Z. Zellforsch. 96, 206--211 (1969)

Axonal Protrusions in the Small Multiple Endings in the Extraocular Muscles of the Rat H. TERXVXINEN* Department of Anatomy, University of Helsinki, Helsinki 17, Finland Received December 23, 1968

Summary. A special type of myoneural junction has been observed in the extraocular muscles of the rat with electron microscopy. These axon terminals are derived from unmyelinated nerves and contain synaptic vesicles and mitochondria. The terminals are invested by teloglia cells and separated by a synaptic cleft of about 500 A from a slow-type muscle fibre. From the nerve ending a pseudopod-like evagination projects into the muscle cell. The membranes of this evagination and the muscle cells are only separated by a narrow cleft of about 100 A, which is devoid of the basement membrane-like material typical of ordinary myoneural junctions. The evagination contains fewer axonal vesicles than other regions of the terminal axoplasm and the postsynaptic part of the muscle plasma membrane in this special region does not exhibit the postsynaptic thickening characteristic of ordinary myoneural junctions. The extraocular muscle fibres in the r a t are i n n e r v a t e d n o t only b y o r d i n a r y m y o n e u r a l j u n c t i o n s (motor end plates) derived from m y e l i n a t c d nerves b u t also b y m y o n e u r a l j u n c t i o n s derived from u n m y e l i n a t e d nerves called " s m a l l multiple e n d i n g s " , since these endings are very small a n d 3 - - 2 0 endings i n n e r v a t e a single muscle fibre (TERXVXINEN, 1968a). The physiological f u n c t i o n of these multiple endings has been a m a t t e r of m u c h confusion i n the literature (cf. TERXVXI~EN, 1968a). Their fine s t r u c t u r e resembles t h a t of the cholinergic excitatory synapse (TERXVXINEN, 1968a) a n d t h e y exhibit a n acetylcholinesterase a c t i v i t y both in the light microscope (TERXVXINEN, 1968a) a n d the electron microscope (TERXV~NEN, 1968b). T h e y are therefore likely to be cholinergic excitatory endings, p r e s u m a b l y of the g a m m a t y p e (KuPFER, 1960; WOLTER, 1964). The present work describes o b s e r v a t i o n s showing a special kind of m y o n e u r a l c o n t a c t a m o n g these small multiple endings i n the r a t extraocular muscles.

Material and Methods The rectus superior, medialis and lateralis muscles of adult Sprague-Dawley rats were removed under ether anaesthesia and fixed immediately for 2--3 hours at 4~ C with 2.5% glutaraldehyde buffered to pH 7.2 with phosphate (SABATINIet al., 1963). The muscles were stored at 4~ C for 1--4 days in 0.25 M sucrose and then fixed with 1% OS20s in the phosphate buffer for 2 hours, dehydrated with graded ethyl alcohol series, transferred to propylenetetroxide and embedded in Epon 812 (LUFT, 1961). Sections were counterstained in grids with lead citrate (REYNOLDS, 1963) for 10--15 minutes and micrographed with a Philips EM-200 electron microscope. * The author thanks Professor ANTTI TELKKX, M.D., Head of the Electron Microscope Laboratory, University of Helsinki, for permission to use the facilities of the laboratory.

Small Multiple Endings

207

Results As described earlier (TER~V)~INEN, 1968a), the small multiple endings in the rat extraocular muscles are derived from unmyelinated nerves making synaptic contacts with a slow-type muscle fibre, the terminals being either separated form each other along the course of the axon or grouped close together. The axon terminals are apposed to the thickened postsynaptic membrane of the muscle fibre and contain a few larger dense-core vesicles among the common 500 A synaptic vesicles. The primary synaptic cleft measured about 400--1,000 A and the secondary synaptic clefts were irregular and sparse or even absent. The fine structure of three out of a total 126 small multiple endings examined were observed to differ markedly from the structure of ordinary motor myoneural junctions. I n these three endings, the terminal axon sent a kind of axonal protrusion or foot, which invaginated the postsynaptic membrane of the muscle fibre (Fig. 1). The synaptie axoplasm contained numerous " e m p t y " vesicles measuring about 500/~, a few dense-cored vesicles measuring about 1,000 A and some mitochondria, like those earlier observed in ordinary small multiple endings. The main axon terminal was separated from the electron-dense postsynaptic membrane of the muscle fibre by a cleft which had the usual width of about 500 A, whereas the cleft between the axonal protrusion and the muscle plasma membrane measured only about 100/~ or even less (Figs. 2, 3). The number of ordinary 500 A axonal vesicles was reduced in the invaginated axonal foot (Figs. 2, 3) compared with other parts of the terminal axoplasm, although vesicular material of very low electron density and faintly visible membranes were present. In addition, the thickness of the muscle plasma membrane was not increased in this region (Figs. 2, 3), as it was in ordinary postsynaptic regions of the same ending (Fig. 2). I n these protruding areas of close membrane apposition no basement membrane-like material was seen (Figs. 2, 3), while it was present in the synaptic cleft between the axon and muscle plasma membranes (Figs. l, 2). The location of the axonal foot was not constantly related to particular structures of the muscle fibre, and coated vesicles and multivesicular bodies were observed (Figs. 2, 3) as in myoneural junctions of other skeletal muscles (DORING, 1967; NICKEL et al., 1967; NICKEL and WASER, 1968). Although the transversely oriental tubules of the sarcoplasmic reticulum were present in the muscle fibres in which these endings were observed (Fig. 1), the fine structure of the muscle fibres was of the slow type (PEACItEYand HUXLEY, 1962; HEss, 1965; PAGE, 1965; FORSMAN and MATTER, 1968) with small mitochondria and a depleted amount of sarcoplasmic reticulum which only irregularly separates the muscle fibrils (Fig. 1). Discussion Unique features were observed in 3 out of 126 small myoneural junctions derived from unmyelinated nerves ("small multiple endings", TER~V)~INEN, 1968a), these endings possessing protrusions of the synaptic axon terminal in close contact with the postsynaptic muscle fibre. These regions were characterized by a narrow cleft (about 100 A), absence of basemement membrane-like material

208

H. TERi~Vi4INE~: Small Multiple Endings

Fig. 1. A small axon terminal (A) is seen apposed to the slow-type muscle fibre (SF) close to its nucleus (/V). I n the axon, an axoplasmic protrusion (P) into the muscle fibre is present. The terminal is invested by the teloglial cell (TC) b u t there is no spindle sheath. The muscle fibre contains little sarcoplasmic reticulum (SR) compared with the fast-fibre (FF), and the sarcoplasmic mitochondria are smaller and usually fewer (although mitochondrial aggregation is seen close to the muscle nucleus in this micrograph). The sarcoplasmic reticulum only irregularly separates the muscle fibrils b u t transverse tubules (T) can be seen. • 18,000

Fig. 2. In the axoplasm of the axon terminal (A) some mitochondria, numerous e m p t y vesicles measuring approximately 500 A and two dense-core vesicles measuring a b o u t 1,000 A (arrow) are seen. Note t h a t there are only a few of the common 500 A vesicles in the invagin a t e d protrusion (P), although vesicular material with hardly visible membranes can be seen. Amorphous basement-membrane-like material (BM) such as is present between the axon membrane (AM) and the postsynaptic membrane of the muscle fibre (PSM) is not seen in the space measuring only a b o u t 100 A between the axonal protrusion (P) and the muscle plasma membrane. Note also t h a t the muscle plasma membrane apposed to the protrusion does not possess its normal increased electron density. TC teloglial cell; M VB multivesiculated body; C V coated vesicle; N nucleus. • 34,000

Fig. 3. The basement membrane-like material (BM) covering the muscle fibre is lacking between the axonal protrusion (P) and the postsynaptic membrane of the muscle fibre (PSM), which is of normal thickness. Here, in contrast to the situation in Fig. 2, the axonal protrusion is not close to the muscle nucleus b u t close to muscle mitochondria. M V B multivesiculated body; TC teloglial cell. • 26,000

210

H. TER);VXINE~:

a n d of the p o s t s y n a p t i c electron density, a n d a r e d u c e d a m o u n t of s y n a p t i c vesicles w i t h h a r d l y visible m e m b r a n e s . A l t h o u g h the p r e s e n t work does n o t w a r r a n t a decision on t h e precise function of these protrusions, some possibilities can be suggested. I n t h e fight of t h e a c c e p t e d s t r u c t u r a l basis of chemical t r a n s m i s s i o n (cf. ECCLES, 1964; KATZ a n d MIL~DI, 1965; NASTUK, 1967) t h e features o b s e r v e d s t r o n g l y suggest t h a t these regions of close m e m b r a n e a p p o s i t i o n are n o t concerned in the t r a n s m i s s i o n of e x c i t a t o r y impulses from t h e nerve to the muscle fibre. E n d i n g s of this t y p e h a v e never been described in m a m m a l i a n s t r i a t e d muscle fibres possessing muscle spindles. On the o t h e r hand, s o m e w h a t similar a x o p l a s m i c p r o t r u s i o n s w i t h close m e m b r a n e a p p o s i t i o n h a v e been o b s e r v e d earlier in m y o n e u r a l j u n c t i o n s of t h e frog (KARLSSON, 1962; KARLSSON a n d ANDERSON-CEDERGREN, 1966) b u t only in i n t r a f u s a l muscle fibres a n d from m y e l i n a t e d nerves (KARLSSON a n d ANDERSONCEDERGR~N, 1966). Because there are no muscle spindles in the e x t r a o c u l a r muscles of m a n y m a m m a l s , including t h e r a t (CILIMBARIS,1910; COOPER a n d DANIEL, 1949, 1956; COOPER etal., 1955, 1956; WOLT]~R a n d O'KE]~FE, 1963; WOLTER, 1964), i t m a y be t h a t the endings with p r o t r u s i o n s h a v e a sensory function analogous to muscle spindles.

References

CILIMBARIS, P . A . : Histologische Untersuchungen fiber die Muskelspindeln der Augenmuskeln. Arch. mikr. Anat. 75, 692--747 (1910). COOrER, S., and P.M. DANIEL: Muscle spindles in human extrinsic eye muscles. Brain 7'2, 1--24 (1949). -- - Human muscle spindles. J. Physiol. (Lond.) 188, 1P 2P (1956). - - , and M. FILLENZ: Afferent discharges in response to stretch from the extraocular muscles of the cat and monkey and the innervation of these muscles. J. Physiol. (Lond.) 127, 4 0 0 4 1 3 (1955). - - , and D. WHITTERIDGE: Muscle spindles and other sensory endings in the extrinsic eye muscles; the physiology and anatomy of these receptors and of their connections with the brain stem. Brain 78, 564--583 (1955). DiiRINO, M. v. : ~ber die Feinstruktur der motorischen Endplatte von hSheren Wirbeltieren. Z. Zellforsch. 81, 74--90 (1967). ECCLES, I.C. (editor): The physiology of synapses. Berlin-G5ttingen-Heidelberg: Springer 1964. FORSSMAN, W. G., u. A. MATTER: Zur Klassifizierung der Skelettmuskulatur. In: Verh. der anat. Ges. auf der 62. Verslg. in Marburg (Hrsg. M. WATZKAU. H. VOSS), S. 5--16. Jena: Gustav-Fischer 1968. HESS, A. : The sarcoplasmic reticulum, the T system, and the motor terminals of slow and twitch muscle fibres in the garter snake. J. Cell Biol. 26, 467--476 (1965). KARLSSO~, U. L. : Specialized contact regions of the myoneural motor junction of the frog. Proc. V. Int. Congr. Electron Microscopy 2, U-4. New York: Acad. Press 1962. - - , and E. ANDERSON-CEDERGREN:Motor myoneural junctions in frog intrafusal muscle fibre. J. Ultrastruct. Res. 14, 191--211 (1966). KATZ, B., and R. MYLEDI: The quantal release of transmitter substances. In: Studies in physiology (eds. D.R. CURTm and A.K. MCI~TYRE), p. 118--125. Berlin-HeidelbergNew York: Springer 1965. KUPFER, C.: Motor innervation of extraocular muscle. J. Physiol. (Lond.) 153, 522--530 (1960). LUFT, J. H. : Improvements in epoxy resin embedding methods. J. biophys, biochem. Cytol. 9, 4 0 9 4 1 4 (1961).

Small Multiple Endings

211

NASTUK, W. L. : F u n d a m e n t a l aspects of neuromuscular transmission. Invest. Ophthal. 6,

235--251 (1967). NICKEL, E., A. VOGEL u. P. G. WASER: Coated vesicles in der Umgebung der neuro-muscul~iren Synapsen. Z. Zellforsch. 78, 261--266, (1967). - - , u. P. G. WASER: Elektronenmikroskopische Untersuchungen am Diaphragma der Maus nach einseitiger Phrcnikotomie. Z. Zellforsch. 88, 278--296 (1968). PAGE, S. G.: A comparison of the fine structures of frog slow and twitch muscle fibres. J. Cell Biol. 477--497 (1965). PEACHEY, L . D . , and A. F. HUXLEY: Structural identification of twitch and slow striated muscle fibres. J. Cell Biol. 13, I77--186 (1962). REYNOLDS, E. S.: The use of lead citrate at high p H as an electron-opaque stain in electron microscopy. J. Cell Biol. 17, 208--212 (1963). SABATINI,D. D., K. BENSC//, a n d R . J . BARRNETT: Cytochemistry and electron microscopy. The preservation of cellular ultrastructure and enzymatic activity b y aldehyde fixation. J. Cell Biol. 17, 19--58 (1963). TER)iV.~.I~EN, H. : Electron microscopic and histochemical observations on different types of nerve endings in the extraocular muscles of the rat. Z. Zellforsch. 90, 372--388 (1968a). - - Electron microscopic localization of acetylcholinesterase in small multiple endings in the extraocular muscles of the rat. in press, Experientia (Basel) (1968b). WOLTER, J. R. : Thin nerves with simple endings containing cholinesterase in striated h u m a n eye muscle. Neurology (Minneap.) 14, 283--286 (I964). - - , a n d N. T. O'KEEFE: Localization of nerve endings in relation to cholinesterase deposits in normal h u m a n eye muscles. Invest. Ophthal. 2, 558--566 (1963). Dr. H. TER.~V~.INEN Dept. of Anatomy, University of Helsinki Siltavuorenpenger 20 Helsinki 17, Finland