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Number and diameter distribution of myelinated afferent fibers innervating the paws of the cat and monkey
EXPERIMENTAL
NEUROLOGY
48, 261-274 (1975)
Number and Diameter Fibers Innervating
Distribution of Myelinated the Paws of the Cat and
Afferent Monkey
A. MATSUMOTOAND S.MORI' Departwacnt
of Physiology, Received Srptcmbcr
Hokkaido
Vuivcrsity
19, 1974;
Sclzool
revision
rcccivcd
of Medicine, March
Sapporo,
Japarz
5, 1975
The number and the diameter distribution of the myelinated cutaneous fibers innervating fore and hind paw were histologically examined in the cat and monkey. In five cats, the superficial peroneal nerve innervating the dorsal surface of the hind paw and the superficial plantar nerve innervating the palmar surface were composed of 2668-2950 fibers, 40-440/O of which were group II fibers. On the other hand, the superficial radial nerve innervating the dorsal surface of the fore paw and the sensory branch of median nerve innervating the palmar surface were composed of 3270-3680 fibers, of which 67-72s were group II fibers. Therefore, it was found that the cutaneous fore paw nerves contained more group II fibers than the hind paw cutaneous nerves. In five monkeys the percentages of group II fibers composing the superficial peroneal nerve and superficial plantar nerve (28933374 fibers) were the same as those of the hind paw nerves in the cat. On the other hand, the percentages of group II fibers of the median nerve (41734472 fibers) were 75-78%, about 10% larger than those of the superficial radial nerve (3596-3821 fibers) whose values were 66669%. Therefore, in the monkey, the forepaw nerves innervating the palmar surface also contain more group II afferent fibers compared to the ones innervating the dorsal one (hairy skin).
INTRODUCTION Recently Mori, Reynolds and Brookhart (16) demonstrated that the stability of trained dogs during quiet stance diminished after local anesthetic blockade of the afferents from the paws. It was furthermore observed that the degrees of diminution of the forepaw blocked animals were 1 The authors express their gratitude to Professor M. Kato for his encouragement during the course of the experiment and for reading the manuscript. They express their thanks to Professor T. Nakanishi (Department of Anatomy, Asahikawa Medical College) and Dr. T. Miyagishi (Department of Psychiatry, Hokkaido University School of Medicine) for their criticism during the preparation of the manuscript. Dr. Mori’s present address is the Department of Physiology, Asahikawa Medical College, Asahikawa. 261 Copyright All rights
@ 1975 by Academic Press, Inc. of reproduction in any form reserved.
262
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AND
MORI
almost as pronounced as they were with quadripedal anesthesia, but were characteristically much less pronounced when only the hind paws were affected. These observations suggested that the feedback inputs from the paws are not only one of the important factors for the stability of animals, but also the individual feedback inputs from the forepaws and hind paws may send functionally different informations from the receptors to the central nervous system. Therefore, in this paper we attempted to study whether the components of sensory receptors were different both for the fore paw and hind paw cutaneous nerves, since it may be possible to estimate roughly the receptor type from the diameters of the afferent fibers (2, 4, 11). This study was also prompted by the previous observations (14, 15) that in the forelimb cutaneous nerves of the cat, dorsal root reflex, for the generation of which the afferent inputs from the touch and pressure receptors greatly contribute (6, 21)) could be elicited at the normal temperature in contrast to that elicited in the hind limb cutaneous nerves at the reduced temperature. We also studied how the distribution pattern of afferent fibers would phylogenetically change by comparing the results obtained from the cat, dog and monkey. METHODS Five cats, two dogs and five monkeys (two Macaca &us, three Macaca were employed. Under nembutal anesthesia, the following four nerves were dissected and removed for approximately 10 mm at the levels of dorsal and palmar paws: branch of superficial radial nerve innervating the dorsal surface of the forepaw; sensory branch of median nerve innervating the palmar surface of the forepaw; superficial peroneal nerve innervating the dorsal surface of the hind paw; and superficial plantar nerve innervating the palmar surface of the hind paw. In two cats, the ulnar nerves innervating the parts of dorsal and palmar surfaces of the forepaw were also dissectedand removed. After removing the nerves, each cut end was fixed on a glass rod with thin silk thread to prevent deformation of the nerves. After fixation and staining in osmic acid (1% solution, Merk Co.) for 24 hr (4, 12), each nerve was dehydrated slowly by immersion for 12 hr in each of a series of graded alcohols. It was then immersed in chloroform for 4 hr, and embedded in the paraffin wax. Serial sections of the nerve were cut at 10 pm. These specimens were photographed with a magnification of 1000x. The films thus made were projected to a screen so that 1 pm corresponded to 1 mm on the screen. The diameters of the fibers outside of the myelin sheath were measured for the fibers constituting the individual nerves. When a myelin sheath showed deformity, we excluded the fiber from meamulatta)
AFFERENT
INNERVATION
OF
PAWS
263
surement. However, when the myelin sheath was slightly ovoid in shape, we measured the diameter at the intermediate axis between the longest and shortest ones. In this experiment, about 97-98s of all myelinated fibers constituting each nerve were measured. The question of shrinkage of the nerve fibers through the process of fixation and dehydration was also examined. For this, the diameters of several nerve fibers, selected at random, were measured both in fresh frozen-sections and in paraffin cross-sections of the same nerve. As the result, it was found that the fixation and dehydration caused an average decrease in the fiber diameters of 2.4 -C 0.7% (n = 120). There was no correlation between the percentages of shrinkage and the diameters. RESULTS An example of the transverse section of the myelinated nerve fibers obtained from the cat is shown in Fig. 1 at three magnifications: 45 x ; 112.5 x ; and 450x. The nerve was enclosed with the epineurium, and consisted of the several fiber bundles. Each fiber bundles was encapsulated with the perineurium, and contained about the 200-1300 myelinated fibers across animals of different species. Before drawing the distribution graph of the diameters of these myelinated fibers, we examined the following problems to exclude the source of measurement error. The first question was the degree of possible variation of the diameters in the course of the fibers due to the technique of preparation or other causes. In order to investigate this problem, measurements were made of 100 consecutive 10 pm paraffin sections of the superficial radial nerve obtained from the cat. The diameters of the several selected fibers were measured in each section. The distribution of the diameters measured from a single fiber is shown in Fig. 2. It was observed that the standard deviation (SD) was 0.4 pm in a modal value of 7.5 pm fiber, and the coefficient of variation was 5.3%. Similar examination was also made in the smaller fiber (2.3 pm fiber) and in the larger one (12.5 pm fiber). In such cases those coefficients were 7.4% in the former and 3.7% in the latter. The coefficients of variations thus obtained in these measurements were in a range of 3-8s across fibers of various diameters. The second problem was the tapering of the nerve fibers. Concerning this problem, Eccles and Sherrington (9) reported that diameters of myelinated fibers decrease as they pass peripherally. Accordingly, we examined the degree of tapering in the fiber diameter between the proximal and distal end of the nerve as follows. The superficial radial nerve (length: 10 mm) used for embedding was cut into three pieces, and each piece was placed side by side in the paraffin block. Thus from these samples, it was determined whether the largest fiber tapered or branched in the course
264
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AND
MORI
magnification of FIG. 1. Cross-section of the cat superficial radial nerve at original 45X (left) and 112.5X (right). All the nerve bundles composing the superficial radial nerve are shown on the left, and one bundle is enlarged on the right. Part of one bundle is shown below at magnification of 450X.
of the preparation. As a result, the diameter of the largest fiber in the peripheral segment of the preparation was found to be not smaller than that of the largest fiber in the middle and proximal segments, and no evidence of the tapering or branching was found in any of the preparations. Therefore the variation of distribution patterns which was caused
AFFERENT
INNERVATION
FIG. 2. Distribution of diameter measurements cat followed through 100 serial sections of 10 pm.
OF
PAWS
26.5
is shown on a single fiber of the
by the tapering was considered to be negligible within the course of fibers used for the preparation. The palmar surface of the paw is covered with the hairless skin, and the dorsal surface is covered with the hairy skin. The latter contains hair follicle receptors in addition to other sensory receptors which could be also observed in the former. Therefore, when investigating the diameter
0
2
4
6 Fiber
diiwter
FIG. 3. A. Distribution of the diameters of myelinated fibers ia the superficial peroneal nerve which innervates the dorsal surface of the hind paw. B. Distribution of the diameters of myelinated fibers in the superficial plantar nerve which innervates the palmar surface of the hind paw. These results were obtained in the nerves of the cat. The number of myelinated fibers measured was 2950 in the superficial plantar nerve and 2710 in the superficial peroneal nerve.
266
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MORI
distribution patterns of the fore and hind paw cutaneous nerves, we also studied whether such a difference in the components of sensory receptors is related to those distribution patterns of the paw nerves innervating the dorsal and palmar surfaces. Hind Paw Nerves Innervating the Dorsal and Palwmr Surfaces in the Cat. Myelinated fibers of the hind paw cutaneous nerves were counted in five cats. The superficial peroneal nerve innervating the dorsal surface of the hind paw was innervated by the 2800-3300 myelinated fibers, and the superficial plantar nerve innervating the palmar surface was innervated by the 3100-3400 fibers. Figure 3 shows the representative example of the distribution histograms. The diameters of both nerves distributed in the range from 1 to 12 pm, and had bimodal distributions with peaks in 2 and 7 pm. From the relationship between fiber diameters and conduction velocities (12, 20)) it is known that the groups of fibers in excess of 5 pm diameter correspond to group II afferent fibers with conduction velocities higher than 30 m/set, and the other groups with less than 5 pm diameter correspond to group III afferent fibers. Therefore, the proportion of group II fibers to all myelinated fibers was used for comparison of the distribution histograms of the individual nerves. In both distribution histograms of Fig. 3, group II fibers were 44.3% of all myelinated fibers (2710 fibers) A %
20 -
I IO -
0-
2
4
6
a
0
12
14
p
-Fiber
2
4
6
a
12
diameter
FIG. 4. A. Distribution of the diameters of myelinated fibers in the superficial radial nerve which innervates the dorsal surface of the fore paw. B. Distribution of the diameters of myelinated fibers in the median nerve which innervates the palmar surface of the fore paw. These results were obtained in the nerves of the cat. The number of myelinated fibers measured was 3680 in the superficial radial nerve and 3350 in the median nerve.
AFFERENT
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B
I
.
2
.
.
4
.
.
6
.
.
B
.
.
W
.
,..a
12
I4
(
FIG. 5. Same as in Fig. 3 except that these distribution diagrams the nerves of the monkey. The number of myelinated fibers measured superficial peroneal nerve and 3374 in the superficial plantar nerve.
were obtained in was 3017 in the
in the superficial peroneal nerve, and 42.5% in the superficial plantar nerve (2950 fibers). The results obtained in five cats are summarized in Table 1. In this table, the numbers of all myelinated fibers composing the individual nerves were 2668-2855 in the superficial peroneal nerve, and 2731-2950 in the superficial plantar nerve; and the percentages of group II fibers were 40.9-44.3% in the superficial peroneal nerve, and 40X-43.6% in the superficial plantar nerve. These values were not different between both nerves (the level of significance: 5%). Fore Pazv Nerves Innervating the Dorsal and Palmar Surfaces in the Cat. Since the feedback signals of the fore paw afferent fibers seem to play an important role for the control of fine forelimb movement, whose function is more highly developed compared to that of the hind limb (lo), we examined how such a characteristic is reflected in the fiber distribution pattern of the forepaw nerve. Figure 4 shows the distribution histograms of the superficial radial nerve innervating the dorsal surface of the fore paw and that of the median nerve innervating the palmar surface. The fiber diameters also distributed in the range from 1 to 15 pm with peaks in 2-3 and 8 pm. Group II fibers were 67.3% of all myelinated fibers (3,680 fibers) in the superficial radial nerve, and 69.6% in the median nerve (3350 fibers). These values were not significantly different between both nerves at the level of 5% (Table 1). On the other hand, comparison of the results obtained from the fore paw nerves and from the hind paw nerves disclosed the following two facts.
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AND
MORI
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The fore paw cutaneous nerves (superficial radial nerve, median nerve) contain more myelinated fibers than the hind paw cutaneous nerves (superficial peroneal nerve, superficial plantar nerve). Total numbers of the myelinated fibers composing the individual nerves were 3270-3650 in the forepaw nerves, but were 2668-2950 in the hind paw nerves. The percentages of group II fibers in the forepaw cutaneous nerves were about 22-30s larger than those of group II fibers in the hind paw cutaneous nerves. Their percentages were 66.1-72.5s in the former, but were 40.&44.3% in the latter. Since the superficial radial nerve and median nerve do not send any branches to the fifth digit and lateral side of the fourth digit of the fore paw (S), we made a similar examination on the ulnar nerves innervating these regions in two cats. In one cat, the percentage of group II fibers of the ulnar nerve innervating the dorsal surface was 65.8% (total number : 379 fibers) and that of group II fibers of the ulnar nerve innervating the palmar surface was 67.3% (total number : 516 fibers). Therefore, the above described characteristics of the fiber histograms were maintained in the fiber histograms of the ulnar nerve. Similar histological examination was also made in two dogs. In one dog, the number of all myelinated fibers composing the individual nerves and the percentages of group II fibers were 3715 and 65.1% in the superficial radial nerve, and were 3592 and 67.37 o in the median nerve. On the other hand, they were 2835 and 40.77 o in the superficial peroneal nerve, and were 3210 and 43.1% in the superficial plantar nerve. This result was similar to those observed in the cat. Hind Paw Nerves Innervating the Dorsal and Palmar Surfaces in the Monkey. Figure 5 shows the representative example of the fiber distribution histograms in the hind paw cutaneous nerves, and the results obtained from five monkeys are summarized in Table 2. It was observed that the superficial peroneal nerve was composed of 3017 fibers, and the percentage of group II fibers was 42.9% ; and that the superficial plantar nerve was composed of 3374 fibers, and the percentage of group II fibers was 41.3%. These values were not significantly different between both nerves at the level of 5% (Table 2). Comparison of these results with the results obtained from the cat disclosed that, although the fibers composing the hind paw cutaneous nerves of the monkey were more numerous than those of the cat, the percentages of group II fibers were similar to those of group II fibers in the cat (Fig. 3). Fore Paw Nerves Innervating the Dorsal and Pahar Surfaces in the Monkey. Similar examination was also made in the fore paw nerves of
five monkeys. Figure 6 shows that the superficial radial nerve is composed of 35% fibers, and the percentage of group II fibers is 67.1% ; and that the median nerve is composed of 4472 fibers and the percentage of group
270
MATSUMOTO
AND
MORI
A oo-
2
4
6
6
IO
12
14 Faber diameter
FIG. 6. Same as in Fig. 4 except that these distribution diagrams were obtained in the nerves of the monkey. The number of myelinated fibers measured was 3596 in the superficial radial nerve and 4472 in the median nerve.
II fibers is 77.0%. These numbers and percentages were significantly different between both nerves at the level of 5% (Table 2). Therefore, it follows that in the monkey, the fore paw cutaneous nerves innervating the palmar surface (merian nerve) contain more numerous group II fibers than those innervating the dorsal one (superficial radial nerve). Furthermore, comparison of these results obtained from the cat disclosed the following two facts: The percentage of group II fibers was about the sameboth for the superficial radial nerves of the cat and monkey, although the total number of myelinated fibers composing the superficial radial nerve of the monkey was more numerous than those of the cat. On the other hand, these percentages and the total number of the median nerve in the monkey were larger than those in the cat, and significantly different at the level of 5 %. DISCUSSION In order to understand whether the components of receptor types are different between the forepaw cutaneous nerves (superficial radial nerve, median nerve) and the hind paw cutaneous nerves (superficial peroneal nerve, superficial plantar nerve), the number and diameter distribution diagrams of the individual nerves were examined in the cat, dog and monkey. Anatomically, the median nerve at the paw level consists of the cutaneous nerve innervating the palmar surface of the fore paw and the
AFFERENT
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part of lumbrical muscle branches (8). Because of this fact, when dissecting and removing the sensory branch of median nerve as the preparation, those muscle branches were carefully excluded as far as possible, The possibility that these preparations contain such muscle branches and modify the distribution histogram was excluded based on the following two facts. The population of group I muscle fibers in excess of 12 pm and with peaks in 16-18 pm (2) was not observed in the distribution histogram of the median nerve (Fig. 4 and Fig 6). In electrophysiological examination using two spinal cats, stimulation of the distal cut end of ventral roots (C6, C7, or CS) did not elicit the response from the proximal cut end of the median nerve which was dissected at the same level as that used for the preparation. In the cutaneous nerves of these animals innervating the hind and fore paw, the diameters of myelinated fibers distributed bimodally with peaks in 2-3 and 7-8 pm. They are typical for the cutaneous nerves and correspond with the characteristic distribution of the physiologically different group II and group III afferent fibers (2, 9, 13, 19). Furthermore, the percentages of group II fibers to all myelinated fibers were 40-44s in the hind paw cutaneous nerves. This result was the same as the result of Jar-rig (13) in which about 40% of myelinated fibers composing the superficial plantar nerve were group II fibers in the cat. However, the percentages of group II fibers were 6679% in the fore paw cutaneous nerves, and these values were 22-370/o larger than those in the hind paw cutaneous ones. Therefore it is obvious that a large number of group II cutaneous fibers innervates the surface of fore paw region compared with that of the hind paw region. This result was also in contrast with the general observation (4) that, in cutaneous nerves, the range of diameter and mean diameter of the fibers within group II and group III varies little from nerve to nerve. The discrepancy may stem from the fact that such a result was obtained only from the number and diameter distribution patterns of the hind limb nerve fibers. By electrophysiological investigation, it has been shown that group II cutaneous fibers are mainly composed of fibers from touch and pressure receptors and hair follicle receptors (1, 3, 5, 7, 11, 22, 23). The above described results, therefore, indicate that these receptors distribute on the surface of the forepaw more densely than on that of the hind paw. This particular dense distribution of the receptors on the forepaw must send multimodal feedback signals to the central nervous system. This histological observation seems to coincide well with the previous observation on the dog (16). Although, in the cat and dog, the distribution patterns of the forepaw cutaneous nerves innervating the palmar surfaces (median nerve) were not significantly different from those of the ones innervating the dorsal
AFFERENT
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surfaces (superficial radial nerve), in the monkey, the percentages of group II fibers of the median nerves (77.0 t 0.9%) were about 10% larger than those of the superficial radial nerves (67.7 * 1.0%) whose values were similar to those of the fore paw cutaneous nerves in the cat and dog. Therefore the touch and pressure receptors, which are innervated by these group II fibers, distribute on the palmar surface of the fore paw much more densely than the dorsal surface (hairy skin). Such a dense distribution of these receptors may play an important role for the motor control of the complex forepaw (hand) movement such as “grasping,” which is peculiar to the monkey. Concerning such a complex fore limb movement of the monkey compared to the cat and dog, Rosen and Asanuma (17, 18) demonstrated that a colony of cells in the monkey motorsensory cortex receives tactile inputs from the part of the limb to which the colony projects. They furthermore hypothesized that the somatosensory inputs from the fore limb to the corresponding areas of the motor cortex are utilized as feed#back informations thus enabling to monitor the accuracy of the fore limb movement, rather than for the initiation of the movement. Our observation does not permit that either of these possibilities is justified, but revealed that the phylogenetically developed motor function is related to the distribution pattern of cutaneous receptors. REFERENCES 1. ANDERSEN, P. J. C. ECCLES, and T. A. SEARS. 1964. Cortically evoked depolarization of primary afferent fibers in the spinal cord. J. Newopkysiol. 27: 63-77. 2. BARD, P. 1958. “Medical Physiology,” p. 1018. C. V. Mosby, St Louis. 3. BISHOP, G. H. 1943. Responses to electrical stimulation of single sensory unit of skin. J. Newophysiol. 6: 361382. 4. BOYD, I. A., and M. R. DAVEY. 1968. “Composition of Peripheral Nerves,” p. 32. E. & S. Livingstone Ltd., Edinburgh. 5. BURGESS, P. R., D. PETIT, and R. M. WARREN. 1968. Receptor types in cat hairy skin supplied by myelinated fibers. J. Nezwophysiol. 31: 833-848. 6. BROOKS, C. McC., and K. KOIZUMI. 1956. Origin of dorsal root reflex. J. Neurophysiol. 19 : 61-74. 7. BROWN, A. G., and A. IGCO. 1967. A quantitative study of cutaneous receptors and afferent fibers in the cat and rabbit. J. Physiol. (Lolzdon) 193: 707-733. 8. CROUCH, J. E. 1969. “Text Atlas of Cat Anatomy,” p. 237. 1st ed., Lea & Febiger, Philadelphia. 9. ECCLES, J. C., and C. S. SHERRINGTON. 1930. Numbers and contraction values of individual motor units examined in some muscles of the limb. Proc. Roy. Sac. London Ser. 106: 326341. 10. HOUK, J., and E. HENNEMAN. 1968. Feedback control of movement and posture, p. 1681. In “Medical Physiology.” V. B. Mountcastle [Ed.]. C. V. Mosby, St. Louis. An analysis of fiber diameter and 11. HUNT, C. C. and A. K. MCINTYRE. 1960. receptor characteristics of myelinated cutaneous afferent fibers in cat. J. Physiol. (London) 153 : 99-112.
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12. HURSR, J. B. 1939. Conduction velocity and diameter of nerve fibers. Amer. 1. Physiol. 127 : 131-139. 13. JANIG, W. 1971. The afferent innervation of the central pad of the cat’s hind foot. Brain Res. 28: 203-216. 14. MATSUMOTO, A., and S. MORI. 1973. Studies on post-tetanic depression of the dorsal root reflex in the forelimb skin nerves of the cat. Brain Res. 63: 384388. 15. MORI, S., and A. MATSUMOTO. 1972. The effects of stimulation of nerves to neck muscle upon flexor reflex in the forelimb. Brain Res. 43: 645-648. 16. MORI, S., P. J. REYNOLDS, and J. M. BROOKHART. 1970. The contribution of pedal afferents to postural control in the dog. Amer. J. Physiol. 218: 726-734. 17. ROSEN, I., and H. ASANUMA. 1972. Peripheral afferent inputs to the forelimb area of the motor cortex : input-output relations. Exp. Brain Res. 14: 257273. 18. ROSEN, I., and H. ASANUMA. 1973. Natural stimulation of group I activated cells in the cerebral cortex of the awake cat. Exp. Bruin Res. 16: 247-254. 19. SASAOKA, S. 1939. Uber das Kaliber der markhaltigen Nervengasern im Gelenkast. Jap. J. Med. Sci. 7: 315-322. 20. TASAKI, I., K. ISHII, and H. ITO. 1949. On the relation between the conductionrate, the fiber diameter and the internodal distance of the medullated nerve fiber. Jap. J. Med. Sk., III Biophysics 9: 189-199. 21. TOENNIES, J. F. 1936. Conditioning of afferent impulses by reflex discharge over the dorsal roots. J. Newophysiol. 2: 515-525. 22. WEDDELL, G. 1941. The pattern of cutaneous innervating in relation to cutaneous sensibility. J. Anat. (Londolz) 75: 346-367. 23. WEDDELL, G. 1941. The multiple innervation of sensory spots in the skin. J. Anat. (London)
75:
441-446.
NEUROLOGY
48, 261-274 (1975)
Number and Diameter Fibers Innervating
Distribution of Myelinated the Paws of the Cat and
Afferent Monkey
A. MATSUMOTOAND S.MORI' Departwacnt
of Physiology, Received Srptcmbcr
Hokkaido
Vuivcrsity
19, 1974;
Sclzool
revision
rcccivcd
of Medicine, March
Sapporo,
Japarz
5, 1975
The number and the diameter distribution of the myelinated cutaneous fibers innervating fore and hind paw were histologically examined in the cat and monkey. In five cats, the superficial peroneal nerve innervating the dorsal surface of the hind paw and the superficial plantar nerve innervating the palmar surface were composed of 2668-2950 fibers, 40-440/O of which were group II fibers. On the other hand, the superficial radial nerve innervating the dorsal surface of the fore paw and the sensory branch of median nerve innervating the palmar surface were composed of 3270-3680 fibers, of which 67-72s were group II fibers. Therefore, it was found that the cutaneous fore paw nerves contained more group II fibers than the hind paw cutaneous nerves. In five monkeys the percentages of group II fibers composing the superficial peroneal nerve and superficial plantar nerve (28933374 fibers) were the same as those of the hind paw nerves in the cat. On the other hand, the percentages of group II fibers of the median nerve (41734472 fibers) were 75-78%, about 10% larger than those of the superficial radial nerve (3596-3821 fibers) whose values were 66669%. Therefore, in the monkey, the forepaw nerves innervating the palmar surface also contain more group II afferent fibers compared to the ones innervating the dorsal one (hairy skin).
INTRODUCTION Recently Mori, Reynolds and Brookhart (16) demonstrated that the stability of trained dogs during quiet stance diminished after local anesthetic blockade of the afferents from the paws. It was furthermore observed that the degrees of diminution of the forepaw blocked animals were 1 The authors express their gratitude to Professor M. Kato for his encouragement during the course of the experiment and for reading the manuscript. They express their thanks to Professor T. Nakanishi (Department of Anatomy, Asahikawa Medical College) and Dr. T. Miyagishi (Department of Psychiatry, Hokkaido University School of Medicine) for their criticism during the preparation of the manuscript. Dr. Mori’s present address is the Department of Physiology, Asahikawa Medical College, Asahikawa. 261 Copyright All rights
@ 1975 by Academic Press, Inc. of reproduction in any form reserved.
262
MATSIJMOTO
AND
MORI
almost as pronounced as they were with quadripedal anesthesia, but were characteristically much less pronounced when only the hind paws were affected. These observations suggested that the feedback inputs from the paws are not only one of the important factors for the stability of animals, but also the individual feedback inputs from the forepaws and hind paws may send functionally different informations from the receptors to the central nervous system. Therefore, in this paper we attempted to study whether the components of sensory receptors were different both for the fore paw and hind paw cutaneous nerves, since it may be possible to estimate roughly the receptor type from the diameters of the afferent fibers (2, 4, 11). This study was also prompted by the previous observations (14, 15) that in the forelimb cutaneous nerves of the cat, dorsal root reflex, for the generation of which the afferent inputs from the touch and pressure receptors greatly contribute (6, 21)) could be elicited at the normal temperature in contrast to that elicited in the hind limb cutaneous nerves at the reduced temperature. We also studied how the distribution pattern of afferent fibers would phylogenetically change by comparing the results obtained from the cat, dog and monkey. METHODS Five cats, two dogs and five monkeys (two Macaca &us, three Macaca were employed. Under nembutal anesthesia, the following four nerves were dissected and removed for approximately 10 mm at the levels of dorsal and palmar paws: branch of superficial radial nerve innervating the dorsal surface of the forepaw; sensory branch of median nerve innervating the palmar surface of the forepaw; superficial peroneal nerve innervating the dorsal surface of the hind paw; and superficial plantar nerve innervating the palmar surface of the hind paw. In two cats, the ulnar nerves innervating the parts of dorsal and palmar surfaces of the forepaw were also dissectedand removed. After removing the nerves, each cut end was fixed on a glass rod with thin silk thread to prevent deformation of the nerves. After fixation and staining in osmic acid (1% solution, Merk Co.) for 24 hr (4, 12), each nerve was dehydrated slowly by immersion for 12 hr in each of a series of graded alcohols. It was then immersed in chloroform for 4 hr, and embedded in the paraffin wax. Serial sections of the nerve were cut at 10 pm. These specimens were photographed with a magnification of 1000x. The films thus made were projected to a screen so that 1 pm corresponded to 1 mm on the screen. The diameters of the fibers outside of the myelin sheath were measured for the fibers constituting the individual nerves. When a myelin sheath showed deformity, we excluded the fiber from meamulatta)
AFFERENT
INNERVATION
OF
PAWS
263
surement. However, when the myelin sheath was slightly ovoid in shape, we measured the diameter at the intermediate axis between the longest and shortest ones. In this experiment, about 97-98s of all myelinated fibers constituting each nerve were measured. The question of shrinkage of the nerve fibers through the process of fixation and dehydration was also examined. For this, the diameters of several nerve fibers, selected at random, were measured both in fresh frozen-sections and in paraffin cross-sections of the same nerve. As the result, it was found that the fixation and dehydration caused an average decrease in the fiber diameters of 2.4 -C 0.7% (n = 120). There was no correlation between the percentages of shrinkage and the diameters. RESULTS An example of the transverse section of the myelinated nerve fibers obtained from the cat is shown in Fig. 1 at three magnifications: 45 x ; 112.5 x ; and 450x. The nerve was enclosed with the epineurium, and consisted of the several fiber bundles. Each fiber bundles was encapsulated with the perineurium, and contained about the 200-1300 myelinated fibers across animals of different species. Before drawing the distribution graph of the diameters of these myelinated fibers, we examined the following problems to exclude the source of measurement error. The first question was the degree of possible variation of the diameters in the course of the fibers due to the technique of preparation or other causes. In order to investigate this problem, measurements were made of 100 consecutive 10 pm paraffin sections of the superficial radial nerve obtained from the cat. The diameters of the several selected fibers were measured in each section. The distribution of the diameters measured from a single fiber is shown in Fig. 2. It was observed that the standard deviation (SD) was 0.4 pm in a modal value of 7.5 pm fiber, and the coefficient of variation was 5.3%. Similar examination was also made in the smaller fiber (2.3 pm fiber) and in the larger one (12.5 pm fiber). In such cases those coefficients were 7.4% in the former and 3.7% in the latter. The coefficients of variations thus obtained in these measurements were in a range of 3-8s across fibers of various diameters. The second problem was the tapering of the nerve fibers. Concerning this problem, Eccles and Sherrington (9) reported that diameters of myelinated fibers decrease as they pass peripherally. Accordingly, we examined the degree of tapering in the fiber diameter between the proximal and distal end of the nerve as follows. The superficial radial nerve (length: 10 mm) used for embedding was cut into three pieces, and each piece was placed side by side in the paraffin block. Thus from these samples, it was determined whether the largest fiber tapered or branched in the course
264
MATSUMOTO
AND
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magnification of FIG. 1. Cross-section of the cat superficial radial nerve at original 45X (left) and 112.5X (right). All the nerve bundles composing the superficial radial nerve are shown on the left, and one bundle is enlarged on the right. Part of one bundle is shown below at magnification of 450X.
of the preparation. As a result, the diameter of the largest fiber in the peripheral segment of the preparation was found to be not smaller than that of the largest fiber in the middle and proximal segments, and no evidence of the tapering or branching was found in any of the preparations. Therefore the variation of distribution patterns which was caused
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FIG. 2. Distribution of diameter measurements cat followed through 100 serial sections of 10 pm.
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is shown on a single fiber of the
by the tapering was considered to be negligible within the course of fibers used for the preparation. The palmar surface of the paw is covered with the hairless skin, and the dorsal surface is covered with the hairy skin. The latter contains hair follicle receptors in addition to other sensory receptors which could be also observed in the former. Therefore, when investigating the diameter
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FIG. 3. A. Distribution of the diameters of myelinated fibers ia the superficial peroneal nerve which innervates the dorsal surface of the hind paw. B. Distribution of the diameters of myelinated fibers in the superficial plantar nerve which innervates the palmar surface of the hind paw. These results were obtained in the nerves of the cat. The number of myelinated fibers measured was 2950 in the superficial plantar nerve and 2710 in the superficial peroneal nerve.
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distribution patterns of the fore and hind paw cutaneous nerves, we also studied whether such a difference in the components of sensory receptors is related to those distribution patterns of the paw nerves innervating the dorsal and palmar surfaces. Hind Paw Nerves Innervating the Dorsal and Palwmr Surfaces in the Cat. Myelinated fibers of the hind paw cutaneous nerves were counted in five cats. The superficial peroneal nerve innervating the dorsal surface of the hind paw was innervated by the 2800-3300 myelinated fibers, and the superficial plantar nerve innervating the palmar surface was innervated by the 3100-3400 fibers. Figure 3 shows the representative example of the distribution histograms. The diameters of both nerves distributed in the range from 1 to 12 pm, and had bimodal distributions with peaks in 2 and 7 pm. From the relationship between fiber diameters and conduction velocities (12, 20)) it is known that the groups of fibers in excess of 5 pm diameter correspond to group II afferent fibers with conduction velocities higher than 30 m/set, and the other groups with less than 5 pm diameter correspond to group III afferent fibers. Therefore, the proportion of group II fibers to all myelinated fibers was used for comparison of the distribution histograms of the individual nerves. In both distribution histograms of Fig. 3, group II fibers were 44.3% of all myelinated fibers (2710 fibers) A %
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FIG. 4. A. Distribution of the diameters of myelinated fibers in the superficial radial nerve which innervates the dorsal surface of the fore paw. B. Distribution of the diameters of myelinated fibers in the median nerve which innervates the palmar surface of the fore paw. These results were obtained in the nerves of the cat. The number of myelinated fibers measured was 3680 in the superficial radial nerve and 3350 in the median nerve.
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FIG. 5. Same as in Fig. 3 except that these distribution diagrams the nerves of the monkey. The number of myelinated fibers measured superficial peroneal nerve and 3374 in the superficial plantar nerve.
were obtained in was 3017 in the
in the superficial peroneal nerve, and 42.5% in the superficial plantar nerve (2950 fibers). The results obtained in five cats are summarized in Table 1. In this table, the numbers of all myelinated fibers composing the individual nerves were 2668-2855 in the superficial peroneal nerve, and 2731-2950 in the superficial plantar nerve; and the percentages of group II fibers were 40.9-44.3% in the superficial peroneal nerve, and 40X-43.6% in the superficial plantar nerve. These values were not different between both nerves (the level of significance: 5%). Fore Pazv Nerves Innervating the Dorsal and Palmar Surfaces in the Cat. Since the feedback signals of the fore paw afferent fibers seem to play an important role for the control of fine forelimb movement, whose function is more highly developed compared to that of the hind limb (lo), we examined how such a characteristic is reflected in the fiber distribution pattern of the forepaw nerve. Figure 4 shows the distribution histograms of the superficial radial nerve innervating the dorsal surface of the fore paw and that of the median nerve innervating the palmar surface. The fiber diameters also distributed in the range from 1 to 15 pm with peaks in 2-3 and 8 pm. Group II fibers were 67.3% of all myelinated fibers (3,680 fibers) in the superficial radial nerve, and 69.6% in the median nerve (3350 fibers). These values were not significantly different between both nerves at the level of 5% (Table 1). On the other hand, comparison of the results obtained from the fore paw nerves and from the hind paw nerves disclosed the following two facts.
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The fore paw cutaneous nerves (superficial radial nerve, median nerve) contain more myelinated fibers than the hind paw cutaneous nerves (superficial peroneal nerve, superficial plantar nerve). Total numbers of the myelinated fibers composing the individual nerves were 3270-3650 in the forepaw nerves, but were 2668-2950 in the hind paw nerves. The percentages of group II fibers in the forepaw cutaneous nerves were about 22-30s larger than those of group II fibers in the hind paw cutaneous nerves. Their percentages were 66.1-72.5s in the former, but were 40.&44.3% in the latter. Since the superficial radial nerve and median nerve do not send any branches to the fifth digit and lateral side of the fourth digit of the fore paw (S), we made a similar examination on the ulnar nerves innervating these regions in two cats. In one cat, the percentage of group II fibers of the ulnar nerve innervating the dorsal surface was 65.8% (total number : 379 fibers) and that of group II fibers of the ulnar nerve innervating the palmar surface was 67.3% (total number : 516 fibers). Therefore, the above described characteristics of the fiber histograms were maintained in the fiber histograms of the ulnar nerve. Similar histological examination was also made in two dogs. In one dog, the number of all myelinated fibers composing the individual nerves and the percentages of group II fibers were 3715 and 65.1% in the superficial radial nerve, and were 3592 and 67.37 o in the median nerve. On the other hand, they were 2835 and 40.77 o in the superficial peroneal nerve, and were 3210 and 43.1% in the superficial plantar nerve. This result was similar to those observed in the cat. Hind Paw Nerves Innervating the Dorsal and Palmar Surfaces in the Monkey. Figure 5 shows the representative example of the fiber distribution histograms in the hind paw cutaneous nerves, and the results obtained from five monkeys are summarized in Table 2. It was observed that the superficial peroneal nerve was composed of 3017 fibers, and the percentage of group II fibers was 42.9% ; and that the superficial plantar nerve was composed of 3374 fibers, and the percentage of group II fibers was 41.3%. These values were not significantly different between both nerves at the level of 5% (Table 2). Comparison of these results with the results obtained from the cat disclosed that, although the fibers composing the hind paw cutaneous nerves of the monkey were more numerous than those of the cat, the percentages of group II fibers were similar to those of group II fibers in the cat (Fig. 3). Fore Paw Nerves Innervating the Dorsal and Pahar Surfaces in the Monkey. Similar examination was also made in the fore paw nerves of
five monkeys. Figure 6 shows that the superficial radial nerve is composed of 35% fibers, and the percentage of group II fibers is 67.1% ; and that the median nerve is composed of 4472 fibers and the percentage of group
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A oo-
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FIG. 6. Same as in Fig. 4 except that these distribution diagrams were obtained in the nerves of the monkey. The number of myelinated fibers measured was 3596 in the superficial radial nerve and 4472 in the median nerve.
II fibers is 77.0%. These numbers and percentages were significantly different between both nerves at the level of 5% (Table 2). Therefore, it follows that in the monkey, the fore paw cutaneous nerves innervating the palmar surface (merian nerve) contain more numerous group II fibers than those innervating the dorsal one (superficial radial nerve). Furthermore, comparison of these results obtained from the cat disclosed the following two facts: The percentage of group II fibers was about the sameboth for the superficial radial nerves of the cat and monkey, although the total number of myelinated fibers composing the superficial radial nerve of the monkey was more numerous than those of the cat. On the other hand, these percentages and the total number of the median nerve in the monkey were larger than those in the cat, and significantly different at the level of 5 %. DISCUSSION In order to understand whether the components of receptor types are different between the forepaw cutaneous nerves (superficial radial nerve, median nerve) and the hind paw cutaneous nerves (superficial peroneal nerve, superficial plantar nerve), the number and diameter distribution diagrams of the individual nerves were examined in the cat, dog and monkey. Anatomically, the median nerve at the paw level consists of the cutaneous nerve innervating the palmar surface of the fore paw and the
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part of lumbrical muscle branches (8). Because of this fact, when dissecting and removing the sensory branch of median nerve as the preparation, those muscle branches were carefully excluded as far as possible, The possibility that these preparations contain such muscle branches and modify the distribution histogram was excluded based on the following two facts. The population of group I muscle fibers in excess of 12 pm and with peaks in 16-18 pm (2) was not observed in the distribution histogram of the median nerve (Fig. 4 and Fig 6). In electrophysiological examination using two spinal cats, stimulation of the distal cut end of ventral roots (C6, C7, or CS) did not elicit the response from the proximal cut end of the median nerve which was dissected at the same level as that used for the preparation. In the cutaneous nerves of these animals innervating the hind and fore paw, the diameters of myelinated fibers distributed bimodally with peaks in 2-3 and 7-8 pm. They are typical for the cutaneous nerves and correspond with the characteristic distribution of the physiologically different group II and group III afferent fibers (2, 9, 13, 19). Furthermore, the percentages of group II fibers to all myelinated fibers were 40-44s in the hind paw cutaneous nerves. This result was the same as the result of Jar-rig (13) in which about 40% of myelinated fibers composing the superficial plantar nerve were group II fibers in the cat. However, the percentages of group II fibers were 6679% in the fore paw cutaneous nerves, and these values were 22-370/o larger than those in the hind paw cutaneous ones. Therefore it is obvious that a large number of group II cutaneous fibers innervates the surface of fore paw region compared with that of the hind paw region. This result was also in contrast with the general observation (4) that, in cutaneous nerves, the range of diameter and mean diameter of the fibers within group II and group III varies little from nerve to nerve. The discrepancy may stem from the fact that such a result was obtained only from the number and diameter distribution patterns of the hind limb nerve fibers. By electrophysiological investigation, it has been shown that group II cutaneous fibers are mainly composed of fibers from touch and pressure receptors and hair follicle receptors (1, 3, 5, 7, 11, 22, 23). The above described results, therefore, indicate that these receptors distribute on the surface of the forepaw more densely than on that of the hind paw. This particular dense distribution of the receptors on the forepaw must send multimodal feedback signals to the central nervous system. This histological observation seems to coincide well with the previous observation on the dog (16). Although, in the cat and dog, the distribution patterns of the forepaw cutaneous nerves innervating the palmar surfaces (median nerve) were not significantly different from those of the ones innervating the dorsal
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surfaces (superficial radial nerve), in the monkey, the percentages of group II fibers of the median nerves (77.0 t 0.9%) were about 10% larger than those of the superficial radial nerves (67.7 * 1.0%) whose values were similar to those of the fore paw cutaneous nerves in the cat and dog. Therefore the touch and pressure receptors, which are innervated by these group II fibers, distribute on the palmar surface of the fore paw much more densely than the dorsal surface (hairy skin). Such a dense distribution of these receptors may play an important role for the motor control of the complex forepaw (hand) movement such as “grasping,” which is peculiar to the monkey. Concerning such a complex fore limb movement of the monkey compared to the cat and dog, Rosen and Asanuma (17, 18) demonstrated that a colony of cells in the monkey motorsensory cortex receives tactile inputs from the part of the limb to which the colony projects. They furthermore hypothesized that the somatosensory inputs from the fore limb to the corresponding areas of the motor cortex are utilized as feed#back informations thus enabling to monitor the accuracy of the fore limb movement, rather than for the initiation of the movement. Our observation does not permit that either of these possibilities is justified, but revealed that the phylogenetically developed motor function is related to the distribution pattern of cutaneous receptors. REFERENCES 1. ANDERSEN, P. J. C. ECCLES, and T. A. SEARS. 1964. Cortically evoked depolarization of primary afferent fibers in the spinal cord. J. Newopkysiol. 27: 63-77. 2. BARD, P. 1958. “Medical Physiology,” p. 1018. C. V. Mosby, St Louis. 3. BISHOP, G. H. 1943. Responses to electrical stimulation of single sensory unit of skin. J. Newophysiol. 6: 361382. 4. BOYD, I. A., and M. R. DAVEY. 1968. “Composition of Peripheral Nerves,” p. 32. E. & S. Livingstone Ltd., Edinburgh. 5. BURGESS, P. R., D. PETIT, and R. M. WARREN. 1968. Receptor types in cat hairy skin supplied by myelinated fibers. J. Nezwophysiol. 31: 833-848. 6. BROOKS, C. McC., and K. KOIZUMI. 1956. Origin of dorsal root reflex. J. Neurophysiol. 19 : 61-74. 7. BROWN, A. G., and A. IGCO. 1967. A quantitative study of cutaneous receptors and afferent fibers in the cat and rabbit. J. Physiol. (Lolzdon) 193: 707-733. 8. CROUCH, J. E. 1969. “Text Atlas of Cat Anatomy,” p. 237. 1st ed., Lea & Febiger, Philadelphia. 9. ECCLES, J. C., and C. S. SHERRINGTON. 1930. Numbers and contraction values of individual motor units examined in some muscles of the limb. Proc. Roy. Sac. London Ser. 106: 326341. 10. HOUK, J., and E. HENNEMAN. 1968. Feedback control of movement and posture, p. 1681. In “Medical Physiology.” V. B. Mountcastle [Ed.]. C. V. Mosby, St. Louis. An analysis of fiber diameter and 11. HUNT, C. C. and A. K. MCINTYRE. 1960. receptor characteristics of myelinated cutaneous afferent fibers in cat. J. Physiol. (London) 153 : 99-112.
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12. HURSR, J. B. 1939. Conduction velocity and diameter of nerve fibers. Amer. 1. Physiol. 127 : 131-139. 13. JANIG, W. 1971. The afferent innervation of the central pad of the cat’s hind foot. Brain Res. 28: 203-216. 14. MATSUMOTO, A., and S. MORI. 1973. Studies on post-tetanic depression of the dorsal root reflex in the forelimb skin nerves of the cat. Brain Res. 63: 384388. 15. MORI, S., and A. MATSUMOTO. 1972. The effects of stimulation of nerves to neck muscle upon flexor reflex in the forelimb. Brain Res. 43: 645-648. 16. MORI, S., P. J. REYNOLDS, and J. M. BROOKHART. 1970. The contribution of pedal afferents to postural control in the dog. Amer. J. Physiol. 218: 726-734. 17. ROSEN, I., and H. ASANUMA. 1972. Peripheral afferent inputs to the forelimb area of the motor cortex : input-output relations. Exp. Brain Res. 14: 257273. 18. ROSEN, I., and H. ASANUMA. 1973. Natural stimulation of group I activated cells in the cerebral cortex of the awake cat. Exp. Bruin Res. 16: 247-254. 19. SASAOKA, S. 1939. Uber das Kaliber der markhaltigen Nervengasern im Gelenkast. Jap. J. Med. Sci. 7: 315-322. 20. TASAKI, I., K. ISHII, and H. ITO. 1949. On the relation between the conductionrate, the fiber diameter and the internodal distance of the medullated nerve fiber. Jap. J. Med. Sk., III Biophysics 9: 189-199. 21. TOENNIES, J. F. 1936. Conditioning of afferent impulses by reflex discharge over the dorsal roots. J. Newophysiol. 2: 515-525. 22. WEDDELL, G. 1941. The pattern of cutaneous innervating in relation to cutaneous sensibility. J. Anat. (Londolz) 75: 346-367. 23. WEDDELL, G. 1941. The multiple innervation of sensory spots in the skin. J. Anat. (London)
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441-446.