SYNAPSE 9~239-243 (1991)
200 kD Neurofilament Protein and Synapse Elimination in the Rat Soleus Muscle R.L.RODEN, S.P. DONAHUE, G.A. SCHWARTZ, J.G. WOOD, AND A.W. ENGLISH Department of Anatomy and Cell Biology and Neuroscience Program, Emory University School of Medicine, Atlanta, Georgia 30322
KEY WORDS
Muscles/innervation, Development, Postnatal
ABSTRACT We studied the distribution and appearance of the phosphorylated form of the 200 kD neurofilament protein in the rat soleus muscle during the period of postnatal synapse elimination. Unlike many muscles, the appearance of singly innervated muscle cells in soleus occurs well after myogenesis has been completed, so that synapses are eliminated from a stable population of muscle cells. Immunoreactivity to the 200 kD neurofilament protein is present in the terminals of neuromuscular synapses of animals at all postnatal ages from 0 to 21 days. Before postnatal day 10, when physiological studies indicate that all soleus muscle cells receive more than one synaptic input, as many as 30% of soleus muscle cells contain phosphorylated 200 kD neurofilament protein immunoreactivity in only one synaptic terminal. At older ages the number of polyneuronally innervated muscle cells observed using immunostaining is similar to that observed physiologically. These findings suggest that not all developing neuromuscular synapses contain phosphorylated 200 kD neurofilament protein, and that those terminals lacking it comprise most of those eliminated early in the postnatal period. We conclude that the presence of phosphorylated 200 kD neurofilament protein might be highly correlated with the survival of motor nerve terminals during postnatal neuromuscular synapse elimination. gastrocnemius (LG) muscle contained immunoreactivity to the phosphorylated form of the 200k NF during At birth all muscle cells receive synaptic inputs from the period of synapse elimination. The LG muscle was more than one motoneuron, but during the next 3 weeks chosen primarily because postnatal synapse eliminaof postnatal life, all but one of these inputs is eliminated tion occurs over an extended period. However, LG un(e.g.,Brown et al., 1976).Although the actual process by dergoes tremendous myogenesis during this period; the which these synaptic terminals are lost is not well number of muscle cells triples in the first 8 days of known, it is generally accepted that the surviving input postnatal life (Donahue et al., 1988b). Thus synapses becomes stabilized and the eliminated inputs do not onto LG muscle fibers could be stabilized either because (Changeux and Danchin, 1976). One feature of neurons they survive the period of synapse elimination or bethat might be related to their mechanical stability is the cause they were used t o innervate newly appearing relative rigidity of their cytoskeletons. Neurofilaments muscle fibers. are one constituent of the axonal cytoskeleton that have In contrast to LG, myogenesis in the soleus (SOL) been implicated in mechanical stabilization (Hoffmanet muscle is complete by the third postnatal day (P3) al., 1987), as neurofilaments are considered more met- (Narusawa et al., 1987),but all SOL muscle cells remain abolically stable than other cytoskeletal elements innervated by more than one motor axon until P9-10 (Mitchison and Kirschner, 1988): Of the three compo- (Brown et al., 1976). Thus synapse elimination in SOL nent proteins that make up neurofilaments, the 200 kD takes place on a stable population of muscle cells. The neurofilament protein (200k NF) has received the most purpose of this study was to examine the expression of attention as a potential stabilizer. Its later appearance the phosphorylated form of the 200k NF in neuromusduring development (e.g., Carden et al., 1987; Donahue cular synapses in SOL during the period of synaptic et al., 1988a) and its localization to the radial cross- stabilization. A preliminary report has been made arms of the neurofilaments (Hirokawa et al., 1984)have (Roden et al., 1988). been used to suggest that the appearance of the 200k NF Received June 15,1990; accepted in revised form April 8,1991. might impart axonal stability (e.g., Carden et al., 1987). R.L. Roden’s present address is Department of Physiology, University of In an attempt to extend this argument to the neuromus- Colorado, Denver, CO. cular synapse, we (Donahue et al., 1988a)demonstrated S.P.Donahue’spresent address is Department of Ophthalmology, University of that nearly all of the synaptic inputs to the lateral Pittsburgh School of Medicine,Pittsburgh, PA 15260. INTRODUCTION
0 1991 WILEY-LISS, INC.
240
R.L. RODEN ET AL.
MATERIALS AND METHODS Neonatal Fischer 344 rats were obtained from our local breeding colony. Adults in the colony were obtained from Charles River Laboratories. The day of birth was designated postnatal day zero (PO). Soleus muscles were studied from rat pups ranging in age from PO to P21. Muscles from at least two animals were examined at each age used. Pups aged
200 kD Neurofilament Protein and Synapse Elimination in the Rat Soleus Muscle R.L.RODEN, S.P. DONAHUE, G.A. SCHWARTZ, J.G. WOOD, AND A.W. ENGLISH Department of Anatomy and Cell Biology and Neuroscience Program, Emory University School of Medicine, Atlanta, Georgia 30322
KEY WORDS
Muscles/innervation, Development, Postnatal
ABSTRACT We studied the distribution and appearance of the phosphorylated form of the 200 kD neurofilament protein in the rat soleus muscle during the period of postnatal synapse elimination. Unlike many muscles, the appearance of singly innervated muscle cells in soleus occurs well after myogenesis has been completed, so that synapses are eliminated from a stable population of muscle cells. Immunoreactivity to the 200 kD neurofilament protein is present in the terminals of neuromuscular synapses of animals at all postnatal ages from 0 to 21 days. Before postnatal day 10, when physiological studies indicate that all soleus muscle cells receive more than one synaptic input, as many as 30% of soleus muscle cells contain phosphorylated 200 kD neurofilament protein immunoreactivity in only one synaptic terminal. At older ages the number of polyneuronally innervated muscle cells observed using immunostaining is similar to that observed physiologically. These findings suggest that not all developing neuromuscular synapses contain phosphorylated 200 kD neurofilament protein, and that those terminals lacking it comprise most of those eliminated early in the postnatal period. We conclude that the presence of phosphorylated 200 kD neurofilament protein might be highly correlated with the survival of motor nerve terminals during postnatal neuromuscular synapse elimination. gastrocnemius (LG) muscle contained immunoreactivity to the phosphorylated form of the 200k NF during At birth all muscle cells receive synaptic inputs from the period of synapse elimination. The LG muscle was more than one motoneuron, but during the next 3 weeks chosen primarily because postnatal synapse eliminaof postnatal life, all but one of these inputs is eliminated tion occurs over an extended period. However, LG un(e.g.,Brown et al., 1976).Although the actual process by dergoes tremendous myogenesis during this period; the which these synaptic terminals are lost is not well number of muscle cells triples in the first 8 days of known, it is generally accepted that the surviving input postnatal life (Donahue et al., 1988b). Thus synapses becomes stabilized and the eliminated inputs do not onto LG muscle fibers could be stabilized either because (Changeux and Danchin, 1976). One feature of neurons they survive the period of synapse elimination or bethat might be related to their mechanical stability is the cause they were used t o innervate newly appearing relative rigidity of their cytoskeletons. Neurofilaments muscle fibers. are one constituent of the axonal cytoskeleton that have In contrast to LG, myogenesis in the soleus (SOL) been implicated in mechanical stabilization (Hoffmanet muscle is complete by the third postnatal day (P3) al., 1987), as neurofilaments are considered more met- (Narusawa et al., 1987),but all SOL muscle cells remain abolically stable than other cytoskeletal elements innervated by more than one motor axon until P9-10 (Mitchison and Kirschner, 1988): Of the three compo- (Brown et al., 1976). Thus synapse elimination in SOL nent proteins that make up neurofilaments, the 200 kD takes place on a stable population of muscle cells. The neurofilament protein (200k NF) has received the most purpose of this study was to examine the expression of attention as a potential stabilizer. Its later appearance the phosphorylated form of the 200k NF in neuromusduring development (e.g., Carden et al., 1987; Donahue cular synapses in SOL during the period of synaptic et al., 1988a) and its localization to the radial cross- stabilization. A preliminary report has been made arms of the neurofilaments (Hirokawa et al., 1984)have (Roden et al., 1988). been used to suggest that the appearance of the 200k NF Received June 15,1990; accepted in revised form April 8,1991. might impart axonal stability (e.g., Carden et al., 1987). R.L. Roden’s present address is Department of Physiology, University of In an attempt to extend this argument to the neuromus- Colorado, Denver, CO. cular synapse, we (Donahue et al., 1988a)demonstrated S.P.Donahue’spresent address is Department of Ophthalmology, University of that nearly all of the synaptic inputs to the lateral Pittsburgh School of Medicine,Pittsburgh, PA 15260. INTRODUCTION
0 1991 WILEY-LISS, INC.
240
R.L. RODEN ET AL.
MATERIALS AND METHODS Neonatal Fischer 344 rats were obtained from our local breeding colony. Adults in the colony were obtained from Charles River Laboratories. The day of birth was designated postnatal day zero (PO). Soleus muscles were studied from rat pups ranging in age from PO to P21. Muscles from at least two animals were examined at each age used. Pups aged
