Cell Tiss. Res. 188, 341-344 (1978) Cell and Tissue Research �9 by Springer-Verlag 1978

Short Communication

Further Immunofluorescence-Microscopic Evidence for Myosin in Various Peripheral Nerves*

K. Unsicker and D. Drenckhahn

Department of Anatomy University of Kiel, Kiel, Federal Republic of Germany

U. Gr6schel-Stewart

Department of Zoology, TH Darmstadt, Darmstadt, Federal Republic of Germany

Summary. An indirect immunofluorescence microscopic technique using antibodies from rabbits against highly purified myosin from chicken gizzard was applied to various peripheral nerves (cranial nerves V, VII, X). Myosin- specific immunoreactivity was found in the axoplasm, in Schwann cells, in the perineural sheath and in vascular walls.

Key words: Peripheral nerves - Myosin - Immunofluorescence microscopy.

Introduction

In previous studies we have shown that sciatic nerve and lumbar ventral and dorsal root axons of the rat contain proteins which are antigenically identical with actin and myosin from smooth muscle of chicken gizzard (Unsicker et al., 1977; Drenckhahn et al., 1977). This was established by using an immunofluorescence- microscopic technique with antibodies against purified actin and myosin from chicken gizzard (Gr6schel-Stewart et al., 1976, 1977), which allow the localization of very small amounts of these contractile proteins in frozen sections. To test the validity of the concept that myosin is a general constituent of peripheral nerves, we have extended our investigation to a variety of peripheral nerves, such as trigeminal, facial and vagal nerve. The positive results reported in this study lend further support to the idea that actin and myosin might be involved in axoplasmic transport.

Send reprint requests to: Dr. K. Unsicker, Department of Anatomy, University of Kiel, D-2300 Kiel, Federal Republic of Germany

* This work was supported by grants from Deutsche Forschungsgemeinschaft (Un 34/4, Dr 91/1, Ste 105/19). Thanks are due to Ursula K6nig, Renate Steffens and Christine Mahlmeister for skilful technical assistance.

0302-766X/78/0188/0341/$01.00

342 K. Unsicker et al.

Materials and Methods

The following nerves taken from adult rats (Hanover-Wistar) of either sex in pentobarbital anaesthesia were rapidly frozen in liquid nitrogen: trigeminal nerve near the trigeminal ganglion, facial nerve in the parotid plexus and vagus nerve (cervical portion).

The following sera were used: antibodies (IgG-fractions) from rabbits against (a) myosin from chicken gizzard (0.5 or 1 mg/ml), (b) myosin from human pectoralis muscle (1 mg/ml). Control incubations were performed using (a) the rabbit antibody against chicken gizzard myosin absorbed to the antigen (1 mg/ml) and (b) rabbit pre-immunoglobulin (1 mg/ml). 5 [~m thick frozen sections were incubated (30 min) with one of the sera listed above, rinsed in PBS (30 rain), incubated with the FITC- labeled goat anti-rabbit IgG (30 rain), and, after washing in PBS (30 min), mounted in glycerol - 0.1 M glycine buffer (1:3) pH 9.6 and viewed under a Zeiss Universal fluorescence microscope fitted with epi- illuminescence.

Results and Discussion

Cross and longitudinal sections through main trunks and branches of the cranial nerves V, VII, and X revealed a brilliant fluorescence in the axoplasm after incubation with antibodies against gizzard myosin. This fluorescence was not observed, when the antigen-absorbed myosin antibody, rabbit-immunoglobulin or FITC labeled goat anti-rabbit IgG alone were applied (Fig. 1).

Incubation with striated muscle myosin antibodies did not yield specific axoplasmic staining. Axonal myelin sheaths were absolutely negative, but Schwann cells exhibited a clear gizzard-type myosin-specific fluorescence. The perineural sheath was also fluorescent (Fig. 1).

In all instances, the fluorescence was weaker than that seen in the wall of small nervous blood vessels.

The present results confirm and extend previous immmunofluorescence- microscopical observations on the presence of actin and myosin in peripheral nerves (Drenckhahn et al., 1977; Unsicker et al., 1977). These findings are corroborated by recent morphological and biochemical results, which give strong evidence in favor of actin and myosin in nervous tissue. An actomyosin-like protein, which has Mg + +- and Ca + +-activated ATPase activity and can be superpre- cipitated, has been isolated from brain (Berl et al., 1972; Puszkin and Berl, 1972; Puszkin et al., 1968). According to Puszkin et al. (1972), about 10 % of the protein of synaptosomal fractions is actomyosin. Myosin has also been extracted from cultured neuroblastoma cells (Miller and Kuehl, 1976). By heavy meromyosin binding and immunofluorescence staining actin has been identified in cultured neuroblastoma cells (Chang and Goldman, 1973; Isenberg et al., 1977) and in rat brain neurons (Le Beux and Willemot, 1975).

To day, we have no direct evidence as to the functional significance of actin and myosin in neurons. However, it is the only contractile system so far identified in these cells. This led Bray (1973) to suggest that it might be responsible for the movement of the growth cone of the growing neurite. The association of the actomyosin-like protein with synaptosomes would be in line with the hypothesis that it may function in exocytosis for the release of neurotransmitters at nerve endings (Bed et al., 1973) as well as in chromaffln cells (Burridge and Phillips, 1975; Creutz, 1977). This hypothesis receives support from the observation that

Myosin in Peripheral Nerves 343

Fig. 1 A-D. Immunofluorescence micrographs of rat vagal A and facial B-D nerves after indirect staining with anti-smooth muscle myosin A-C and anti-smooth muscle myosin absorbed with smooth muscle myosin D. A myosin-specifc immunofiuorescence staining is seen in the axoplasm (arrows). Arrowheads point to Schwann cells, the cells of the perineural sheath and a blood vessel. Myoepithelial (me) and the apical borders of acinar cells (ac) in the parotid gland B show a clear specific immunofluorescence, while striated muscle fibres (SM) do not react with the antiserum to smooth muscle myosin. The control section D shows a very low background fluorescence only. A x 750; B • 600; C x 375; D x 600

344 K. Unsicker et al.

cytochalasin B, which interferes with actin filaments in general, not only impairs neuronal movements in tissue culture (Wessels et al., 1971), but also inhibits the actin activated Mg § § of rat brain synaptosomal actomyosin (Nicklas and Berl, 1974) and the release of noradrenaline and dopamine fl-hydroxylase from sympathetic nerve endings (Thoa et al., 1972).

At present we do not know anything as to the precise location of the myosin molecules in nerves. An adequate electron microscopical immunocytochemical technique, which has recently been developed in our department, will contribute to settle this problem.

References

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(Lond.) 254, 256-259 (1975) Chang, C.-M., Goldman, R.D.: The localization of actin-like fibers in cultured neuroblastoma cells as

revealed by heavy meromyosin binding. J. Cell Biol. 57, 867-874 (1973) Creutz, C.E.: Isolation, characterization and localization of bovine adrenal medullary myosin. Cell Tiss.

Res. 178, 17-38 (1977) Drenckhahn, D., Unsicker, K., Grfschel-Stewart, U.: Immunocytochemical demonstration of myosin

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Grrschel-Stewart, U., Schreiber, J., Mahlmeister, Ch., Weber, K.: Production of specific antibodies to contractile proteins, and their use in immunofluorescence microscopy. I. Antibodies to smooth and striated chicken muscle myosin. Histochemistry 46, 229-236 (1976)

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Unsicker, K., Drenckhahn, D., Grfschel-Stewart, U., Schumacher, U., Griesser, G.H.: Immunohis- tochemical evidence for myosin in peripheral nerves and spinal cord of the rat. Neuroscience (in press)

Wessels, N.K., Spooner, B.S., Ash, J.F., Bradley, M.O., Ludena, M.A., Taylor, E.L., Wrenn, J.T., Yamada, K.M.: Microfilaments in cellular and developmental, processes. Science 171, 135-143 (1971)

Accepted December 19, 1977