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Localization of normal and reinnervated mammalian muscle spindles for microscopy
240
SHORT COMMUNICATIONS
Localization of normal and reinnervated mammalian muscle spindles for microscopy Localization of mammalian muscle spindles for electron microscopy has been achieved by random selection of tissue blocks or dissection in the living muscle 1,2,7-11, 13,16,17. These procedures are largely dependent on chance, and they impose the risk of damage to the spindle as well as restrictions on recording the receptor's afferent discharge. Alternately, several procedures described for localization of spindles utilizing electrophysiological techniques ~,4,6 have not included systematic histological confirmation, or, by their nature, cannot easily be used with normal muscle tissue. We describe, here, (1) a method of localizing a muscle spindle for both electrophysiological and morphological study and (2) an application for this method in examining normally innervated and reinnervated mammalian spindles. Experiments on 4 adult cats form the basis of this report. Three animals remained intact; the fourth underwent denervation of the left tenuissimus muscle 6 weeks prior to the acute experiment. Using halothane anesthesia, denervation was accomplished by freezing the nerve with dry ice for 5 min, 1 cm proximal to its entry into the muscle. This method of denervation results in degeneration of all nerve fibers distal to the freeze, yet causes minimal disorganization within the epineurium (Fig. 2B). For acute experiments designed to localize muscle spindles, each animal was anesthetized with sodium pentobarbital (35 mg/kg, i.p.); the spinal cord was exposed and all nerves in the hindlimb were sectioned except the nerve to the tenuissimus muscle. This muscle was exposed along its length, care being taken to keep it warm and moist with blood supply intact. Recording electrodes were placed along carefully dissected filaments of ipsilateral L7 or SI dorsal roots. Units were identified by their constant amplitude and time-locked discharge with stimulation of the nerve to the tenuissimus muscle. Each afferent was identified as to its spindle origin by a pause in firing during shortening of the muscle; the unit's conduction velocity was subsequently calculated. Localization of a muscle spindle was achieved by noting the area of muscle from which a high frequency unit discharge could be elicited with discrete mechanical stimulation. By applying a lengthwise, moving deformation with a glass probe, we found a burst in spindle firing with each back and forth stroke along only one small length of muscle, usually less than 2 mm in extent. When similar back and forth strokes were applied on either side of the optimal response area, bursts in firing were obtained only during the stroke away from that area giving the maximal response (Fig. 1A). Upon localizing the point of maximal response to the mechanical stimulus, that area, centered in a block of tissue 5-6 mm long and 1-2 mm in width, was carefully cut from the muscle. Each block was fixed in 4 ~ paraformaldehyde in Dalton's solution at 4°C (1 h) and post-fixed in 2 ~ osmium tetroxide in Dalton's solution at 4°C (1 h). The tissue was then dehydrated and embedded in Araldite. Sequential 1 /~m sections were cut and stained with a 0.5 ~ toluidine blue solution. These sections were Brain Research, 39 (1972) 240-244
SHORT COMMUNICATIONS
241
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Fig. 1. A, Spindle afferent unit responses corresponding to localized sliding deformations along 3 adjacent areas (stipple) of the tenuissimus muscle. Conduction velocity of the unit was 91 m/sec. The scaled, cross-section drawings show the portion of the muscle spindle located in respective thirds of the excised segment (broken line). Dots along tenuissimus show the relative positions along the muscle for 10 separate, localized, unit responses and indicate whether one ( • ) or two (O) spindles were found in the excised tissue. B, Conduction velocities for the 10 localized units, and the number and longitudinal extent of the muscle spindle(s) observed in the corresponding biopsied tissue.
studied with the light m i c r o s c o p e to find the muscle spindle within the block. U l t r a thin sections, stained with u r a n y l acetate a n d l e a d citrate, were studied a n d p h o t o g r a p h e d with a Philips-200 electron m i c r o s c o p e . Ten spindles were localized in 4 tenuissimus muscles (Fig. 1B). Their relative positions a l o n g the length o f the muscle are shown in the c o m p o s i t e d r a w i n g in Fig. 1A. A t least one spindle, b u t never m o r e t h a n two, c o u l d be l o c a t e d in each b i o p s i e d block. I f two spindles were seen in a block, it was possible to p r e s u m e f r o m which one the afferent response h a d been elicited since one was usually centered within the biopsied tissue. Spindles c o u l d be localized regardless o f whether the unit's conduction velocity i n d i c a t e d the fiber t e r m i n a t e d p e r i p h e r a l l y as a p r i m a r y o r s e c o n d a r y ending. Six weeks f o l l o w i n g d e n e r v a t i o n it is quite possible to r e c o r d f r o m afferent units with c o n d u c t i o n velocities in the g r o u p I o r I I range which give typical spindle afferent Brain Research, 39 (1972) 240-244
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Fig. 2. A, Neurogram and electromyogram recorded from the cut S1 dorsal root (lower trace) and medial head of the gastrocnemius (MG) muscle (upper trace) upon stimulating the nerve to the MG, before (1) and after (2, 3) freezing the nerve. Stimulating electrode proximal to freeze in 2 and distal in 3. B, Photomicrograph showing Wallerian degeneration found in tenuissimus nerve 4 days following freeze. C-E, Electron micrographs obtained from the equatorial and juxtaequatorial region of 3 different muscle spindles localized in the tenuissimus muscle of an undenervated animal (C) and one 6 weeks subsequent to denervation (D, E). Sensory ending-like structures (SE) are contiguous to nuclear bag (C, E) or nuclear chain (D) intrafusal muscle fibers, containing centrally located muscle nuclei (N). Basement membrane (arrows) overlies muscle fiber and sensory ending, but is not interposed in the cleft between them.
SHORT COMMUNICATIONS
243
responses to contraction and phasic lengthening of the tenuissimus muscle. Prior to 4 weeks, such responses could not be elicited in our denervated animals. When these functionally reinnervated spindles are examined with the electron microscope, a few sensory terminals of normal configuration are found on nuclear chain intrafusal fibers (Fig. 2D). There are, however, numerous structures suggestive of sensory terminals in appearance and distribution, but with a consistent, distinctly abnormal configuration. These structures are seen exclusively in the equatorial and juxtaequatorial regions of the spindle and almost entirely on nuclear bag fibers (Fig. 2E). As with typical sensory endings, each of these terminal-like structures is partially depressed in the muscle fiber; a basement membrane covers the free surface of the terminal but is not seen in the gap between sensory terminal and intrafusal muscle fiber. In contrast to the ultrastructural configuration of normal sensory endings (Fig. 2C), the abnormal terminal-like structures appear flattened and highly osmophilic. Within these structures, in addition to plentiful, closely packed ribosomes, dense mitochondria and characteristic vesicles, there are occasional myelin figures and aggregates of heavily stained cellular debris. Like the normally innervated spindle, no motor terminals have been found in the equatorial regions of the reinnervated spindle. Study of motor innervation in juxtaequatorial and polar regions of the spindles is not included in this report. Studies with the light microscope have demonstrated that the capsule and intrafusal fibers of muscle spindles in adult mammals are relatively resistant to destruction of their sensory and motor nervesS,is and that the spindle can be reinnervated by regenerating nerve fibers 12,19. The functional integrity of muscle spindles reinnervated in the adult animal has been reported 15. Reinnervated atypical muscle spindles, resuiting after denervation in the newborn rat, give a slowly adapting discharge to maintained stretch of the muscle 14. In the present study, by 6 weeks after denervation, muscle spindles were again partially functional. Only a few sites on intrafusal muscle fibers of these spindles showed the usual morphological pattern of sensory innervation. We can be certain that the nerve supply to the muscle was completely destroyed by the denervation procedure (Fig. 2A, B). It appears, therefore, that the normal sensory terminals found after 6 weeks in the denervated spindles are regenerated sensory endings. The dense, flattened structures may be sensory endings which are returning to normal at the time of fixation. The presence of a few normal endings on the nuclear chain fibers only, and the presence o f the dense endings, predominantly on the nuclear bag fibers, suggests that the reinnervation may occur on the chain fibers first. This interpretation is consistent, so far, with results of additional experiments being conducted in our laboratory on animals studied at other times after denervation.
We wish to thank Scott Morrill (electronics), Robert Barrett and Gail Chambers (illustrations), Geraldean Dunkerley and Martha Monks (manuscript) for assistance in the course of this work. This study was supported by Bureau of Medicine and Surgery Work Unit Brain Research, 39 (1972) 24(I-244
244
SHORT COMMUNICATIONS
MR011.01.01-7014. Dr. De Santis was a Research Associate of the National Research Council. Opinions or conclusions contained in this report are those of the authors and do not necessarily reflect the views or the endorsement of the Navy Department. Naval Aerospace Medical Research Laboratory, Naval Aerospace Medical Institute, Naval Aerospace Medical Center, Pensacola, Fla. 32512 (U.S.A.)
M A R K DE SANTIS* WILLIAM O. WHETSELL, JR. K A R E N FRANCIS
1 ADAL, M. N., The fine structure of the sensory region of cat muscle spindles, J. Ultrastruct. Res., 26 (1969) 332-353. 2 ADAL, M. N., AND BARKER, D., The fine structure of cat fusimotor endings, J. Physiol. (Lond.), 192 (1967) 50P-52P. 3 BESSOU, P., AND LAPORTE, Y., Responses from primary and secondary endings of the same neuromuscular spindle of the tenuissimus muscle of the cat. In D. BARKER (Ed.), Symposium on Muscle Receptors, Hong Kong Univ. Press, Hong Kong, 1962, p. 105. 4 BONSETT, C. A., AND ABREU, B. E., Skeletal muscle studies by means of combined electromyography and needle biopsy. II. The muscle spindle, Neurology (Minneap.), 13 (1963) 327-330. 5 BoYo, I. E., The structure and innervation of the nuclear bag muscle fibre system and the nuclear chain muscle fibre system in mammalian muscle spindles, Phil. Trans. B, 245 (1962) 81-136. 6 BRIDGMAN, C. F., AND ELDRED, E., Hypothesis for a pressure-sensitive mechanism in muscle spindles, Science, 143 (1964) 481 482. 7 CHENG, K., AND BREININ, G. M., A comparison of the fine structure of extraocular and interosseus muscles in the monkey, Invest. Ophthalmol., 5 (1966) 535-549. 8 CORVAJA,N., AND MARINOZZI, V., Close appositions and junctions of plasma membranes of intrafusal fibres in mammalian muscle spindles, Pfliigers Arch. ges. Physiol., 296 (1967) 337-345. 9 CORVAJA,N., MARINOZZI, V., AND POMPEIANO, O., Muscle spindles in the lumbrical muscle of the adult cat. Electron microscopic observations and functional considerations, Arch. ital. Biol., 107 (1969) 365-545. 10 DURING, M. VON, UND ANDRES, K . H . , Zur Feinstruktur der Muskelspindel yon Mammalia, Anat. Anz., 124 (1969) 566 573. l l GRUNER, J. E., La structure fine du fuseau neuromusculaire humain, Rev. neurol., 104 (1961) 490-507. 12 GUTMANN, E., Reinnervation of muscle by sensory nerve fibres, J. Anat. (Lond.), 79 (1945) 1-8. 13 HENNIG, G., Die Nervenendigungen der Rattenmuskelspindel imelektronen- und phasenkontrastmikroskopischen Bild, Z. Zellforsch., 96 (1969) 275-294. 14 HNiK, P., Functional characteristics of free nerve endings and atypical spindles after muscle reinnervation in very young rats, Physiol. bohernoslov., 13 (1964) 216-219. 15 HOMMA, S., Peripheral nerve regeneration and muscle receptor reconstruction, Electroenceph. din. Neurophysiol., 27 (1969) 720. 16 LANDON, D. N., Electron microscopy of muscle spindles. In B. L. ANDREWS (Ed.), Symposium on Control and Innervation of Skeletal Muscle, Livingstone, Edinburgh, 1966, p. 96. 17 MERRILLEES,N. C., The fine structure of muscle spindles in the lumbrical muscles of the rat, J. biophys, biochem. CytoL, 7 (1960) 725-742. 18 TOWER, S. S., Atrophy and degeneration in the muscle spindle, Brain, 55 (1932) 77-90. 19 ZHENEVSKAYA,R. P., AND UMNOVA, M. M., Degeneration and restoration of sensory nerve endings in skeletal muscle, Arkh. Anat. Gistol. Embriol., 49 (1965) 3 11 (in Russian). (Accepted January 12th, 1972)
* Present address: Department of Anatomy, Georgetown University, Washington, D.C. 20007, U.S.A.
Brain Research, 39 (1972) 240-244
SHORT COMMUNICATIONS
Localization of normal and reinnervated mammalian muscle spindles for microscopy Localization of mammalian muscle spindles for electron microscopy has been achieved by random selection of tissue blocks or dissection in the living muscle 1,2,7-11, 13,16,17. These procedures are largely dependent on chance, and they impose the risk of damage to the spindle as well as restrictions on recording the receptor's afferent discharge. Alternately, several procedures described for localization of spindles utilizing electrophysiological techniques ~,4,6 have not included systematic histological confirmation, or, by their nature, cannot easily be used with normal muscle tissue. We describe, here, (1) a method of localizing a muscle spindle for both electrophysiological and morphological study and (2) an application for this method in examining normally innervated and reinnervated mammalian spindles. Experiments on 4 adult cats form the basis of this report. Three animals remained intact; the fourth underwent denervation of the left tenuissimus muscle 6 weeks prior to the acute experiment. Using halothane anesthesia, denervation was accomplished by freezing the nerve with dry ice for 5 min, 1 cm proximal to its entry into the muscle. This method of denervation results in degeneration of all nerve fibers distal to the freeze, yet causes minimal disorganization within the epineurium (Fig. 2B). For acute experiments designed to localize muscle spindles, each animal was anesthetized with sodium pentobarbital (35 mg/kg, i.p.); the spinal cord was exposed and all nerves in the hindlimb were sectioned except the nerve to the tenuissimus muscle. This muscle was exposed along its length, care being taken to keep it warm and moist with blood supply intact. Recording electrodes were placed along carefully dissected filaments of ipsilateral L7 or SI dorsal roots. Units were identified by their constant amplitude and time-locked discharge with stimulation of the nerve to the tenuissimus muscle. Each afferent was identified as to its spindle origin by a pause in firing during shortening of the muscle; the unit's conduction velocity was subsequently calculated. Localization of a muscle spindle was achieved by noting the area of muscle from which a high frequency unit discharge could be elicited with discrete mechanical stimulation. By applying a lengthwise, moving deformation with a glass probe, we found a burst in spindle firing with each back and forth stroke along only one small length of muscle, usually less than 2 mm in extent. When similar back and forth strokes were applied on either side of the optimal response area, bursts in firing were obtained only during the stroke away from that area giving the maximal response (Fig. 1A). Upon localizing the point of maximal response to the mechanical stimulus, that area, centered in a block of tissue 5-6 mm long and 1-2 mm in width, was carefully cut from the muscle. Each block was fixed in 4 ~ paraformaldehyde in Dalton's solution at 4°C (1 h) and post-fixed in 2 ~ osmium tetroxide in Dalton's solution at 4°C (1 h). The tissue was then dehydrated and embedded in Araldite. Sequential 1 /~m sections were cut and stained with a 0.5 ~ toluidine blue solution. These sections were Brain Research, 39 (1972) 240-244
SHORT COMMUNICATIONS
241
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Fig. 1. A, Spindle afferent unit responses corresponding to localized sliding deformations along 3 adjacent areas (stipple) of the tenuissimus muscle. Conduction velocity of the unit was 91 m/sec. The scaled, cross-section drawings show the portion of the muscle spindle located in respective thirds of the excised segment (broken line). Dots along tenuissimus show the relative positions along the muscle for 10 separate, localized, unit responses and indicate whether one ( • ) or two (O) spindles were found in the excised tissue. B, Conduction velocities for the 10 localized units, and the number and longitudinal extent of the muscle spindle(s) observed in the corresponding biopsied tissue.
studied with the light m i c r o s c o p e to find the muscle spindle within the block. U l t r a thin sections, stained with u r a n y l acetate a n d l e a d citrate, were studied a n d p h o t o g r a p h e d with a Philips-200 electron m i c r o s c o p e . Ten spindles were localized in 4 tenuissimus muscles (Fig. 1B). Their relative positions a l o n g the length o f the muscle are shown in the c o m p o s i t e d r a w i n g in Fig. 1A. A t least one spindle, b u t never m o r e t h a n two, c o u l d be l o c a t e d in each b i o p s i e d block. I f two spindles were seen in a block, it was possible to p r e s u m e f r o m which one the afferent response h a d been elicited since one was usually centered within the biopsied tissue. Spindles c o u l d be localized regardless o f whether the unit's conduction velocity i n d i c a t e d the fiber t e r m i n a t e d p e r i p h e r a l l y as a p r i m a r y o r s e c o n d a r y ending. Six weeks f o l l o w i n g d e n e r v a t i o n it is quite possible to r e c o r d f r o m afferent units with c o n d u c t i o n velocities in the g r o u p I o r I I range which give typical spindle afferent Brain Research, 39 (1972) 240-244
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Fig. 2. A, Neurogram and electromyogram recorded from the cut S1 dorsal root (lower trace) and medial head of the gastrocnemius (MG) muscle (upper trace) upon stimulating the nerve to the MG, before (1) and after (2, 3) freezing the nerve. Stimulating electrode proximal to freeze in 2 and distal in 3. B, Photomicrograph showing Wallerian degeneration found in tenuissimus nerve 4 days following freeze. C-E, Electron micrographs obtained from the equatorial and juxtaequatorial region of 3 different muscle spindles localized in the tenuissimus muscle of an undenervated animal (C) and one 6 weeks subsequent to denervation (D, E). Sensory ending-like structures (SE) are contiguous to nuclear bag (C, E) or nuclear chain (D) intrafusal muscle fibers, containing centrally located muscle nuclei (N). Basement membrane (arrows) overlies muscle fiber and sensory ending, but is not interposed in the cleft between them.
SHORT COMMUNICATIONS
243
responses to contraction and phasic lengthening of the tenuissimus muscle. Prior to 4 weeks, such responses could not be elicited in our denervated animals. When these functionally reinnervated spindles are examined with the electron microscope, a few sensory terminals of normal configuration are found on nuclear chain intrafusal fibers (Fig. 2D). There are, however, numerous structures suggestive of sensory terminals in appearance and distribution, but with a consistent, distinctly abnormal configuration. These structures are seen exclusively in the equatorial and juxtaequatorial regions of the spindle and almost entirely on nuclear bag fibers (Fig. 2E). As with typical sensory endings, each of these terminal-like structures is partially depressed in the muscle fiber; a basement membrane covers the free surface of the terminal but is not seen in the gap between sensory terminal and intrafusal muscle fiber. In contrast to the ultrastructural configuration of normal sensory endings (Fig. 2C), the abnormal terminal-like structures appear flattened and highly osmophilic. Within these structures, in addition to plentiful, closely packed ribosomes, dense mitochondria and characteristic vesicles, there are occasional myelin figures and aggregates of heavily stained cellular debris. Like the normally innervated spindle, no motor terminals have been found in the equatorial regions of the reinnervated spindle. Study of motor innervation in juxtaequatorial and polar regions of the spindles is not included in this report. Studies with the light microscope have demonstrated that the capsule and intrafusal fibers of muscle spindles in adult mammals are relatively resistant to destruction of their sensory and motor nervesS,is and that the spindle can be reinnervated by regenerating nerve fibers 12,19. The functional integrity of muscle spindles reinnervated in the adult animal has been reported 15. Reinnervated atypical muscle spindles, resuiting after denervation in the newborn rat, give a slowly adapting discharge to maintained stretch of the muscle 14. In the present study, by 6 weeks after denervation, muscle spindles were again partially functional. Only a few sites on intrafusal muscle fibers of these spindles showed the usual morphological pattern of sensory innervation. We can be certain that the nerve supply to the muscle was completely destroyed by the denervation procedure (Fig. 2A, B). It appears, therefore, that the normal sensory terminals found after 6 weeks in the denervated spindles are regenerated sensory endings. The dense, flattened structures may be sensory endings which are returning to normal at the time of fixation. The presence of a few normal endings on the nuclear chain fibers only, and the presence o f the dense endings, predominantly on the nuclear bag fibers, suggests that the reinnervation may occur on the chain fibers first. This interpretation is consistent, so far, with results of additional experiments being conducted in our laboratory on animals studied at other times after denervation.
We wish to thank Scott Morrill (electronics), Robert Barrett and Gail Chambers (illustrations), Geraldean Dunkerley and Martha Monks (manuscript) for assistance in the course of this work. This study was supported by Bureau of Medicine and Surgery Work Unit Brain Research, 39 (1972) 24(I-244
244
SHORT COMMUNICATIONS
MR011.01.01-7014. Dr. De Santis was a Research Associate of the National Research Council. Opinions or conclusions contained in this report are those of the authors and do not necessarily reflect the views or the endorsement of the Navy Department. Naval Aerospace Medical Research Laboratory, Naval Aerospace Medical Institute, Naval Aerospace Medical Center, Pensacola, Fla. 32512 (U.S.A.)
M A R K DE SANTIS* WILLIAM O. WHETSELL, JR. K A R E N FRANCIS
1 ADAL, M. N., The fine structure of the sensory region of cat muscle spindles, J. Ultrastruct. Res., 26 (1969) 332-353. 2 ADAL, M. N., AND BARKER, D., The fine structure of cat fusimotor endings, J. Physiol. (Lond.), 192 (1967) 50P-52P. 3 BESSOU, P., AND LAPORTE, Y., Responses from primary and secondary endings of the same neuromuscular spindle of the tenuissimus muscle of the cat. In D. BARKER (Ed.), Symposium on Muscle Receptors, Hong Kong Univ. Press, Hong Kong, 1962, p. 105. 4 BONSETT, C. A., AND ABREU, B. E., Skeletal muscle studies by means of combined electromyography and needle biopsy. II. The muscle spindle, Neurology (Minneap.), 13 (1963) 327-330. 5 BoYo, I. E., The structure and innervation of the nuclear bag muscle fibre system and the nuclear chain muscle fibre system in mammalian muscle spindles, Phil. Trans. B, 245 (1962) 81-136. 6 BRIDGMAN, C. F., AND ELDRED, E., Hypothesis for a pressure-sensitive mechanism in muscle spindles, Science, 143 (1964) 481 482. 7 CHENG, K., AND BREININ, G. M., A comparison of the fine structure of extraocular and interosseus muscles in the monkey, Invest. Ophthalmol., 5 (1966) 535-549. 8 CORVAJA,N., AND MARINOZZI, V., Close appositions and junctions of plasma membranes of intrafusal fibres in mammalian muscle spindles, Pfliigers Arch. ges. Physiol., 296 (1967) 337-345. 9 CORVAJA,N., MARINOZZI, V., AND POMPEIANO, O., Muscle spindles in the lumbrical muscle of the adult cat. Electron microscopic observations and functional considerations, Arch. ital. Biol., 107 (1969) 365-545. 10 DURING, M. VON, UND ANDRES, K . H . , Zur Feinstruktur der Muskelspindel yon Mammalia, Anat. Anz., 124 (1969) 566 573. l l GRUNER, J. E., La structure fine du fuseau neuromusculaire humain, Rev. neurol., 104 (1961) 490-507. 12 GUTMANN, E., Reinnervation of muscle by sensory nerve fibres, J. Anat. (Lond.), 79 (1945) 1-8. 13 HENNIG, G., Die Nervenendigungen der Rattenmuskelspindel imelektronen- und phasenkontrastmikroskopischen Bild, Z. Zellforsch., 96 (1969) 275-294. 14 HNiK, P., Functional characteristics of free nerve endings and atypical spindles after muscle reinnervation in very young rats, Physiol. bohernoslov., 13 (1964) 216-219. 15 HOMMA, S., Peripheral nerve regeneration and muscle receptor reconstruction, Electroenceph. din. Neurophysiol., 27 (1969) 720. 16 LANDON, D. N., Electron microscopy of muscle spindles. In B. L. ANDREWS (Ed.), Symposium on Control and Innervation of Skeletal Muscle, Livingstone, Edinburgh, 1966, p. 96. 17 MERRILLEES,N. C., The fine structure of muscle spindles in the lumbrical muscles of the rat, J. biophys, biochem. CytoL, 7 (1960) 725-742. 18 TOWER, S. S., Atrophy and degeneration in the muscle spindle, Brain, 55 (1932) 77-90. 19 ZHENEVSKAYA,R. P., AND UMNOVA, M. M., Degeneration and restoration of sensory nerve endings in skeletal muscle, Arkh. Anat. Gistol. Embriol., 49 (1965) 3 11 (in Russian). (Accepted January 12th, 1972)
* Present address: Department of Anatomy, Georgetown University, Washington, D.C. 20007, U.S.A.
Brain Research, 39 (1972) 240-244