisternal distention in membrane-glycogen complexes of rabbit extraocular muscle.

Arrays of glycogen layers alternating with the double membranes of flattened cisternae were observed in apparently normal muscle. The cisternae of such arrays appeared in both nondistended and evenly distended forms. The distended cisternae retained their structural integrity in contrast to a frequently seen disruption of those that were not distended. Cisternal distention was related to the extent of glycogen deposition on membranes that were partially glycogen-bearing. Randomly organized membrane—glycogen complexes were also seen in which the cisternae consistently appeared in a distended form.

Morphological changes associated with the myoneural junction in extraocular muscle of the dystrophic mouse

SummaryAbnormalities of the muscle fiber in the sub-junctional region were observed in association with both normal and disrupted terminal axons. The most frequently observed myopathic change was the presence of extensive areas of densely packed vacuoles beneath the junction. Fibers containing such conglomerations of vacuoles also manifested swollen sarcoplasmic reticulum. Degeneration of terminal axons was evidenced by swelling, a decrease in the number of synaptic vesicles, and fragmented mitochondria. These findings suggest an involvement of the myoneural junction in the etiology of muscular dystrophy in the extraocular muscles of the mouse.

Scanning electron microscopy of etched Epon extraocular muscle sections.

The internal fine structure of extraocular muscle cells was observed with scanning electron microscopy. One micrometre Epon sections were etched with an iodine acetone solution. When adhered to carbon coated glass, such specimens may be viewed without surface coating. Such preparations provide information about the fibrillar organization of the muscle fibre, as well as its transverse tubular system and M line.

Intermitochondrial junctions in the extraocular muscle of the rat

SummaryIntermitochondrial junctions with a spacing of 17–21 nm were observed in the superior rectus muscle of a rat. Periodic rounded densities are aligned midway between the apposed outer mitochondrial membranes at some of these junctions. Such densities have a diameter of about 8–10 nm and a center-to-center spacing of about 26–30 nm. These junctions occur in cases where one mitochondrial profile is enclosed within another or where two profiles are interlocked so that their combined overall form has a smoothly contoured profile. Intermitochondrial junctions seem not to have been previously described in muscle, but have been reported in other kinds of tissues. In agreement with those previous reports, the presently observed intermitochondrial junctions usually involve mitochondria that display atypical features indicative of tissue abnormality or stress. Such junctions were never observed in normal extraocular muscle.

Membrane—glycogen complexes in rabbit extraocular muscle

Analysis of 432 electron micrographs of membrane-glycogen complexes revealed that: (1) Golgi apparatus is closely associated with 4.2% of the complexes, such associations occurring irrespective of the degree of glycogen loading in the complex. (2) Apparent ribosomes are seen in association with about 30% of the complexes, either attached to membranes or enclosed between cisternae. (3) In longitudinal sections of the muscle fibers, complexes may form columns which extend for as much as 40 microns along the fiber. (4) Various cytoplasmic organelles may become enclosed within a complex. (5) Some cisternae of a complex may assume the form of randomly oriented tubules, in contrast to the typical systematic array of flattened cisternae. (6) Some cisternae of a complex may become distended in a wide and uneven manner, in contrast to the typical narrow and even distension.

The distribution of membrane-glycogen complexes in the orbital surface layer of rabbit superior rectus

The distribution of membrane--glycogen complexes along the length of individual muscle fibers was compared among three fiber populations in the orbital surface layer of rabbit superior rectus. These three populations were (a) 61 singly innervated fibers (SIFs), (b) 10 multiply innervated fibers of relatively constant 10 micrometer diameter (10 micrometer MIFs), and (c) 22 multiply innervated fibers which are of about 5 micrometer diameter toward the middle of their length and of about 15 micrometer diameter toward their proximal and distal segments (5--15 micrometer MIFs). The orbital surface layer was sampled by electron microscopy at 68 sequential locations. Membrane--glycogen complexes were not seen in any of the 1738 samples of the SIFs. In the MIFs, such complexes were observed in 14% of the 1541 samples. However, both the 10 micrometer MIFs and 5--15 micrometer MIFs displayed a preferential concentration of membrane--glycogen complexes toward their distal fiber portions, and such complexes were seen in about 50% of the MIF samples near the beginning of the muscles distal third. In the distal portion of 5--15 micrometer MIFs, there was a direct relationship between their increasing fiber diameter and their increasing frequency of occurrence of membrane--glycogen complexes.