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LIM kinase 1 (LIMK1) is a cytoplasmic protein kinase that is highly expressed in neurons. In transfected cells, LIMK1 binds to the cytoplasmic tail of neuregulins and regulates the breakdown of actin filaments. To identify potential functions of LIMK1 in vivo, we have determined the subcellular distribution of LIMK1 protein within neurons of the rat by using immunomicroscopy. At neuromuscular synapses in the adult hindlimb, LIMK1 was concentrated in the presynaptic terminal. However, little LIMK1 immunoreactivity was detected at neuromuscular synapses before the 2nd week after birth, and most motoneuron terminals were not strongly LIMK1 immunoreactive until the 3rd week after birth. Thus, LIMK1 accumulation at neuromuscular synapses coincided with their maturation. In contrast, SV2, like many other presynaptic terminal proteins, can be readily detected at neuromuscular synapses in the embryo. Similar to its late accumulation at developing synapses, LIMK1 accumulation at regenerating neuromuscular synapses occurred long after these synapses first formed. In the adult ventral spinal cord, LIMK1 was concentrated in a subset of presynaptic terminals. LIMK1 gradually accumulated at spinal cord synapses postnatally, reaching adult levels only after P14. This study is the first to implicate LIMK1 in the function of presynaptic terminals. The concentration of LIMK1 in adult, but not nascent, presynaptic terminals suggests a role for this kinase in regulating the structural or functional characteristics of mature synapses.

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To determine if ciliary neurotrophic factor (CNTF) is involved in the response to spinal cord injury, we studied changes in the expression of CNTF and that of its receptor, CNTF-receptor alpha (CNTFR alpha), in the rat spinal cord after a unilateral spinal cord hemisection. Using in situ hybridization, we found that CNTFR alpha mRNA levels in spinal cord motoneurons increased dramatically by 1 day after hemisecting the spinal cord at T2. This increase in expression was present only in motoneurons caudal, but not rostral, to the lesion. In addition, we detected increased levels of CNTF mRNA in the spinal cord white matter, also by 1 day following injury. Unlike CNTFR alpha, however, the increase in CNTF mRNA was evident both rostral and caudal to the lesion. Levels of both CNTF and CNTFR alpha mRNA declined between 1 and 5 days, and by 10 days they were not significantly different from normal animals. These findings suggest that CNTF may play a local role in the response to spinal cord injury.

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Ciliary neurotrophic factor (CNTF) has been shown to promote the survival of motoneurons, but its effects on axonal outgrowth have not been examined in detail. Since nerve growth factor (NGF) promotes the outgrowth of neurites within the same populations of neurons that depend on NGF for survival, we investigated whether CNTF would stimulate neurite outgrowth from motoneurons in addition to enhancing their survival. We found that CNTF is a powerful promoter of neurite outgrowth from cultured chick embryo ventral spinal cord neurons. An effect of CNTF on neurite outgrowth was detectable within 7 hours, and at a concentration of 10 ng/ml, CNTF enhanced neurite length by about 3- to 4-fold within 48 hours. The neurite growth-promoting effect of CNTF does not appear to be a consequence of its survival-promoting effect. To determine whether the effect of CNTF on spinal cord neurons was specific for motoneurons, we analyzed cell survival and neurite outgrowth for motoneurons labeled with diI, as well as for neurons taken from the dorsal half of the spinal cord, which lacks motoneurons. We found that the effect of CNTF was about the same for motoneurons as it was for neurons from the dorsal spinal cord. The responsiveness of a variety of spinal cord neurons to CNTF may broaden the appeal of CNTF as a candidate for the treatment of spinal cord injury or disease.

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The restoration of a normal pattern of neural connectivity following nerve injury depends upon the selective reinnervation of appropriate postsynaptic targets. Previous studies suggest that, in the neuromuscular system, recognition between regenerating motoneurons and target muscles depends upon the positions of origin of the motoneurons and muscles. In axolotls, portions of the motor pools of adjacent muscles overlap. We found that, following removal of a pair of adjacent hindlimb muscles, anterior and posterior iliotibialis, many regenerating iliotibialis motor axons invaded foreign muscles. A more anterior foreign muscle, puboischiofemoralis internus, received greater innervation from anterior iliotibialis motoneurons, whereas a more posterior muscle, iliofibularis, received greater innervation from posterior iliotibialis motoneurons. Furthermore, anterior iliotibialis motoneurons that reinnervated puboischiofemoralis internus occupied the rostral portion of anterior iliotibialis motor pool, which overlaps that of puboischiofemoralis internus. Anterior iliotibialis motoneurons that reinnervated iliofibularis occupied the caudal portion of the anterior iliotibialis motor pool, which overlaps that of iliofibularis. When both anterior and posterior iliotibialis were damaged so that their myofibers were permanently destroyed, the rostrocaudal origins of the motoneurons that reinnervated them were virtually the same, suggesting that the motoneurons had difficulty distinguishing between the myofiberless iliotibialis muscles. However, some iliotibialis motoneurons invaded puboischiofemoralis internus instead of their myofiberless targets. Puboischiofemoralis internus received more innervation from the anterior iliotibialis motoneurons than the positionally less appropriate posterior iliotibialis motoneurons. These data are consistent with the hypothesis that selective reinnervation of muscle depends upon a system of recognition cues based on position.

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We analyzed the fiber-type composition of the soleus muscle in rats and mice to determine whether the adult proportion of fiber types is fixed soon after birth or whether it changes during postnatal maturation. We examined muscles from animals varying in age from 1 week to 1 year using monoclonal antibodies that distinguish between fast and slow isoforms of myosin heavy chains. In cross sections of unfixed muscle containing profiles of all myofibers in the muscle, we counted the fibers that stained with antibodies to fast myosin, and in adjacent sections, those that stained positive with an antibody to slow myosin. We also counted the total number of fibers in each section. Rat soleus contained about 2500 myofibers, and mouse about 1000 at all ages studied, suggesting that myogenesis ceases in soleus by 1 week after birth or sooner. In mouse soleus, the relative proportions of fibers staining positive with fast and slow myosin antibodies were similar at all ages studied, about 60%-70% being fast and 30%-40% slow. In rat soleus, however, the proportions of fast antibody-positive and slow antibody-positive fibers changed dramatically during postnatal maturation. At 1 week after birth, about 50% of rat soleus fibers stained with fast myosin antibodies, whereas between 1 and 2 months this value fell to about 10%. In mouse, about 10% of fibers at 1 week, but none at 1 year, reacted with both fast and slow antibodies, whereas in rat, fewer than 3% bound both antibodies to a significant degree at 1 week. It is puzzling why, in rat soleus, the majority of apparently fast fibers present at 1 week is converted to a slow phenotype, whereas in mouse soleus the predominant change appears to be the suppression of fast myosin expression in a subset of fibers that expresses both myosin types at 1 week. It is possible that this may be related to differences in size and the amount of body growth between these two species.

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The structure of individually identified neuromuscular junctions (NMJs) in mouse lateral gastrocnemius (LG) muscles was studied on 2 or more occasions over 3-6 months. Presynaptic motor nerve terminals and their underlying acetylcholine receptors were stained in living animals with the fluorescent dye 4-(4-diethylaminostyryl)-N-methylpyridinium iodide) and tetramethylrhodamine isothiocyanate-conjugated alpha-bungarotoxin (R alpha BTX), respectively, and visualized by video-enhanced fluorescence microscopy. The overall shape of most NMJs changed very little over this time except for enlargement of some junctions attributable to growth of the animals. A few junctions did, however, change appreciably: over 3-6 months about 15% underwent modifications such as additions to, or losses from, their original configuration. The frequency and extent of changes in LG NMJs were substantially less than in a similar study of NMJs from mouse soleus (Wigston, 1989). These findings, together with those from other laboratories, indicate a correlation between the extent of NMJ remodeling and the fiber-type composition of a muscle: NMJs in muscles consisting of predominantly fast-twitch myofibers appear to undergo less remodeling than NMJs in muscles containing a substantial fraction of slow-twitch fibers. Since fast- and slow-twitch muscles and their motoneurons exhibit strikingly different patterns of electrical activity, these findings suggest that structural remodeling at mammalian NMJs may depend on the amount of impulse activity experienced by motoneurons, their target muscle, or individual synaptic terminals.

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Motoneurons seem to require contact with their target muscle even after embryogenesis is complete, but the consequences of target-deprivation during postnatal development are poorly understood. To examine the fate of motoneurons separated from their targets postnatally, we labeled the motoneurons that innervate the biceps brachii muscle with the retrograde tracer Fluorogold and then separated them from their muscle by amputating the forelimb. Fluorogold was subsequently found within motoneurons, as well as within much smaller cells that were identified as microglia. The number of labeled microglial cells steadily increased with time following limb amputation, while the number of labeled motoneurons declined. The magnitude of this response depended on the age of the animal: the younger the animal at the time of the amputation, the greater the number of labeled microglia and the more extensive the neuronal loss. To ensure that the response to amputation was caused by target deprivation, rather than by the injury itself, the nerve to the biceps muscle was cut or crushed. In this way, axons were transected but target access was only temporarily denied. After the nerve was cut, motoneurons began to reinnervate the muscle within 3 weeks but, just as after amputation, the spinal cord subsequently contained labeled microglia and a reduced number of motoneurons. In contrast, after nerve crush, reinnervation began within 4 d and there was no evidence of motoneuron death. Our results demonstrate that target-deprivation causes motoneurons to be lost in an age- and time-dependent manner, and indicate a critical period after axotomy during which motoneurons must reinnervate their target in order to survive. Further, we provide evidence that microglial cells may phagocytose dying motoneurons. The approach we used would provide a convenient assay for testing candidate motoneuron growth factors in animals where in vivo studies of the embryo are difficult.

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The stability of neuromuscular junctions (NMJs) was studied in soleus muscles of adult mice by labeling acetylcholine receptors in vivo with rhodamine alpha-bungarotoxin. Identified NMJs were examined in living animals by low-light-level fluorescence microscopy on 2 or 3 occasions separated by up to 6 months. Many NMJs appeared identical each time they were viewed except for overall enlargement probably related to growth of the animal. Forty-four percent of NMJs, however, changed their shape over 6 months; these changes consisted mostly of small deletions or additions to part of the initial configuration. NMJs in adult soleus appeared to be less malleable than suggested by earlier studies but more plastic than NMJs in another muscle, the mouse sternomastoid, in which virtually no remodeling was observed using similar methods to the present study (Lichtman et al., 1987a). Thus, the degree of remodeling at NMJs may vary among different muscles, perhaps depending on their pattern of use.

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The selective reinnervation of muscles suggests that muscles have intrinsic recognition cues that promote selective synaptogenesis. For example, the anterior and posterior heads of the axolotl iliotibialis (ILT) muscle are preferentially reinnervated by their original motoneurons even after surgically exchanging them. The nature and location of cues that promote such selectivity are unknown, although previous work suggests that the muscle fibers themselves might harbor the relevant molecules. To address this question, we removed anterior and posterior ILT muscles, destroyed their myofibers by surgically damaging them and treating them with bupivacaine, and reimplanted them in either a normal or a reversed anterior/posterior orientation. After the regenerated myofibers became innervated, we stimulated different spinal nerves and recorded the synaptic potentials evoked in muscle fibers. Our results showed that if the muscles were removed, damaged, and reimplanted in their original positions, the segmental origin of inputs to the regenerated myofibers was similar to that seen in normal muscles and in muscles reimplanted with their myofibers intact. However, muscles that were removed and damaged but regenerated in new positions were innervated differently from normal muscles and from muscles whose myofibers survived transplantation. Thus, the site at which a muscle regenerates has an influence on the source of the muscle's reinnervation. Nevertheless, the innervation of muscles that regenerated after transplantation to a foreign site was not strictly appropriate for the new position, but was biased towards the muscle's original innervation pattern. Therefore, some, but not all, of the cues that reflect the original identity of the transplanted muscles survive the replacement of its myofibers.

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The motoneurons innervating 3 hindlimb extensor muscles, anterior and posterior iliotibialis and iliofibularis, were studied separately by retrograde labeling with HRP. The motor pools for these 3 muscles overlapped to such an extent that individual motoneurons between ventral roots 16 and 17 could not be assigned unambiguously to one pool or another. Thus, conventional retrograde labeling could not identify particular axolotl motoneurons. Instead, a double retrograde-labeling technique was employed to mark the motoneurons innervating a particular muscle, the left posterior iliotibialis. Either diamidino yellow (DY) or HRP satisfactorily labeled axolotl motoneurons for at least 3 months in vivo. After labeling, both anterior and posterior iliotibialis muscles were removed from the injected limb and replaced with their counterparts from the opposite limb, in reversed anterior-posterior orientation. Several weeks later, a second marker (DY or HRP) injected into the posterior iliotibialis muscle in its new, more anterior, position labeled the neurons that reinnervated this muscle; the number of neurons labeled with both first and second tracers gave an indication of the selectivity of reinnervation. Using this approach, we have found that the majority of neurons reinnervating a particular muscle are members of that muscle's original motor pool.

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The segmental pattern of motor innervation of hindlimb muscles in the axolotl was studied before and after reinnervation. To ascertain the specificity of reinnervation, the four spinal nerves innervating the hindlimb were severed and allowed to regenerate. The segmental origin of axons reinnervating particular muscles was then determined by intracellular recording from muscle fibers. Muscles were reinnervated in a specific manner: From the outset, the axons reinnervating each muscle originated largely from segmentally appropriate spinal nerves in the proper proportions, suggesting that a reliable mechanism of selective synapse regeneration exists even in mature axolotls. To examine the selectivity of reinnervation, individual muscles were transplanted to novel positions within the limb and the specificity of their reinnervation determined. Even after being moved to new positions, muscles were reinnervated for the most part by axons of appropriate segmental origin. Therefore, cues must exist on or within limb muscles that regenerating motor axons recognize and use to discriminate between different muscles during synapse formation. These results suggest that one of the mechanisms that promote the reestablishment of correct connections during reinnervation of axolotl limbs may be the selective formation of synapses with appropriate target cells.

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Eight sensory structures (campaniform sensilla), appearing identical in the light and scanning electron microscopes, are found in specific locations on the wings of Drosophila. Their axons enter one of 2 central tracts, a medial one or a lateral one. The topographic arrangement of the sensilla on the wing is not reflected in this central projection pattern. There is, however, a strict correlation between the time when a sensillum develops and the path its axon follows: The 4 sensilla whose axons form the medial projection are born and differentiate early during the development of the wing, while the other 4 sensilla, all of which project laterally, arise during a second wave of differentiation. This time-related projection pattern remains stable in the face of a variety of genetically induced alterations in the precise number and location of sensilla.

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We transplanted external intercostal muscles from one of several thoracic (T) levels to the neck of adult rats. The cervical sympathetic trunk, which innervates the superior cervical ganglion, was cut, and its proximal end was apposed to the muscle. Preganglionic axons in the trunk reinnervated muscle fibers in the transplants. We determined the segmental origin of synaptic inputs to transplanted muscles by recording intracellularly from muscle fibers while stimulating individual ventral roots which supply axons to the trunk. In one series of experiments, T2 or T8 muscles were transplanted from the thorax to the neck of the same rat. While T2 and T8 muscles were reinnervated to a similar extent, they differed in the segmental origin of the innervation they received: T2 muscles received more inputs from rostral segments (T1 and T2) than did T8 muscles, and T8 muscles received more inputs from caudal segments (T4 to T6) than did T2 muscles. This difference between reinnervation of T2 and T8 muscles was detected both 2 to 4 weeks and 10 to 14 weeks after surgery. In a separate series, using rats of an inbred strain, T3, T4, or T5 muscles were transplanted from one rat to a separate host. Again, the average segmental origin of inputs to transplants from different levels differed systematically: it was most rostral to T3 muscles, intermediate to T4 muscles, and most caudal to T5 muscles. Finally, T3 and T5 muscles were soaked in a myotoxin, Marcaine, before reimplantation. This treatment kills muscle fibers but not myoblastic satellite cells; therefore, muscle fibers were replaced by regeneration. Marcaine-treated T3 and T5 muscles were successfully reinnervated but did not differ significantly in the segmental origin of their inputs. Our results show that adult mammalian muscles can be selectively reinnervated, and they raise the possibility that the selectivity is based on some positional quality that matches axons and muscles from corresponding segments. However, while differences among muscles survive denervation and transplantation, their expression or accessibility may change during regeneration.

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The ciliary ganglion has been examined in young adult (1.5-2 months) and aged rats (24-28 months). In young adult rats the ganglion contains about 200 neurones, each of which is innervated by 1-4 preganglionic axons (mean, 2.2). In these animals about 2-3 X 10(3) synaptic boutons were observed per square millimetre in thin sections of the ganglion; synapses were located mainly on finger-like extensions of cell bodies and on dendrites. The size of the ganglion cell population, the number of inputs per ganglion cell, the density of synaptic boutons and the amplitude of post-synaptic potentials recorded intracellularly did not differ significantly between the young and aged animals. It thus appears that the neuronal population of the ciliary ganglion and the cholinergic synapses received by ganglion cells are remarkably stable well into old age.

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The size and arrangement of the set of neurones innervated by individual preganglionic axons (the neural unit) has been investigated in the superior cervical ganglion of the guinea-pig. 1. Based on the ratio of preganglionic neurones to ganglion cells, and the average number of axons contacting each ganglion cell, we estimated that individual preganglionic axons innervate on the order of 50-200 superior cervical ganglion cells. 2. Of 562 pairs of ganglion cells examined with intracellular recording, forty-seven (8.4%) were innervated by one or more common axons. 3. Pairs of ganglion cells innervated by the same axon were not necessarily near each other. Although nearby cells were more likely to share innervation than neurones far apart, cells sharing innervation were often found several hundred micrometers apart, and were occasionally separated by the largest dimension of the ganglion (about 1-2 mm). 4. The incidence of cell pairs that shared innervation from more than one axon was greater than expected from the frequency of pairs sharing at least one axon. 5. Extracellular recordings from small fascicles of the cervical sympathetic trunk showed that preganglionic axons from different segmental levels intermingle extensively en route to the superior cervical ganglion. 6. Taken together, these findings support the view that sets of ganglion cells are innervated in common not because of any special topographic relationship within the ganglion, but because they share one or more properties that make them especially attractive to particular preganglionic axons.

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1. Individual neurones in the guinea-pig superior cervical ganglion were studied to determine whether they are innervated by preganglionic axons with similar conduction velocities. 2. Latencies of synaptic responses recorded intracellularly in ganglion cells after stimulation of individual ventral roots varied from 28 to 430 msec. Most of this variability arose from differences in preganglionic conduction velocity. 3. The twelve different axons that on average innervate each ganglion cell tended to have broadly similar conduction velocities; a neurone receiving a rapidly conducting input was usually contacted by other rapidly conducting axons, and vice versa. 4. Preferential innervation of individual neurones by axons with similar conduction velocities was evident even when only axons arising from the same spinal segment were compared. Thus preferential innervation by axons of similar conduction velocity cannot be simply a manifestation of segmental preferences. 5. These results suggest that the mature pattern of innervation in mammalian sympathetic ganglia reflects the functional as well as the positional qualities of the synaptic partners.

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1. The supracoracoideus (s.c.) muscle of the axolotl shoulder is innervated by two nerves, the anterior and posterior s.c. nerves. The posterior nerve was induced to make synapses outside its normal territory in the muscle by removing a segment of the anterior nerve. Intracellular recording indicated that the efficacy of transmission from posterior nerve terminals outside their normal territory increased over several weeks prior to the return of the anterior nerve. 2. The anterior nerve reinnervated its muscle by 40-50 days after the operation, and quickly made synapses throughout the muscle. The posterior nerve territory subsequently returned to its original size and location over 3-6 months. 3. Transplantation of either of two completely foreign nerves into s.c. muscles with enlarged posterior nerve territories resulted in a similar return of the posterior nerve territory to its normal size when anterior nerve regeneration was prevented. 4. These results suggest that the advantage which newly regenerated native nerves have over sprouted foreign nerves is not the quality of 'nativeness' but rather the smaller number of synapses they support. In this view, sprouted nerves compete less effectively because they initially support more synapses per neurone than regenerating nerves.

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The posterior supracoracoideus nerve of the axolotl, Ambystoma mexicanum, was induced to make synapses outside its normal muscle territory. Muscle fibres with inputs from both native and foreign nerves were studied during the period of suppression of foreign transmission and in only 8% of fibres were foreign and native terminals found within 120 micrometer of each other. A combined cholinesterase/silver staining technique revealed non-innervated endplates in foreign-innervated fibres just prior to the return of the native nerve. These results suggest a mechanism enabling suppression of foreign synapses at some distance from native synapses probably beginning with the reinnervation of empty sites by the native nerve.

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