Neuroscience Letters, 132 (1991) 65~8 © 1991 Elsevier Scientific Publishers Ireland Ltd. All fights reserved 0304-3940/91/$ 03.50 ADONIS 030439409100611Y

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Protein kinase C fl-subtype-immunoreactive motor and sensory nerves in rat muscle spindle

Marke Hietanen-Peltola, Leena Rechardt and Markku Pel to-Huikko

Department of Biomedical Sciences, University of Tampere, Tampere (Finland)

(Received 17 June 1991; Revised version received 15 July 1991; Accepted 17 July 1991)

Key words: Protein kinase C fl-subtype; Skeletal muscle; Muscle spindle; Annulospiral ending; Immunofluorescence

The localization of protein kinase C ]/-subtype-like immunoreactivity (PKC-p-LI) was studied in the muscle spindles of rat neck muscles. In the equatorial regions of muscle spindles PKC-p-LI was detected in spiral sensory nerve endings surrounding intrafusal muscle fibers. In polar regions single PKC-B-immunoreactive (IR) nerve fibers were found between intrafusal muscle fibers and some PKC-p-IR motor nerve endings were seen on the surface of intrafusal fibers. These results suggest that PKC-p may be involved in regulation of muscle contraction and tonus by modulating the sensitivity of afferent and efferent innervation of muscle spindles.

Protein kinase C (PKC) is an enzyme which has im- portant roles in cellular transduction of various extracel- lular signals [17]. PKC is involved in the regulation of gene expression, cell proliferation and differentiation and several metabolic processes [18]. PKC was first iden- tified in 1977 and since then it has been found in many mammalian tissues [14, 31]. Several subtypes of PKC (i.e. ct, Ell, ,82, 7, ~, e, 0 have been identified by molecular cloning [9, 12, 20-22], and probably each of these sub- types has a specific function [1, 15, 19, 30].

PKC-like immunoreactivity (LI) and PKC mRNAs have been localized in several areas of rat central and peripheral nervous system [1, 3, 7, 11, 24, 25, 27]. We have previously demonstrated PKC-i-LI in rat moto- neurons, motor nerves and in myoneural junctions [6]. PKC-fl-LI has also been detected in the neurons of rat dorsal root ganglia [24]. The present study was con- ducted to examine whether PKC-i-immunoreactive (IR) sensory or motor nerves are present in rat muscle spin- dies.

Adult male Sprague-Dawley rats (2-3 months old) were used for this study. Rats were anesthetized with intraperitoneal injection of chloralhydrate (350 mg/kg) and perfused transcardially with 100 ml of physiologic saline followed by 4% paraformaldehyde or 4% parafor- maldehyde with 0.2% picric acid in phosphate buffer (0.1

Correspondence: M. Hietanen-Peltola, Department of Biomedical Sciences, University of Tampere, Box 607, 33101 Tampere, Finland.

M, pH 7.3). The neck muscles were excised and immersed in the same fixative for an additional 3 h. The tissues were cryoprotected with 20% sucrose and sec- tioned in Leitz 1720 cryostat at 10 gm. Some specimens were serially sectioned throughout the length of muscle spindles at 150 gm intervals. The sections were mounted on gelatin coated glass slides and indirect immunofluor- escence method was used to demonstrate PKC-fl-LI. A rabbit antibody raised against a synthetic PKC-peptide sequence shared by the ill- and ff2-subspecies was kindly provided by Drs. Iadarola and Roth [26]. The antibody was diluted (1:80) in phosphate-buffered saline (PBS) containing 0.3% Triton and 1% BSA. The sections were incubated with the primary antibody for 12-24 h at 4°C and after several washes the sections were incubated in FITC-conjugated goat anti-rabbit serum (Boehringer Mannheim, Mannheim, F.R.G.) diluted 1:100 for 30 min at 37°C. The sections were embedded in the mixture of glycerol and PBS containing 0.1% phenylenediamide and examined with a Nikon Microphot-FX miscroscope equipped for epitluorescence with proper filter combina- tions. The controls included normal rabbit serum and the antibody preabsorbed with 10/tg of synthetic PKC-t peptide in 1 ml of diluted antibody. No staining was observed in the controls.

PKC-t-LI was seen in nerve bundles and fibers between muscle fibers and in myoneural junctions on the surface of unlabeled extrafusal muscle fibers (Fig. 1). PKC-t-IR nerve bundles were seen close to muscle spin-

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dies and they could be traced entering the intracapsular

space (Figs. 2 and 3). In equatorial regions of muscle spindles PKC-p-LI was seen to surround the intrafusal muscle fibers. In longitudinally cut spindles it could be seen that PKC-p-LI was located in spiral nerve fibers surrounding the intrafusal muscle fibers (Figs. 3 and 4). PKC-p- IR nerve fibers seemed to surround all intrafusal muscle fibers with similar density. Some PKC-p- IR spi- ral nerve fibers were seen on the surface of intrafusal fibers also in the juxtaequatorial regions (Fig. 5). Some PKC-p- IR nerve fibers were seen between intrafusal fibers in juxtaequatorial and polar regions and flattened PKC-p- IR nerve endings were occasionally seen on the surface of intrafusal fibers in the polar regions of muscle spindles (Figs. 5 and 6). Intrafusal muscle fibers and the spindle capsule did not exhibit PKC-p-LI.

This study provides immunohistochemical evidence for the presence of PKC-p-LI in efferent and afferent nerve fibers of rat muscle spindles. The efferent innerva- tion of muscle spindle is exerted by 7- and p-efferent nerve fibers [5]. In polar regions the PKC-p- IR nerve fibers and nerve endings found on the surface of intrafu- sal fibers probably represent their efferent nerve compo- nent. We have earlier demonstrated PKC-p-LI in the neurons of ventral spinal cord, in motor nerves and in myoneural junctions [6]. p-skeletofusimotor axons to muscle spindles are branches of motor nerves innervat- ing extrafusal muscle fibers [13], so it is probable that they also contain PKC-p-LI. On the other hand, the p- efferent innervation of muscle spindles is sparse, and for example muscle spindles of rat soleus muscle rarely receive p-innervation [2]. Thus, most PKC-p- IR endings seen in polar regions may represent terminals of 7-effer- ent fibers.

There are two kinds of sensory endings in each muscle spindle; the pr imary (annulospiral) endings and the

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secondary (flowerspray) endings [5]. The spiral PKC-fl- IR nerve fibers surrounding the intrafusal muscle fibers

in equatorial regions are likely to be pr imary sensory endings. The secondary sensory endings are also said to form spiral- or half circle-like structures around some intrafusal muscle fibers [16, 29]. Thus, the PKC-fl-IR spi- ral nerve fibers in the juxtaequatorial regions are possi- bly secondary sensory endings.

Electron microscopic studies on dorsal horn neurons have shown that PKC-fl-LI is associated with Golgi- complexes and the cell membrane [15]. It has been pro- posed that PKC-fl may also be involved in Golgi func- tions such as protein processing and intracellular vesicle t ransport [10, 28]. The present results suggest that PKC- fl may have a role in cell membrane functions and vesicle traffic also in the efferent and the afferent nerves of mus- cle spindle.

Electrophysiological studies have revealed that PKC may be involved in sensory transmission in dorsal root ganglion [4, 32] and an immunocytochemical study has demonstrated the presence of PKC-fl-LI in the large neu- rons of dorsal root ganglia [24]. In neurons PKC regu- lates calcium, potassium and chloride channels and acti- vation of PKC increases the spontaneous and stimulated neurotransmitter release from nerve cells [8, 18, 23, 33]. Thus, in muscle spindle PKC-fl may modulate the mus- cle tonus and contraction and the activity of stretch re- flex by affecting the threshold level o f afferent nerves and the transmitter release from efferent nerve endings.

1 Ase, K., Saito, N., Shearman, M.S., Kikkawa, U., Ono, Y., Igar- ashi, K., Tanaka, C. and Nishizuka, Y., Distinct cellular expression of ill- and fill-subspecies of protein kinase C in rat cerebellum, J. Neurosci., 8 (1988) 3850-3856.

2 Boyd, I.A. and Gladden, M.H., Review. In I.A. Boyd and N.H. Gladden (Eds.), The Muscle Spindle, Stockton Press, New York, 1985, 415 pp.

Fig. 1. Protein kinase C-fl-immunoreactive (PKC-fl-IR) nerve fibers are seen between extrafusal muscle fibers. One PKC-p-IR motor endplate (arrow) is localized on the surface of a muscle fiber (m). Bar = 30 ,urn.

Fig. 2. PKC-fl-IR nerve fibers are seen to surround 4 intrafusal muscle fibers (small arrows) in a muscle spindle. A PKC-fl-IR nerve bundle (big arrow) is seen entering the intracapsular space. Bar = 60/tin.

Fig. 3. In equatorial region of muscle spindle PKC-fl-LI is seen in spiralized nerve fibers around intrafusal fibers (big arrows). Small PKC-fl-IR nerve bundles (small arrows) can be seen in the intracapsular space. Bar = 25/Jm.

Fig. 4. A PKC-fl-IR primary afferent fiber (arrow) forms nerve spirals around an intrafusal muscle fiber in equatorial region of muscle spindle. Bar = 25/tm.

Fig. 5. Cross section of a muscle spindle in juxtaequatorial region. PKC-fl-IR nerve fibers (big arrows) are seen between the intrafusal muscle fibers (asterisks). Thin PKC-fl-IR nerve fiber (small arrows) surround one of the intrafusal fibers. The spindle capsule (c) is unlabeled. Bar = 25/~m.

Fig. 6. In polar region of muscle spindle 4 intrafusal fibers (asterisks) are seen between the extrafusal muscle fibers (m). PKC-fl-IR motoric nerve endings (arrows) are seen on the surface of an intrafusal muscle fiber. Bar = 15 gm.

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