THE ANATOMICAL RECORD 214~198-203(1986) Unmyelinated Axons in the Feline Trigeminal Motor Root M h T E N RISLING, KAJ FRIED, CLAES HILDEBRAND, AND ANNA CUKIERMAN Department of Anatomy, Karolinska Institutet, Box 60400, S-104 01 Stockholm, Sweden ABSTRACT The trigeminal motor root was studied in the electron microscope at different proximodistal levels in eight adult cats. Counts at a level halfway between the trigeminal ganglion and the pontine junction showed that the root contains about 9% (n = approximately 300) unmyelinated axon profiles at this level. Small groups of unmyelinated axons occur on both sides of the PNS-CNS border, in the surrounding pia mater, and in perivascular spaces of the CNS compartment. Examination of serial sections from the PNS-CNS transitional region showed that some unmyelinated axons actually cross the PNS-CNS border. The functional significance of these fibres remains unknown. Both in the cat and in man spinal ventral roots contain significant numbers of unmyelinated sensory axons, many of which relay nociceptive information from peripheral receptive fields (see Coggeshall, 1980). It has been proposed that ventral roots channel these axons into the CNS, which would contradict the law of Magendie (see Coggeshall, 1980). As judged from recent electron microscopic (EM) studies of the PNS-CNS transitional region, however, the law of Magendie is valid (Risling et al., 1984). The EM results show that the unmyelinated axons in the feline ventral root L7 either leave the root and project to the pia mater or loop and course in a peripheral direction. Unmyelinated axons entering the spinal cord through this root have not been found (Risling et al., 1984). The motor root of the trigeminal nerve in the adult cat contains about 10% unmyelinated axons and it has been suggested that it might represent an additional pathway for trigeminal pain (Young, 1978; Young and Stevens, 1979). The trigeminal motor root is only partly analogous to spinal ventral roots, since myelinated proprioceptive axons do enter the CNS through this root (May and Horsley, 1910; Pelletier et al., 1974; Ryu and Kawana, 1985).In view of this principal difference between trigeminal and spinal motor roots, the present study sets out to examine whether unmyelinated axons cross the PNS-CNS border of the trigeminal motor root. MATERIALS AND METHODS Material was taken from eight adult cats aged 1-6 years. The animals were anesthetized with sodium pentobarbital (Mebumal, 40 m g k g i.p.1, artificially ventilated, and perfused with Tyrode’s solution followed by a solution of 5% glutaraldehyde and 0.1 M sucrose in a 300-mOsm phosphate buffer (Carlstedt, 1977a). After perfusion each trigeminal ganglion was removed with its motor and sensory roots attached to a piece of the ventrolateral pons. After postfixation in glutaraldehyde the motor root, including its junction with the pons and portions of the most proximal part of the sensory root, was carefully trimmed out. After buffer rinse, osmica0 1986 ALAN R. LISS. INC. tion, and acetone dehydration the specimens were embedded in Vestopal W (Carlstedt, 1977a). Semithin toluidine blue-stained sections were used for orientation. Thin transverse sections covering the whole motor root were cut a t the following levels: 1)the proximal end of the trigeminal ganglion, 2) approximately halfway between the ganglion and the pons, and 3) the proximal end of the motor root close to the pontine junction. At the latter level the sections included the PNS-CNS transitional region of the sensory root. In addition, a series composed of 1,517 consecutive thin transverse sections were cut from the PNS-CNS transitional region of the motor root. The sections were collected on one-hole copper grids coated with carbon-stabilized Formvar, contrasted with uranyl acetate and lead citrate, and examined in a Philips EM 301 electron microscope. RESULTS In the electron microscope the motor root fascicles were easily identified and differentiated from the sensory root and from the small accessory sensory fascicles on the basis of the markedly different fibre size distributions (Fig. 1) (Young, 1977). At the distal level the motor root formed a rather coherent bundle composed of large and medium-sized myelinated axons and a n occasional neuronal perikaryon. Unmyelinated axons also occurred among the myelinated axons and these were sometimes arranged in large bundles. At the middle level neuronal perikarya were not observed and the bundles of unmyelinated axons tended to be smaller but more numerous. At this level approximately 9% of all axon profiles were unmyelinated (range 6.8-11.0%, n = 3). The total number of unmyelinated axon profiles per motor root varied between 231 and 305. At proximal levels, which included the glial fringe (Berthold and Carlstedt, 19771, unmyelinated axons surrounded by astrocytic processes formed islands between myelinated PNS-type axons, with or without the participation of Schwann cell-like profiles (Fig. 2a,b). On the CNS side Received March 1, 1985; accepted September 10, 1985. 199 TRIGEMINAL MOTOR ROOT Fig. 1. Low-power electron micrograph showing a transverse section from a fairly proximal level of the trigeminal root region. The picture shows fascicles of the motor root (M) partly surrounding the larger sensory root (S).At this level the motor root fascicles remain in the PNS compartment, while the sensory root contains a large core of CNS tissue. Note the relatively high proportion of large myelinated axons in the motor root fascicles compared to the sensory root. x 160. of the PNS-CNS transition a few small groups of unmyelinated axons were observed. At places close to the surface of the CNS these axons were sometimes approached by slender fibrous astrocytic processes and patches of a n electron-dense axolemmal undercoating could be present at such sites (Fig. 2c). In addition, a few small myelinated axons could be observed superficially in the CNS compartment. Occasional small groups of unmyelinated axons were also encountered at deeper levels in the CNS compartment (Fig. 2d). It was not possible to count unmyelinated and small myelinated axons at deeper CNS levels, because the motor root fibres were not clearly demarcated from those of the sensory root (Ryu and Kawana, 1985). The total number of CNS-type axons with diameters below 1 pm was, however, estimated to be less than 100. The results from the series composed of consecutive sections have been illustrated in Figures 3 and 4.Figure 3 shows a bundle of unmyelinated axons which could be followed from the PNS compartment of the motor root into the CNS. The axon shown in Figure 4 was myelinated both in the PNS and the CNS but possessed a n about 20 pm long unmyelinated segment a t the PNSCNS transition. Larger blood vessels within the CNS compartment were often accompanied by one to three perivascular nerve bundles, each of which contained some five to thirty unmyelinated PNS-type axons (Fig. 5a). Finally, the pia mater in the vicinity of the PNS-CNS transition of the trigeminal motor root contained tiny bundles of unmyelinated and small myelinated PNS-type axons (Fig. 5b). DISCUSSION The present results confirm previous evidence that the main trunk of the feline trigeminal motor root contains approximately 10% unmyelinated axon profiles (Young and Stevens, 1979). It has been suggested that these axons are sensory (Young and Kruger, 1981). Our results show that unmyelinated axons are present on both sides of the PNS-CNS transition and at deeper levels in the CNS compartment of the motor root. At least some of these unmyelinated axons actually cross the PNS-CNS border. Larger unmyelinated PNS axons tend to become myelinated as they enter the CNS (Carlstedt, 1977b). Therefore the few myelinated axons with diameters below 1pm which occurred superficially in the CNS compartment, might be unmyelinated on the PNS side of the transition. These findings show that, in contrast to the ventral root L7 (Risling et al., 1984), the trigeminal motor root represents a site where some unmyelinated 200 M. RISLING, K. FRIED, C. HILDEBRAND, AND A. CUKIERMAN Fig. 2. Electron micrographs from transverse sections through the PNS-CNS transitional region in the trigeminal motor root of an adult cat. a) Two profiles resembling unmyelinated axons (*) are associated with both astrocytic processes of the glial fringe and with Schwann cell profiles. ~ 3 5 , 0 0 0b) . This field shows several unmyelinated axons (arrows) and a small myelinated axon in an island composed of CNS tissue (astrocytic processes). Note the surrounding PNS-type large myelinated axons. x 14,000. c) The three unmyelinated axons indicated by asterisks occupy superficial positions in the CNS compartment of the motor root at the level of the pontine surface. The myelinated axon also present is located on the PNS-side of the transition. One of the axons (right) is partly apposed by radially oriented astrocytic processes and exhibits an electron-dense axolemmal undercoating at these sites (arrowheads). ~ 2 4 , 0 0 0d) . This picture illustrates a group of unmyelinated axons (*) at a deeper level in the CNS compartment of the motor root. Note the surrounding astrocytic processes and the CNS-type myelinated axons (top and left) nearby. ~ 2 5 , 0 0 0 . TRIGEMINAL MOTOR ROOT Fig. 3. Electron micrographs from the series of transverse sections through the PNS-CNS transition of the trigeminal motor root. Distally (a = section number 750) a group of unmyelinated axons (arrows) is associated with a Schwann cell in the PNS compartment. More proximally (b = section number 1,151) the group of axons has split into three small clusters of unmyelinated axons (arrows), which are found immediately beneath the surface of the CNS compartment. The last 201 micrograph (c = section number 1,181)shows the same clusters at a deeper position within the CNS compartment. At this proximal level the unmyelinated axons are completely surrounded by astrocytic processes. The long arrow indicates a comparatively large unmyelinated axon in which an patchlike electron-dense axolemmal undercoating was observed at some levels. This axon, however, remained unmyelinated throughout the series. x 12,000. 202 M. RISLING, K. FRIED, C. HILDEBRAND, AND A. CUKIERMAN and small myelinated axons shift from the PNS to the CNS or vice versa. The presence of unmyelinated axons in the trigeminal motor root and on both side of the PNS-CNS transition may be explained in various ways: 1)It is known that serotoninergic brainstem neurons project to pial blood vessels (Chan-Palay, 1976; Edvinsson et al., 1983) and some of these axons might travel to their leptomeningeal targets through cranial nerve roots. Thus, some proportion of the unmyelinated axons in the trigeminal motor root might represent efferent axons from brainstem neurons. 2) Adrenergic, cholinergic, and peptidergic fibres are related to the adventitial surfaces of large cerebral pial arteries (Edvinsson, 1982).The proximal segments of these arteries are among the few intracranial structures which, when stimulated, elicit the sensation of pain (Mayberg et al., 1984). The trigeminal ganglia seem to represent the most important source of afferents from pial blood vessels (Kautzky and Wolter, 1952; Mayberg et al., 1984). By analogy with unmyelinated ventral root axons, some of the unmyelinated axons in the trigeminal motor root might then leave this root and enter the pia mater (Risling et al., 1984). Accordingly, unmyelinated and small myelinated axons unrelated to blood vessels were seen in the pia mater surrounding the pontine entrance of the motor root. 3) Unmyelinated sensory axons might form loops in the motor root in the manner described for cat ventral roots misling et al., 1984). 4) Finally, unmyelinated axons in the trigeminal motor root might, to some extent, represent sensory axons, which enter the CNS together with the large-caliber proprioceptive afferents known to be present (May and Horsley, 1910; Pelletier et al., 1974). The involved fibers are so few, however, that they are not likely to represent a pathway of any major significance for trigeminal nociception. ACKNOWLEDGMENTS This study was supported by grants from the Swedish Medical Research Council (project 37611, the Karolinska Institute, the Swedish Society of Medicine, Magn. Bergvalls stiftelse, and A ke Wibergs stiftelse. We wish to thank Ms. Pippi Lindqvist and Ms. Maria Meier for excellent technical assistance. ~ Fig. 4. Electron micrographs from the series of transverse sections through the PNS-CNS transition of the trigeminal motor root. Figure 4a (section 750) shows a small myelinated axon (*) on the peripheral side of the PNS-CNS border. Note the neighboring astrocytic processes of the glial fringe. More proximally (b = section 867), the axon (*) is surrounded by an inner Schwann cell layer and an outer astrocytic layer and it has lost its myelin sheath. Figure 4c (section 1,211)shows the same axon (*) in a superficial flap of the CNS. Also at this level the axon is unmyelinated. In the last micrograph (d = section 1,347) the axon (*) is surrounded by a CNS-type myelin sheath and is located deeply within the CNS compartment of the motor root. X 17,000. TRIGEMINAL MOTOR ROOT 203 Fig. 5. Electron micrographs from transverse sections through the trigeminal motor root at a level near the pons. a) This bundle of PNStype unmyelinated axons (arrow)is located in the adventitia of a larger blood vessel CL = lumen) within the CNS compartment of the motor root. ~ 6 , 0 0 0b) . This field shows a group of PNS-type unmyelinated axons (arrow) and one small myelinated axon within the pia mater PIN unrelated to blood vessels. The pontine surface can be seen in the lower part of the picture. ~ 3 , 0 0 0 . LITERATURE CITED Kautzky, R., and R. Wolter (1952) Leptomeningeale Aste des Nervus trigeminus. Deutsche Zeitschrift f. Nervenheilknnde, 168.24-29. May, O., and V. Horsley (1910) The mesencephalic root of the fifth nerve. Brain, 33r175-203. Mayberg, M.R., N.T.Zervas, and M.A. Moskowitz (1984) Trigeminal projections to supratentorial pial and dural blood vessels in cats demonstrated by horseradish peroxidase histochemistry. J. Comp. Neurol., 223.46-56. Pelletier, V.A., D.A. 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