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Light and electron microscopic identification of a nerve sprout in muscle of normal adult frog
Neuroscienee Letters, 21 (1981) 261-266
261
© Elsevier/North-Holland Scientific Publishers Ltd.
LIGHT AND ELECTRON MICROSCOPIC IDENTIFICATION OF A NERVE SPROUT IN MUSCLE OF NORMAL ADULT FROG
A. W E R N I G , A.P. A N Z I L and A. BIESER
Max-Planck-lnstitut f o r Psychiatrie, Kraepelinstrasse 2, D-8000 Manchen 40 (F. R. G.) (Received October 24th, 1980; Revised version received October 29th, 1980; Accepted October 29th, 1980)
Recent evidence from this laboratory indicates that axonal sprouting (and regression) occurs in neuromuscular junctions of normal adult frogs. In the present investigation, the appearance of a single nerve branch, which from light microscopy was assumed to be a sprout, was studied in ultrathin serial sections. In confirming the light microscopic evidence small synaptic contacts were found, which showed characteristics of new synapse formation. Unexpectedly, the Schwann cell surrounding the axon extended several microns distally from the axon tip. It appears that nerve sprouting (and regression) is a physiological event in adult frog muscles.
Axonal sprouting of the peripheral motoneuron with synapse formation occurs under a variety of experimental and pathological conditions even in adult muscles [3, 4, 6, 7]. Recently, evidence has been obtained suggesting that nerve sprouting (and regression) might occur regularly in normal adult muscles [1, 12], thus raising the question whether or not this is a physiological event. Fig. 1 shows parts of frog neuromuscular junctions from two neighbouring muscle cells after a combined axon and cholinesterase (ChE) staining [12]. In contrast to the usual appearance where ChE is more or less continuously present along nerve terminal branches (upper fibre in Fig. 1), ChE is restricted to small isolated spots along the nerve branches in the lower part of Fig. 1. Such branches have been regarded as axonal sprouts with newly formed synaptic contacts [12, 13]. In order to prove or disprove the validity of the light microscopic evidence, an ultrastructural study was carried out after a similar structure had been identified under the light microscope. After staining for ChE [9], whole cutaneus pectoris muscle of frog (Rana temporaria) was embedded in epoxy [10]. Following polymerization, the block was filed down to a thin (about 1 - 2 m m thick) rectangular plate so that individual synapses could be identified and photographed in a direct light microscope. For this purpose the block was placed between a glass plate and a coverglass after both surfaces were covered with immersion oil. The block was subsequently trimmed to within a few microns from the point of interest and 60-90 nm sections were cut
262
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Fig, 1. Presumed nerve sprouts and mature neuromuscular contacts in cutaneus pectoris muscle of normal frog (Rana temporaria). Combined axon and ChE stains [12]. Axons are out of focus in some places. Bar = 30 ~m. Fig, 2. Light micrograph and camera lucida drawing of part of a synapse after ChE staining [9]. Besides two long ( > 100 tzm) chains of ChE rings (the most distal part of one is shown) this synapse has several additional branches with continuous ChE stain (not shown). Cutaneus pectoris muscle of Rana temporaria (rump to nose length, 6 cm). Bar - 10 t*nl.
perpendicular to the long axis of the muscle cell. Sections were placed on wide meshed grids (5 sections used per grid) or on slot grids (two sections per grid). With the use of meshed grids it was clear that occasionally the structure of interest was obscured, but it was unlikely that this resulted in a loss of more than 220 nm of continuous length of the block. Later in the study only slot grids were used. By checking repeatedly the distance between the edge of the block and the ChE rings (this was done in a specially adapted microscope allowing visualization of the material while the block remained in the microtome holder) and recording the number and sequence of the sections made, the location of the individual sections could be pinpointed to a sufficiently small area (less than 3 ~m in width) of the light microscope picture. Fig. 2. shows a light micrograph and a camera lucida drawing of the distalmost
Fig. 3. Selected electron micrographs ( A - H ) of transverse sections through the muscle fiber visible in Fig. 2 (lines A - H ) . ChE reaction product shows up as individual electron-dense grains (asterisk); in many places the grains have been lost from the sections and corresponding empty spots are visible (asterisk). The quality of the electron micrographs is that which is to be expected after cytochemical reaction for ChE demonstration. The presence of small amounts of ChE reaction products on and around the Schwann cell profiles of the A and B micrographs (see also Fig. 2) signifies enzyme formation on the part of the Schwann cell, the muscle cell or both. It cannot be ruled out, however, that either the enzyme or the reaction product may have diffused away from sites of ChE production during the histochemical procedure. Arrowheads point out axon profiles. Bar - 0.5 gin,
263
. . . . .
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264 part (to the left) of a chain of presumably newly formed synaptic contacts (ChE without axon stain). Serial sections were made at several locations (indicated by the horizontal bars in Fig. 2); the lines marked A - H in Fig. 2 indicate the positions taken across the chosen sample and are depicted in Fig. 3 A - 3 H . Electron micrograph C in Fig. 3, corresponding to line C in Fig. 2, shows an axon profile in contact with the muscle fibre. The presence of vesicles within the axon, the finding of a subsarcolemmal electron density at the corresponding site of the muscle cell, and the occurrence of ChE reaction product in the space between them, signifies the presence of a synaptic contact. Other than in a mature contact, however, the postsynaptic site is poorly developed. A secondary cleft is missing here and a synaptic gutter is barely suggested. The same was true of all sections obtained from this nerve branch. A small membrane indentation, possibly indicating the beginning of a secondary cleft formation, was present in the second contact (middle of the three ChE rings). The lack of mature secondary clefts identifies the synaptic sites as newly formed ones. A few microns proximal from the area detailed above, the axon (arrows) is reduced to a fraction of its previous diameter (electron micrographs D and E and Fig. 4), being totally encircled by its Schwann cell wrapping. There is a sudden increase in axon diameter and a synaptic contact at the ChE ring occurring just a short distance ,~rom it (no electron micrograph shown) and at line F (electron micrograph F). At both places synapse formation has the same distinctive features as visible in C. In G and H, axon and attendant Schwann cell have once more moved away from the muscle fibre, but ChE reaction product is still present and focal subsarcolemmal electron density as well. The Schwann cell profiles shown in micrographs A and B do not ensheath any axon structure (higher magnification in Fig. 4). From these pictures it is clear that a finger-like Schwann cell cytoplasmic process extends several microns beyond the distalmost end of the axon and that the axon studied terminates in a bulbous enlargement establishing a synaptic contact (electron micrograph C of Fig. 3). A similar extension of the Schwann cell well past the nerve terminal tip was seen at an additional nerve fibre forming new synaptic contacts, but was missing in still another nerve sprout studied (both not shown). From the series of electron micrographs it appears that Schwann cell cytoplasmic layers wrap around the nerve sprout while keeping at a distance from the muscle cell. At some points along the nerve fibre, the axon enlarges and gains direct contacts with the underlying muscle cell through small window-like openings of the Schwann cell sheath. It is conceivable that in the course of maturation the axon enlarges the original bulbous expansions and develops additional ones leaving but small Schwann cell cytoplasmic bridges in between them. The Schwann cell fingers regularly present in mature synapses [5, 11] might thus represent remnants of early synaptogenetic events. Similarly, the active zones and the postsynaptic infoldings might develop at the sites of initial synaptic contacts and their positioning in
265
Fig. 4. Electron micrograph on the left x 86,700) and on the right ( x 50,825) are higher magnifications of electron micrograph A and E, respectively, of Fig. 3; notice tubuli in Schwann cell cytoplasm of micrograph on the left and synaptic vesicles in axon of micrograph on the right.
between the Schwann cell fingers in mature synapses [5, 11] could be related to these early events. The Schwann cell projection extending several microns ahead of the axon terminal could provide a guide for the axon's path of growth and/or induce specific changes at some spots on the underlying muscle cell membrane. Alternatively, the axon could have retracted leaving alone its former Schwann cell layers: mature empty gutters from which nerve and Schwann cell have retracted have recently been observed in normal muscles [12, 13]. In accordance with the observations reported here the Schwann cell closely accompanies the axon tip in embryonic synaptogenesis [2] or during reinnervation [10]. In tissue culture, on the contrary, contacts between nerve and muscle cells apparently can form in the absence of a satellite cell [8].
1 Barker, D. and lp, M.C., Sprouting and degeneration of mammalian motor axons in normal and deafferentiated skeletal muscle, Proc. roy. Soc. B, 163 (1966) 538-544. 2 Bennett, M.R., Florin, T. and Woog, R., The formation of synapses in regenerating mammalian striated muscle, J. Physiol. (Lond.), 238 (1974) 79-92. 3 Bowden, R.E.M. and Duchen, L.W., The anatomy and pathology of the neuromuscular junction. In E. Zaimis (Ed.) Neuromuscular Junction, Springer-Verlag, Berlin, 1976, pp. 23-29. 4 Brown, M.C. and lronton, R., Sprouting and regression of neuromuscular synapses in partially denervated mammalian muscles, J. Physiol. (Lond.), 278 (1978) 325-348. 5 Couteaux, M.R. et P~cot-Dechavassine, M., Donn~es ultrastructurales et cytochimiques sur le m~canisme de libbration de l'ac~tylcholine dans la transmission synaptique, Arch. ital. Biol., 34(1973) 231-262.
266
6 Duchen, L.W. and Strich, S..I., Tile effects of botulinmn toxin on the pattern of innervation ol skeletal muscle in the mouse, Quart. J. exp. Physiol., 53 (1968) 84 89. 7 Duchen, L.W. and Tonge, D.A., The effects of tetanus toxin on neuromuscular transmission and on the morphology of motor endplates in slox~ and fast skeletal muscle of lhe mouse, J. Physiol. (Lond.), 228 (1973) 157-172. 8 Frank, E. and Fischbach, G.D., Early events in neuromuscular junction formation in vitro. Induction of acetylcholine receptor clusters in the postsynaplic membrane and morphology of newly formed synapses, J. Cell Biol., 83 (1979) 143-158. 9 Karnovsky, M.J. and Roots, L., A 'direct-coloring' thiocholine method for cholinesterase, .I. Histochem. Cytochem., 12 (1964) 219-221. 10 Letinsky, M.S., Fischbeck, K.H. and McMahan, U.J., Precision of reinnervation of original postsynaptic sites in frog muscle after a nerve crush, J. Neurocytol., 5 (1976) 691-718. 11 Peper, K., Dreyer, F., Sandri, C., Akert, K. and Moor, H., Structure and ultrastructure of the frog motor endplate, Cell Tiss. Res., 149 (1974) 437 455. 12 Wernig, A., Pecot-Dechavassine, M. and StOver, H., Signs of nerve regression and sprouting in the frog neuromuscular synapse. In J. Taxi (Ed.). Ontogenesis and Functional Mechanisms of Peripheral Synapses, Elsevier/North-Holland Biomedical Press, Amsterdam, 1980, pp. 225-238. 13 Wernig, A., Pecot-Dechavassine and StOver, H., Sprouting and regression of the nerve at the frog neuromuscular junction in normal conditions and after prolonged paralysis with curare, .I. Neurocytol., 9(1980) 277 303.
261
© Elsevier/North-Holland Scientific Publishers Ltd.
LIGHT AND ELECTRON MICROSCOPIC IDENTIFICATION OF A NERVE SPROUT IN MUSCLE OF NORMAL ADULT FROG
A. W E R N I G , A.P. A N Z I L and A. BIESER
Max-Planck-lnstitut f o r Psychiatrie, Kraepelinstrasse 2, D-8000 Manchen 40 (F. R. G.) (Received October 24th, 1980; Revised version received October 29th, 1980; Accepted October 29th, 1980)
Recent evidence from this laboratory indicates that axonal sprouting (and regression) occurs in neuromuscular junctions of normal adult frogs. In the present investigation, the appearance of a single nerve branch, which from light microscopy was assumed to be a sprout, was studied in ultrathin serial sections. In confirming the light microscopic evidence small synaptic contacts were found, which showed characteristics of new synapse formation. Unexpectedly, the Schwann cell surrounding the axon extended several microns distally from the axon tip. It appears that nerve sprouting (and regression) is a physiological event in adult frog muscles.
Axonal sprouting of the peripheral motoneuron with synapse formation occurs under a variety of experimental and pathological conditions even in adult muscles [3, 4, 6, 7]. Recently, evidence has been obtained suggesting that nerve sprouting (and regression) might occur regularly in normal adult muscles [1, 12], thus raising the question whether or not this is a physiological event. Fig. 1 shows parts of frog neuromuscular junctions from two neighbouring muscle cells after a combined axon and cholinesterase (ChE) staining [12]. In contrast to the usual appearance where ChE is more or less continuously present along nerve terminal branches (upper fibre in Fig. 1), ChE is restricted to small isolated spots along the nerve branches in the lower part of Fig. 1. Such branches have been regarded as axonal sprouts with newly formed synaptic contacts [12, 13]. In order to prove or disprove the validity of the light microscopic evidence, an ultrastructural study was carried out after a similar structure had been identified under the light microscope. After staining for ChE [9], whole cutaneus pectoris muscle of frog (Rana temporaria) was embedded in epoxy [10]. Following polymerization, the block was filed down to a thin (about 1 - 2 m m thick) rectangular plate so that individual synapses could be identified and photographed in a direct light microscope. For this purpose the block was placed between a glass plate and a coverglass after both surfaces were covered with immersion oil. The block was subsequently trimmed to within a few microns from the point of interest and 60-90 nm sections were cut
262
~
o
°
~. , ~ D ~ . ~ ' ~ ,
°"
oO "
A
!
FGH
BC DE
0
Fig, 1. Presumed nerve sprouts and mature neuromuscular contacts in cutaneus pectoris muscle of normal frog (Rana temporaria). Combined axon and ChE stains [12]. Axons are out of focus in some places. Bar = 30 ~m. Fig, 2. Light micrograph and camera lucida drawing of part of a synapse after ChE staining [9]. Besides two long ( > 100 tzm) chains of ChE rings (the most distal part of one is shown) this synapse has several additional branches with continuous ChE stain (not shown). Cutaneus pectoris muscle of Rana temporaria (rump to nose length, 6 cm). Bar - 10 t*nl.
perpendicular to the long axis of the muscle cell. Sections were placed on wide meshed grids (5 sections used per grid) or on slot grids (two sections per grid). With the use of meshed grids it was clear that occasionally the structure of interest was obscured, but it was unlikely that this resulted in a loss of more than 220 nm of continuous length of the block. Later in the study only slot grids were used. By checking repeatedly the distance between the edge of the block and the ChE rings (this was done in a specially adapted microscope allowing visualization of the material while the block remained in the microtome holder) and recording the number and sequence of the sections made, the location of the individual sections could be pinpointed to a sufficiently small area (less than 3 ~m in width) of the light microscope picture. Fig. 2. shows a light micrograph and a camera lucida drawing of the distalmost
Fig. 3. Selected electron micrographs ( A - H ) of transverse sections through the muscle fiber visible in Fig. 2 (lines A - H ) . ChE reaction product shows up as individual electron-dense grains (asterisk); in many places the grains have been lost from the sections and corresponding empty spots are visible (asterisk). The quality of the electron micrographs is that which is to be expected after cytochemical reaction for ChE demonstration. The presence of small amounts of ChE reaction products on and around the Schwann cell profiles of the A and B micrographs (see also Fig. 2) signifies enzyme formation on the part of the Schwann cell, the muscle cell or both. It cannot be ruled out, however, that either the enzyme or the reaction product may have diffused away from sites of ChE production during the histochemical procedure. Arrowheads point out axon profiles. Bar - 0.5 gin,
263
. . . . .
• ..... '
~YI~ ,
i",~
264 part (to the left) of a chain of presumably newly formed synaptic contacts (ChE without axon stain). Serial sections were made at several locations (indicated by the horizontal bars in Fig. 2); the lines marked A - H in Fig. 2 indicate the positions taken across the chosen sample and are depicted in Fig. 3 A - 3 H . Electron micrograph C in Fig. 3, corresponding to line C in Fig. 2, shows an axon profile in contact with the muscle fibre. The presence of vesicles within the axon, the finding of a subsarcolemmal electron density at the corresponding site of the muscle cell, and the occurrence of ChE reaction product in the space between them, signifies the presence of a synaptic contact. Other than in a mature contact, however, the postsynaptic site is poorly developed. A secondary cleft is missing here and a synaptic gutter is barely suggested. The same was true of all sections obtained from this nerve branch. A small membrane indentation, possibly indicating the beginning of a secondary cleft formation, was present in the second contact (middle of the three ChE rings). The lack of mature secondary clefts identifies the synaptic sites as newly formed ones. A few microns proximal from the area detailed above, the axon (arrows) is reduced to a fraction of its previous diameter (electron micrographs D and E and Fig. 4), being totally encircled by its Schwann cell wrapping. There is a sudden increase in axon diameter and a synaptic contact at the ChE ring occurring just a short distance ,~rom it (no electron micrograph shown) and at line F (electron micrograph F). At both places synapse formation has the same distinctive features as visible in C. In G and H, axon and attendant Schwann cell have once more moved away from the muscle fibre, but ChE reaction product is still present and focal subsarcolemmal electron density as well. The Schwann cell profiles shown in micrographs A and B do not ensheath any axon structure (higher magnification in Fig. 4). From these pictures it is clear that a finger-like Schwann cell cytoplasmic process extends several microns beyond the distalmost end of the axon and that the axon studied terminates in a bulbous enlargement establishing a synaptic contact (electron micrograph C of Fig. 3). A similar extension of the Schwann cell well past the nerve terminal tip was seen at an additional nerve fibre forming new synaptic contacts, but was missing in still another nerve sprout studied (both not shown). From the series of electron micrographs it appears that Schwann cell cytoplasmic layers wrap around the nerve sprout while keeping at a distance from the muscle cell. At some points along the nerve fibre, the axon enlarges and gains direct contacts with the underlying muscle cell through small window-like openings of the Schwann cell sheath. It is conceivable that in the course of maturation the axon enlarges the original bulbous expansions and develops additional ones leaving but small Schwann cell cytoplasmic bridges in between them. The Schwann cell fingers regularly present in mature synapses [5, 11] might thus represent remnants of early synaptogenetic events. Similarly, the active zones and the postsynaptic infoldings might develop at the sites of initial synaptic contacts and their positioning in
265
Fig. 4. Electron micrograph on the left x 86,700) and on the right ( x 50,825) are higher magnifications of electron micrograph A and E, respectively, of Fig. 3; notice tubuli in Schwann cell cytoplasm of micrograph on the left and synaptic vesicles in axon of micrograph on the right.
between the Schwann cell fingers in mature synapses [5, 11] could be related to these early events. The Schwann cell projection extending several microns ahead of the axon terminal could provide a guide for the axon's path of growth and/or induce specific changes at some spots on the underlying muscle cell membrane. Alternatively, the axon could have retracted leaving alone its former Schwann cell layers: mature empty gutters from which nerve and Schwann cell have retracted have recently been observed in normal muscles [12, 13]. In accordance with the observations reported here the Schwann cell closely accompanies the axon tip in embryonic synaptogenesis [2] or during reinnervation [10]. In tissue culture, on the contrary, contacts between nerve and muscle cells apparently can form in the absence of a satellite cell [8].
1 Barker, D. and lp, M.C., Sprouting and degeneration of mammalian motor axons in normal and deafferentiated skeletal muscle, Proc. roy. Soc. B, 163 (1966) 538-544. 2 Bennett, M.R., Florin, T. and Woog, R., The formation of synapses in regenerating mammalian striated muscle, J. Physiol. (Lond.), 238 (1974) 79-92. 3 Bowden, R.E.M. and Duchen, L.W., The anatomy and pathology of the neuromuscular junction. In E. Zaimis (Ed.) Neuromuscular Junction, Springer-Verlag, Berlin, 1976, pp. 23-29. 4 Brown, M.C. and lronton, R., Sprouting and regression of neuromuscular synapses in partially denervated mammalian muscles, J. Physiol. (Lond.), 278 (1978) 325-348. 5 Couteaux, M.R. et P~cot-Dechavassine, M., Donn~es ultrastructurales et cytochimiques sur le m~canisme de libbration de l'ac~tylcholine dans la transmission synaptique, Arch. ital. Biol., 34(1973) 231-262.
266
6 Duchen, L.W. and Strich, S..I., Tile effects of botulinmn toxin on the pattern of innervation ol skeletal muscle in the mouse, Quart. J. exp. Physiol., 53 (1968) 84 89. 7 Duchen, L.W. and Tonge, D.A., The effects of tetanus toxin on neuromuscular transmission and on the morphology of motor endplates in slox~ and fast skeletal muscle of lhe mouse, J. Physiol. (Lond.), 228 (1973) 157-172. 8 Frank, E. and Fischbach, G.D., Early events in neuromuscular junction formation in vitro. Induction of acetylcholine receptor clusters in the postsynaplic membrane and morphology of newly formed synapses, J. Cell Biol., 83 (1979) 143-158. 9 Karnovsky, M.J. and Roots, L., A 'direct-coloring' thiocholine method for cholinesterase, .I. Histochem. Cytochem., 12 (1964) 219-221. 10 Letinsky, M.S., Fischbeck, K.H. and McMahan, U.J., Precision of reinnervation of original postsynaptic sites in frog muscle after a nerve crush, J. Neurocytol., 5 (1976) 691-718. 11 Peper, K., Dreyer, F., Sandri, C., Akert, K. and Moor, H., Structure and ultrastructure of the frog motor endplate, Cell Tiss. Res., 149 (1974) 437 455. 12 Wernig, A., Pecot-Dechavassine, M. and StOver, H., Signs of nerve regression and sprouting in the frog neuromuscular synapse. In J. Taxi (Ed.). Ontogenesis and Functional Mechanisms of Peripheral Synapses, Elsevier/North-Holland Biomedical Press, Amsterdam, 1980, pp. 225-238. 13 Wernig, A., Pecot-Dechavassine and StOver, H., Sprouting and regression of the nerve at the frog neuromuscular junction in normal conditions and after prolonged paralysis with curare, .I. Neurocytol., 9(1980) 277 303.