Myoneural and intermuscular junctions in a molluscan smooth muscle

Copyright © 1973 by Academic Press, Inc. All rights of reproduction in any form reserved

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M y o n e u r a l and I n t e r m u s c u l a r Junctions in a Molluscan S m o o t h Muscle 1,2 OLIVIA C. MCKENNA a n d JACK ROSENBLUTH

Departments of Physiology and Rehabilitation Medicine, New York University School of Medicine, New York, New York 10016, and Marine Biological Laboratory, Woods Hole, Massachusetts Received June 1, 1972, and in revised form November 6, 1972 The anterior byssus retractor muscles of two bivalve molluscs, Mytilus edMis and Brachidontes demissus, were studied by electron microscopy and fluorescence histochemistry for monoamines to determine whether two nerve ending types, proposed in earlier pharmacological studies, could be distinguished morphologically. By means of fluorescence microscopy a fluorescent compound compatible with 5-hydroxytryptamine can be visualized in beaded nerve fibers within formaldehyde-treated muscle specimens. Examination of the ultrastructure reveals myoneural junctions characterized by vesicle-containing nerve terminals which are consistently separated from muscle cells by a gap of 150-200~. Junctional specializations such as a striated gap material, "thickening" of preor postjunctional membranes, and mitochondria in close apposition to the postjunctional membrane occur only inconsistently, however. The nerve endings exhibit wide variations in the proportion of clear to dense cored vesicles thus preventing a clear visual or statistical separation of the endings into distinct classes. However, a scatter diagram relating the proportions of the two vesicle types to the total number of vesicles per nerve ending profile suggests a possible separation into two groups of nerve endings, one containing relatively few vesicles of which, on the average, roughly half are clear and half dense cored, and the other containing a much larger total number of vesicles per ending of which roughly 90 To are clear. It is proposed that the first type of nerve ending according to this classification is serotonergic and the second type cholinergic. Two forms of intermuscular apposition also occur. In one the apposed membranes are separated along a considerable length by a gap of 100-160 A containing no collagen or other connective tissue elements. In the other, equivalent to the "nexus" of vertebrate smooth muscle, the gap is even narrower (20-40 ~). Either or both of these junctions may underlie electrical coupling in the anterior byssus retractor. 1 A report of this investigation was presented at the annual meeting of the American Association of Anatomists held in April, 1972 (16). 2 This work was supported by Grants NS-09331 and NS-07495 from the National Institutes of Health.

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O n the basis of physiological and pharmacological studies it has been proposed that the anterior byssus retractor muscle of the bivalve mollusc Mytilus (ABRM), receives a dual innervation, one producing a sustained contraction and the "catch" state, and one responsible for relaxation (27, 28). The first nerve type is thought to be cholinergic since acetylcholine has been shown to mimic nerve stimulation in producing depolarization of the muscle membrane and subsequent prolonged contraction of the muscle. The action of both acetylcholine and nerve stimulation can be potentiated by anticholinesterases and blocked with cholinergic antagonists. In addition, an acetylcholine-like compound has been identified within the muscle by bioassay. Comparable evidence has been collected for a serotonergic innervation. After cholinergic blockade 5-hydroxytryptamine (5-HT) produces a fall in tension similar to that produced by electrical stimulation of the muscle and the effect of both can be partially blocked by a 5-HT antagonist (7). A 5-HT like compound has been measured within the muscle by bioassay. The present study was undertaken to determine whether 5-HT could be localized in this muscle in two species, MytiIus edulis and Brachidontes dernissus, by fluorescence histochemistry and to determine whether two types of myoneural junction corresponding to the two transmitters could be identified by electron microscopy.

METHODS

Mytilus edulis was obtained at the Marine Biological Laboratory (MBL) in Woods Hole, Massachusetts and from a New York fish market, and Braehidontes dernissus was collected from Long Island Sound. The mussels were maintained either in running natural sea water at the MBL or in an artificial sea water aquarium at 10-12°C for up to 2 weeks. Fluorescence histochernistry. Pieces from the ABRM were freeze-dried and exposed to formaldehyde fumes according to the histochemical method of Falck and Hillarp for the detection of catecholamines and 5-hydroxytryptamine (11, 12). Other animals were either immersed in 100 mg/100 ml nialamide in sea water at 10-12°C for 2-6 days or immersed in 5 rag/100 ml 5-hydroxytryptamine for 10, 30, or 60 minutes. Dissecting needles were placed between the shells to prevent their closing. After drug treatment, one ABRM was freeze-dried and studied histochemically and the other from the same animal was prepared for electron microscopy. Electron microscopy. The ABRM was fixed either by flooding the extended muscle in situ or by immersing the excised muscle, which contracts upon removal, in the fixative. The muscles were fixed in 4% paraformaldehyde--5% glutaraldehyde in 0.1 M phosphate (pH 7.4) buffer, as described by Karnovsky (14), rinsed in saline or buffer, and postfixed in 1 or 2 % osmium tetroxide in either phosphate or s-collidine buffer at the same pH. Alternatively, the ABRM was fixed in 6% glutaraldehyde in 0.2 M s-collidine buffer with the osmolarity adjusted to 1 200 m0s by the addition of sucrose, subsequently rinsed, and postfixed in osmium tetroxide (4). On one occasion the muscle was fixed in potassium per-

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manganate in 0.1 M phosphate buffer (pH 7.0). All tissues were dehydrated in increasing concentrations of methanol and embedded in Araldite. Silver sections (~700 A) were cut on a Porter-Blum microtome and examined in a Philips 300 electron microscope.

RESULTS The ABRM is composed of densely packed cylindrical muscle cells and is surrounded by an epimysial sheath composed primarily of collagen fibrils, but also containing nerve fibers and scattered muscle cells (Fig. 1). The muscle cells in the sheath resemble those within the body of the muscle but are more widely spaced~ smaller, and do not necessarily run parallel to one another. The innervation of the ABRM is derived from the cerebropedal connectives which travel along the dorsal surface of the muscle. Axons which arise from the connectives are found not only in the epimysium but also within the underlying compact muscle (Fig. 2). Accompanying the axons are glial elements which partially enwrap them and which can be readily identified by their large, homogeneously dense, irregularlyshaped "gliosomes" (19). The latter are very conspicuous in toluidine blue-stained sections viewed by light microscopy.

Fluorescence histochemistry When freeze-dried tissue is treated with formaldehyde fumes, a very dim fluorescence within beaded fibers can be detected, but it fades almost immediately with exposure to ultraviolet light. The fluorescence intensity is so weak that a color cannot be ascribed to it. After mussels are immersed in a nialamide solution for 2 to 6 days and subsequently prepared for histochemistry, faint yellow fluorescent dots joined together by even fainter fine fibers are seen throughout the tissue (Fig. 2 inset). These structures also fade with ultraviolet light exposure. Relatively few fibers are found, and these are distributed all along the length of the muscle, both proximally, distally and within the depths of the muscle as well as along its surface. In general the fluorescent fibers tend to run parallel to the long axis of the muscle cells although occasionally they are seen running at an angle to this axis. No yellow fluoroescent cell bodies have been seen but red autofluorescent cells are occasionally included in the specimen. In control tissue not exposed to formaldehyde fumes, bright gold fluorescent granules are seen scattered randomly throughout the muscle. The fluorescence of the yellow varicose fibers differs from that of the autofluorescent gold granules in color, in its sensitivity to ultraviolet light and in the requirement of formaldehyde treatment before it can be visualized. When animals are pretreated with 5-HT, all the ceils within the tissue including the muscle cells themselves develop a yellow fluorescence which obscures the nerve

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FIG. 1. Lightmicrographof the ABRM. At the peripheryof the musclelies a connectivetissuesheath which includesscatteredslendermuscleprocesses (arrows). × 630. fibers. This observation indicates that in this animal 5-HT is accumulated nonspecifically by cells other than neural elements and consequently localization by radioautography is not feasible.

Ultrastructure of myoneuraljunctions Myoneural junctions have been identified both in the body of the muscle and in the epimysial sheath, which also contains a few muscle cells. However, since previous physiological and pharmacological studies have been concerned exclusively with the body of the muscle, only those endings found within the muscle proper have been illustrated and used in the statistical analysis. Axons with their companion glial processes travel within the endomysial connective tissue parallel to the muscle fibers forming periodic expansions, which usually contain accumulations of vesicles (Fig. 2). In junctional regions the nerve and muscle plasma membranes are closely apposed, with no collagen fibrils or basement lamina interposed between them. The cleft between the respective membranes is 150-200 A wide. A glial process is often, but not always, present along the nonjunctional surface of the nerve ending. Most fre-

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F~G. 2. Electron micrograph showing 2 nerve fibers containing dense-cored and clear vesicles and a companion glial process, which contains large dense gliosomes (G), traveling in the connective tissue space between the muscle cells. x 13 000. Inset: Fluorescence micrograph of a beaded nerve fiber found in the ABRM after pretreatment with nialamide, x 670. FIG. 3. Nerve fibers, with glial processes, running along the surface of a muscle cell forming myoneural junctions. Note the variety of vesicle types present in the 2 nerve endings and their extensive contact with the muscle cell. x 17 000.

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Fins. 4 and 5. Extremes of nerve ending types. FIo. 4. Nerve ending packed with clear vesicles. A few larger vesicles containing punctate material are also present (arrows). In the postjunctional cell a mitochondrion (M) is in close proximity to the muscle membrane and a coated vesicle (C) is seen. x 52 000. FIo. 5. A nerve ending with extensive contact with a muscle cell contains predominantly dense-cored vesicles ranging in diameter from 700 to 1 200 ~. Only a few clear vesicles are present, x 61 000.

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FIc. 6. Nerve ending containing a mixture of clear and dense-cored vesicles, a mitochondrion (M), and some glycogen particles. The dense-cored vesicles vary from 800 to 1 200 A in diameter, and a dense material is present within the synaptic cleft. Glial process contains gliosomes (G). x 46 000.

quently the nerve comes to lie on the muscle cell without producing any change in the contour of the cell and often areas of extensive contact are formed (Fig. 3). Occasionally, however, the nerve ending may form an indentation in the surface of the muscle cell (Figs. 7 and 9). In general the junctional portion of the axon is filled with glycogen, a mixture of clear and dense-cored vesicles, and occasionally a mitochondrion or some microtubules (Figs. 4-9). The clear vesicles, which are usually clustered against the prejunctional membrane, are circular in profile and measure 400-600 A in diameter (mean ~ 500 A). Dense-cored vesicles, which are usually scattered randomly throughout the nerve ending, vary in diameter from 700 to 1 200 A, (mean ~ 1 000 A) and contain material of varying density with a clear halo interposed between it and the vesicle membrane. The diameter of the dense core ranges from 350 to 1 100 A, with a mean of N 800 A. The appearance of both vesicle types is similar in all nerve endings found, but the ratio of clear to dense-cored vesicles varies markedly from one ending to the next. At one extreme are endings containing no dense-cored vesicles and a large number of

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FIG. 7. Nerve ending with a companion glial process (G) forms a junction with a muscle cell in the vicinity of its nucleus. Mitochondria, many free ribosomes, and nuclear pores (arrows) are apparent, x 22 000. Inset: Detail of the same myoneural junction showing material adhering to the cytoplasmic surface of both pre- and postjunctional membranes and a clustering of clear vesicles near the axolemma. Fine dense projections appear to bridge the junctional cleft (arrows). x 120 000.

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Fro. 8. Nerve ending with mixed population of vesicles has a prejunctional membrane which exhibits a distinct coating on its cytoplasmic surface. The muscle cell has a slightly darkened postjunctional membrane. Fine granular material is found in the junctional cleft running parallel to the two membranes (arrow). A hemidesmosome is present nearby (/t). × 55 000.

clear vesicles, and at the other are endings filled primarily with dense-cored vesicles and few clear vesicles. Figure 4 illustrates an ending which contains a great many clear vesicles. A few larger vesicles with punctate material are present, but their relatively low density and coarser granularity distinguishes them from dense-cored vesicles. This third type of vesicle is also found in endings that contain dense-cored vesicles. Figure 5 illustrates the opposite extreme. Only a few clear vesicles are present among a large number of densecored vesicles which vary widely in diameter and density. This ending has an extensive area of contact with the muscle cell, and vesicles appear to be randomly scattered rather than clustered against the prejunctional membrane. Most endings fall between_ these extremes; i.e., they contain a more even mixture of vesicle types (Figs. 6-9). Pretreatment with 5-HT or nialamide has no apparent effect on the relative proportion of clear to dense-cored vesicles.

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FIG. 9. Nerve ending, with its glial covering (G) and containing a variety of vesicle types, forms an indentation in a muscle cell. A mitochondrion (M) is closely applied to the postsynaptic membrane. × 45 000.

Occasionally other junctional specializations can be found. The prejunctional membrane may have a layer of material on its cytoplasmic surface which gives it a denser appearance than the adjacent nonjunctional membrane (Fig. 7 inset and Fig. 8). Such "thickening" of the postjunctional membrane is less frequent (Fig. 7 inset). Within the cleft separating these 2 membranes a slightly dense homogeneous material may be present which sometimes forms a faint line parallel to the pre- and postjunctional membranes (Fig. 8). Rarely, faint hairlike projections bridge the gap between the membranes (Fig. 7 inset). Mitochondria may also occur in the postjunctional cell near the junctional area (Figs. 4 and 7), and in some instances are very closely applied to the postjunctional membrane (Fig. 9). Although examples of these various specializations can be found, none of them appears consistently in relation to the ratio of dense-cored to clear vesicles.

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TOTAL (CLEAR+ DENSE CORED)VESICLES PER PROFILE FIG. 10. Scatter diagram relating total number of clear vesicles to the percentage of clear vesicles in each nerve ending profile: clear vesicles/(clear + dense-cored vesicles) x 100. The dots represent these coordinates for each of fifty-eight nerve ending profiles, and the x's represent estimates of the expected values if the clear and dense-cored vesicles vary independently.

Because of the variability in the proportion of dense-cored to clear vesicles within the nerve ending profiles and the inconsistency of •the1 junctional specializations, a clear separation of the nerve endings into 2 or more populations cannot be made visually as has been done elsewhere (21). Even statistical analysis which takes into account not only the ratio of clear to dense-cored vesicles, but also the absolute number of vesicles in each nerve ending profile, is consistent with the existence of either a continuous spectrum of nerve endings or of two different populations which overlap (see below).

Statistical analysis of the nerve endings The numbers of dense-cored and clear vesicles were counted in each of the fiftyeight nerve ending profiles and tabulated. The raw counts were then corrected by Abercrombie's method (1) for errors due to section thickness (,-~ 700/~) and the difference in the average diameters of the two classes of vesicles (clear, N 500 ~ ; densecored, ,,~ 1 000 A) as follows: the corrected count, in a nerve ending profile, of each type of vesicle = raw count x [section thickness/(vesMe diameter + section thickness)]. The percentage of clear vesicles in each profile [clear vesMes/(clear +dense-cored vesicles) x 100] was next calculated from the corrected vesicle counts, and the results

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TABLE I JOINT DISTRIBUTION OF DENSE-CORED AND CLEAR VESICLES IN F I F T Y - E I G H T NERVE E N D I N G PROFILES a

Clear Vesicles

r-0-3

4--7

0-19 20-39 40-59 > 60 Total

13 (13.4) 3 (3.1) 1 (2.1) 3 (1.4) 20

17 (15.4) 4 (3.6) 2 (2.4) 0 (1.6) 23

Dense Cored Vesicles -~8-11

6 (6.7) 2 (1,6) 1 (1,0) 1 (0.7) 10

~>12

, Total

3 (3.4) 0 (0.8) 2 (0.5) 0 (0.3) 5

39 9 6 4 58

Of each pair of figures, the one on the left is the observed number and the one in parentheses is the number that would be expected assuming independent distribution of dense-cored and clear vesicles. are plotted in a graph of percemage of clear vesicles vs. total number of vesicles per profile. The scatter diagram thus obtained shows that in nerve endings containing relatively few vesicles there is a wide variation in the ratio of clear to dense-cored vesicles whereas in endings containing relatively large numbers of vesicles the clear vesicles compose a large proportion of the total (Fig. 10). Table I illustrates the joint distribution of clear and dense-cored vesicles in the fifty-eight nerve ending profiles. By means of the chi-square test it was determined that the clear and dense-cored vesicles vary independently within each ending (Z2= 13.3, p >0.10). Estimates of the expected values of the percentage of clear vesicles as a function of the total number of vesicles per profile (indicated by x's in Fig. 10) are seen to be distributed similarly to the points derived from the data. Such a scatter diagram could arise from a single population of nerve ending profiles in which the clear and dense-cored vesicles vary independently but in which the maximum number of dense-cored vesicles occurring in any single profile is much smaller than the maximum number of clear vesicles (17 vs. 111, respectively). Thus, with increasing numbers of vesicles per profile, especially beyond 17, the relative proportion of clear vesicles increases continuously, resulting in the type of distribution seen in Fig. 10. Another possible interpretation is that the nerve endings represent a mixture of two populations whose extremes overlap and which cannot be separated by statistical methods. Visual inspection of the graph suggests a division of the points into two groups at approximately T = 30. The population of endings to the left can then be characterized as having relatively few vesicles, of which roughly half are clear. Those at the right, in contrast, contain approximately four times as many vesicles per profile; of these roughly 90 % are clear.

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FIG. 11. Close intermuscular junctions. Three muscle cells (I, 2, 3) are shown. The upper two (1 and 2) are s e p a r a t e d by the typical intercellular space (IS) filled with collag,~n fibrils, and the lower two (2 and 3) are closely apposed ove~ a considerable distance. A hemidesmosome is present in the uppermost cell (1). x 51 000. Upper inset: A focal narrowing of the closely apposed membranes. × 103 000. Lower inset: Detail of a close junction with a cleft of 100-160 A. x 130 000.

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Fto. 12. Intermuscular gap junction. Three cells (1, 2, 3) are present. The center cell (2), which forms a gap junction with the upper cell (1), is presumed to be a muscle cell by the similarity of its Cytoplasmic components to the two muscle cells (1 and 3). The overall width of the apposed membranes is 165 ~. The more typical intercellular space (IS) is filled with collagen fibrils. Fixed in glutaraldehyde and posffixed in OsO,. x 43 000. Upper inset: Detail of a gap junction seen in tissue fixed with formaldehyde and posffixed in OsO4. The gap is ~ 30 ~ wide. x 130 000. Lower inset: Detail of a gap junction seen in tissue fixed with potassium permanganate. × 180 000.

Intermuscular ./unctions The muscle cells within the A B R M run parallel to one a n o t h e r a n d are generally s e p a r a t e d b y a relatively u n i f o r m space of 1 000-4 000 • which contains collagen fibrils. This space is lined b y the b a s e m e n t m e m b r a n e of the cells flanking it. T w o types of closer a p p o s i t i o n of muscle cells have been observed. A t the first, the interm u s c u l a r cleft, which is only 100-160 A wide, contains no collagen fibrils or basem e n t m e m b r a n e m a t e r i a l (Fig. 11). A n y u n d u l a t i o n in one m e m b r a n e is reflected in the a p p o s i n g m e m b r a n e , so t h a t the w i d t h of the j u n c t i o n a l gap varies only slightly. Such regions of a p p o s i t i o n m a y extend 6 # m in the section plane, b u t no c y t o p l a s m i c 30-- 731822 J . Ultrastructure Research

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densities or other structural specializations are associated with them. In one instance a focal narrowing of the 150 ~ intercellular space was observed (Fig. 11, upper inset). In the second type, the gap between the membranes is only 20-40 A wide, again with no visible intercellular material intervening and with no specializations of the apposed membranes (Fig. 12). The extent of the junction in transverse sections of the muscle is ~ 0.4-0.8 #m, and in longitudinal sections ~ l-2/~m. Occasionally (Fig. 12) a process of one muscle cell appears to indent a second cell to form such a junction (cf. 22), and in this case the length along which the respective membranes is apposed may be as much as 2.5-4.5/~m. These 20-40 A gap junctions have been observed between muscle cells in tissue fixed in glutaraldehyde and/or paraformaldehyde, followed by osmium tetroxide, or in potassium permanganate (Fig. 12 insets) and are therefore probably not artifacts of a particular preparative technique.

DISCUSSION

Intermuscular junctions The present study has demonstrated the existence of two specialized types of intermuscular apposition in the ABRM. Regions at which the muscle cell membranes are separated by ~ 20-40 ~ are morphologically equivalent to the "nexuses" (10), or "gap junctions," which are thought to mediate electrical coupling (13). Regions at which the apposed muscle cell membranes are separated by ~ 150 ~ over a large area could also theoretically mediate electrical coupling, depending on the resistance of the apposed membranes even without closer proximity (5). Thus in the ABRM, which appears to be sparsely innervated, either or both of these intermuscular junctions may be responsible for the spread of current, initiated by neural stimulation to noninnervated muscle cells in a manner equivalent to that which occurs in the unitary smooth muscles of vertebrates.

Myoneuraljunctions By means of fluorescence histochemistry, we have shown that the 5-HT, previously measured by bioassay or biochemical analysis (9, 28), is localized to nerve fibers running between the muscle cells. Myoneural junctions visualized by electron microscopy are characterized by a junctional gap of 150-200 ~ containing no collagen fibrils or basement lamina. Other junctional specializations, however, including the cleft material and cytoplasmic coatings on the pre- and postjunctional membranes occur only inconsistently, and, as the statistical analysis shows, the endings can be regarded either as a broad single population or as a mixture of two populations. Although both of these interpretations are possible, the previous pharmacological studies indicating a dual innervation of the A B R M (27, 28) favor the second one. In

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addition, when the endings are separated into two groupings and characterized, they resemble serotonergic and cholinergic nerve endings found in muscles of a variety of species. The first population of nerve endings, which can be characterized as having a relatively small number of vesicles about half of which are dense-cored on average, resembles serotonergic neurites described in other invertebrate nervous systems. In the Aplysia heart, 5-ttT, which has been visualized in nerve fibers by fluorescence histochemistry as well as measured in the perfusate after eardioaccelerator nerve stimulation, has been localized in autoradiographic studies over nerve terminals containing both clear and dense-cored vesicles (8, 24, 25). The ApIysia nerve ending illustrated in plate 4a of reference 25 resembles endings in the ABRM not only with respect to the presence of both vesicle types but also in having a relatively small total number of vesicles within the nerve ending profile. Vesicles similar in diameter and density to those in endings in the ABRM have also been found in the serotonincontaining cell body of the leech (23). On morphological grounds we therefore propose that the first type of ending in the ABRM, as characterized above, is serotonergic. Endings of the second type closely resembles defined cholinergic nerve endings in vertebrate skeletal, smooth and cardiac muscle (6, 20, 26) in that they contain large numbers of vesicles the great majority of which are clear. In the molluscan heart and somatic muscles of Ascaris and Lumbricus, which are known to have a cholinergic innervation, endings with large numbers of clear vesicles can also be found (17, 21, 22). It is proposed therefore that the second type of nerve ending that wehave characterized in the ABRM represents the cholinergic innervation suggested by earlier pharmacological studies. The mixing of both clear and dense-cored vesicles seen in the nerve endings of the ABRM is not an unusual phenomenon. Clear vesicles are found in mammalian peripheral noradrenergic terminals (20) as well as in neurosecretory endings (2) while an occasional dense-cored vesicle is found in cholinergic endings in vertebrate skeletal muscle (6, plates 1 and 4b). The frequency of dense-cored vesicles in endings with a preponderance of clear vesicles appears to be higher in invertebrates, as demonstrated in muscles of blowfly larva (18), crayfish (15), slug (3), and earthworm (21). The occurrence of both vesicle types in all of these different endings raises a question about their function in each case. The clear vesicles found in cholinergic endings pre' sumably represent packets of acetylcholine while in serotonergic terminals they may represent membrane recaptured after neurotransmitter discharge or immature serotonin storage organelles which will develop a dense core and increase in size as 5-HT and its carrier protein accumulate. Conversely, the dense-cored vesicles present in serotonergic neurons most likely are the storage organelles for 5-HT, whereas in cholinergic nerve endings they may be related to the metabolism of the endin~ ~

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contain a t r o p h i c substance. Thus both dense-cored and clear vesicles appear not to be single entities, but rather to have diverse functions depending on their location. The authors wish to express their appreciation to three members of the Department of Environmental Medicine, New York University Medical Center; Dr Bernard Pasternack and Ms Laurel Ehrlich for their advice about the statistical analysis and assistance in its computation, and Dr Bernard Altshuler for helpful discussions on the analysis of the vesicle counts. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28.

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