Quantitative comparison of low- and high-output neuromuscular synapses from a motoneuron of the lobster (Homarus americanus)

Summary Representative examples of lowand high-output neuromuscular synapses between motoneuron and distal accessory flexor muscle of the lobster were selected on the basis of their mean quantal content, and subsequently analysed by serial section electron microscopy. The high-output terminal has twice as many synapses as the low-output terminal. However, since the mean surface area of synapses is significantly smaller in the high-output terminal than in the low-output one, the total synaptic surface area between the two types of terminals is similar. Also, though the high-output terminal possesses a greater number of presynaptic dense bodies than its low-output counterpart, the mean number per synapse is similar for the two terminals. The terminals, however, differ significantly in the size of their dense bodies. Thus both the mean and total surface area of these bodies is greater in the high-output terminal than in the low-output one. Moreover, the mean ratio of dense body area to synaptic area is significantly greater for the high-output terminal than for its low-output counterpart. This difference in dense body area parallels the difference in quantal content of synaptic transmission between the lowand high-output terminals and supports the hypothesis that presynaptic densities represent the ultrastructural correlates of transmitter mobilization and/or release.Supported by grants from the National Research Council and Muscular Dystrophy Association of Canada to C.K. Govind. D.E. Meiss is a post-doctoral fellow of the Muscular Dystrophy Association of Canada. We thank Eva Yap-Chung for her expert and unfailing technical assistance     

Synaptic plasticity at the crayfish opener neuromuscular preparation

The crayfish opener neuromuscular preparation exhibits most of the plasticities yet described for any synapse, including facilitation, long-term potentiation, presynaptic inhibition, and modulation. Since the presynaptic terminals and postsynaptic muscle fibers can both be intracellularly penetrated, one can now more easily examine the cellular/molecular bases for these plasticities. Data from such studies suggest that facilitation may be influenced by something other than residual free calcium and that presynaptic inhibition is produced by a conductance increase to chloride in the terminals of the excitor axon. Several drugs (ethanol, pentobarbital) have significant effects on these synaptic plasticities over concentration ranges which produce obvious behavioral effects in crayfish and mammals. Hence, this preparation should be a useful model system to determine cellular/molecular bases for various synaptic plasticities and the effects of drugs on these plasticities.     

Fine structure of comparable synapses in a mature and larval lobster muscle

Neuromuscular synapses from the single excitor axon to the proximal accessory flexor muscle (PAFM) was studied by serial section electron microscopy in a 1st stage larval (< 0.1 g) and a large adult (6.8 kg) lobster. The adult innervation of a lateral and a medial fiber, physiologically identified as low- and high-output respectively, was similar in the number and mean size of synapses but had significantly larger pre-synaptic dense bars for the high-output synapses. This correlation between quantal transmitter output and pre-synaptic dense bars and the appearance of exocytotic profiles along the dense bars strongly implicates the bars as active sites of transmitter release. Moreover the mature innervation is differentiated on the basis that the percentage of dense bar area to synaptic area is 9% for the low-output type compared to 22% for its high-output counterpart. In the larval PAFM the excitatory axon has not proliferated many branches and the innervation is therefore localized to groups of fibers in the lateral, medial and central regions of the muscle rather than to individual fibers. The lateral and medial sites of innervation representing putative low- and high-output types respectively (because of their location) do not differ in the size and number of pre-synaptic dense bars thereby suggesting a similarity in quantal synaptic transmission. However the percentage of dense bar area to synaptic area is 40% for the lateral site compared to 67% for the medial site. Since this is a trend mimicking the mature innervation it shows an early stage in the differentiation of low-and high-output synapses. Furthermore the main axon provides half of the total innervation in the larval PAFM but none in the adult thereby demonstrating a restructuring of multiterminal innervation.     

Seasonal differences in the physiology and morphology of crayfish motor terminals

The physiology and morphology of identified crayfish motor terminals were compared at different seasons. We examined initial excitatory postsynaptic potential (EPSP) amplitudes, synaptic fatigue, and the frequency of synaptic varicosities along the motor terminals of an identified phasic motoneuron in animals collected over a period of 5 years. The physiology and morphology of identified crayfish motor terminals are different for animals collected in summer and winter. In winter animals, phasic axon motor terminals in the claw closer muscle produce large EPSPs initially, but show dramatic synaptic fatigue during repetitive stimulation. In summer animals, these terminals produce smaller initial EPSPs, but are more fatigue resistant. Due to their greater fatigue resistance, synaptic terminals have a greater over-all capacity for transmitter release in summer animals than do those of winter animals. Morphologically, terminals in summer animals have more synaptic varicosities, this result supports earlier studies that have shown that fatigue-resistant motor terminals have more synaptic varicosities. Experiments in which the electrical activity of the motoneuron was experimentally altered suggest that these differences in motor terminals may be due to seasonal differences in activity.     

Regenerated synaptic terminals on a crayfish slow muscle identify with transplanted phasic or tonic axons

Phasic or tonic nerves transplanted onto a denervated slow superficial flexor muscle in adult crayfish regenerated synaptic connections that displayed large or small excitatory postsynaptic potentials (EPSPs), respectively, suggesting that the neuron specifies the type of synapse that forms (Krause et al., J Neurophysiol 80:994-997, 1998). To test the hypothesis that such neuronal specification would extend to the synaptic structure as well, we examined the regenerated synaptic terminals with thin serial section electron microscopy. There are distinct differences in structure between regenerated phasic and tonic innervation. The phasic nerve provides more profuse innervation because innervation sites occurred more frequently and contained larger numbers of synaptic terminals than the tonic nerve. Preterminal axons of the phasic nerve also had many more sprouts than those of the tonic nerve. Phasic terminals were thinner and had a lower mitochondrial volume than their tonic counterparts. Phasic synapses were half the size of tonic ones, although their active zone-dense bars were similar in length. The density of active zones was higher in the phasic compared with the tonic innervation, based on estimates of the number of dense bars per synapse, per synaptic area, and per nerve terminal volume. Because these differences mirror those seen between phasic and tonic axons in crayfish muscle in situ, we conclude that the structure of the regenerated synaptic terminals identify with their transplanted axons rather than with their target muscle. Therefore, during neuromuscular regeneration in adult crayfish, the motoneuron appears to specify the identity of synaptic connections.     

Differentiation of Nerve Terminals in the Crayfish Opener Muscle and Its Functional Significance

Junctional potentials (jp's) recorded from superficial distal fibers of the crayfish opener muscle are up to 50 times larger than jp' in superficial central fibers when the single motor axon that innervates the muscle is stimulated at a frequency of 1/sec or less. At 80/sec, in contrast, central jp's are up to four times larger than those observed in distal fibers. The tension produced by single muscle fibers of either type is directly proportional to the integral of the time-voltage curve minus an excitation-contraction coupling threshold of 3 mv. Distal fibers therefore produce almost all the total muscle tension at low frequencies of stimulation and central fibers add an increasingly greater contribution as their nerve endings begin to facilitate in response to increased rate of motor discharge. Differentiation of muscle membrane characteristics (input resistance, space constant, time constant) cannot account for these differences in facilitation ratios. The mechanism of neuronal differentiation is not based upon the size or effectiveness of transmitter quanta, since equal sized jp's have equal variances;: mjp sizes and variances are also equal. No differences were found between fiber types in rates of transmitter mobilization, density of innervation, or the relationship between transmitter release and terminal depolarization. Single terminals on distal fibers were found to release transmitter with a greater probability than central terminals. More effective invasion of distal terminals by the nerve impulse at low frequencies can account for the difference.     

Neuronal experience modifies synaptic long-term facilitation

In a crayfish phasic neuromuscular junction, we have demonstrated low-frequency depression (LFD), high-frequency depression (HFD), and long-term facilitation (LTF) in response to different regimens of stimulation. Chronic stimulation of the phasic axon supplying the closer muscle of the claw in Procambarus clarkii resulted in diminished expression of HFD and LTF. Conversely, when impulse production in the phasic motoneuron was reduced by claw immobilization, both HFD and LTF were enhanced. LFD was insensitive to these manipulations. These results provide further evidence for long-term adaptation of the phasic neuromuscular junction to ongoing levels of impulse activity and illustrate the importance of a neuron's past history for synaptic plasticity. The ability of the neuron to adjust its short-term plasticity in response to altered experience constitutes an adaptive response that could be of general significance.     

Synaptic diversity and differentiation: Crustacean neuromuscular junctions

Crustacean motor neurons exhibit a wide range of synaptic responses. Tonically active neurons generally produce small excitatory postsynaptic potentials (EPSPs) at low impulse frequencies, and are able to release much more transmitter as the impulse frequency increases. Phasic neurons typically generate large EPSPs in their target cells, but have less capability for frequency facilitation, and undergo synaptic depression during maintained activity. These differences depend in part upon the neuron's ongoing levels of activity; phasic neurons acquire physiological and morphological features of tonic neurons when their activity level is altered. Molecules responsible for adaptation to activity can be sought in single identified phasic neurons with current techniques. The fact that both phasic and tonic neurons innervate the same target muscle fibers is evidence for presynaptic determination of synaptic properties, but there is also evidence for postsynaptic determination of specific properties of different endings of a single neuron. The occurrence of high- and low-output endings of the same tonic motor neurons on different muscle fibers suggests a target-specific influence on synaptic properties. Structural variation of synapses on individual terminal varicosities leads to the hypothesis that individual synapses have different probabilities for release of transmitter. We hypothesize that structurally complex synapses have a higher probability for release than the less complex synapses. This provides an explanation for the larger quantal contents of high-output terminals (where the proportion of complex synapses is higher), and also a mechanism for progressive recruitment of synapses during frequency facilitation.     

Structural-functional differences of a crab motoneuron to four stomach muscles

A motor unit in the stomach of the blue crab, Callinectes sapidus, consists of four separate muscles involved in different aspects of the trituration and filtering of food. Motor nerve terminals to two of the muscles (CPV7a and GM5) release small amounts of transmitter (low-output) while those to the other two muscles (CV2 and CV3) release between three and five-fold greater amounts (high-output). Structural features underlying the disparity in synaptic strength were analysed with thin serial-section electron microscopy. Nerve terminals were similar in their volume percent of mitochondria, clear vesicles and dense core vesicles among the four muscles. This was also the case for the number and size of synaptic contacts. However, presynaptic dense bars representing active zones were longer and occurred more frequently at high-output synapses than at low-output ones. High-output synapses were also characterized by the close spacing of adjacent dense bars. The longer and more closely spaced dense bars at high-output synapses would be factors in the generation of larger synaptic potentials in these terminals compared to their low-output counterparts. Other factors, however, need to be considered to fully account for the physiological differences in synaptic strength among the four muscles.     

Long-term facilitation and long-term adaptation at synapses of a crayfish phasic motoneuron

Stimulation of the phasic (fast) motor axon of the isolated crayfish claw preparation at relatively low frequency (0.1 Hz) leads to depression of the excitatory junction potential (EJP) recorded from single muscle fibers. When the same stimulation is delivered following depression of the EJP at a higher frequency (5 Hz), a potentiated EJP appears, which is more resistant to low frequency depression. The potentiation appears to be analogous to "long-term facilitation" observed after stimulation of a tonic motor axon in crayfish and crabs. Long-term facilitation can be detected in preparations made from claws of animals in which the phasic motoneuron was stimulated at 5 Hz for 2 h in situ. This effect lasts for at least one day after one conditioning trial. Long-term facilitation is observed after stimulation of decentralized axons in situ, indicating that the change is attributable to local changes in terminal regions of the axon, and does not require the cell body. When electrodes are implanted in situ and the phasic motoneuron stimulated at 5 Hz for 2 h each day, synaptic depression becomes less pronounced and initial EJP amplitude becomes smaller over a period of several days. The latter changes, which adapt the neuron to a more tonic activity pattern, usually require several days for completion. Adaptation of fatigability occurs more rapidly than adaptation of initial EJP amplitude, and once established, remains for many days without further superimposed activity. Long-term adaptation does not occur in decentralized axons. Long-term facilitation and long-term adaptation are different responses of the neuron to enhanced activity. The former can occur in isolated or decentralized axons and leads to enhancement of EJP amplitude for a period of several hours to at least one day after a single episode of conditioning. The latter requires more time to be established, and leads to reduction of initial EJP amplitude and to lessened fatigability which persists for many days.     

Fast-axon synapses of a crab leg muscle

Neuromuscular synapses of the "fast" excitatory axon supplying the main extensor muscle in the leg of the shore crab Pachygrapsus crassipes were studied with electrophysiological and electron-microscopic techniques. Electrical recording showed that many muscle fibers of the central region of the extensor muscle responded only to stimulation of the fast axon, and electron microscopy revealed many unitary subterminal axon branches. Maintained stimulation, even at a low frequency, resulted in depression of the excitatory junctional potentials (EJPs) set up by the fast axon but EJPs of different muscle fibers depressed at different rates, indicating some physiological heterogeneity among the fast-axon synapses. Focal recording at individual synaptic sites on the surfaces of the muscle fibers showed quantal contents ranging from 1.4 to 5.5 at different synapses; these values are relatively high in comparison with similar determinations made in the crayfish opener muscle. Synapse-bearing nerve terminals were generally relatively small in diameter and filiform, with many individual synaptic contact areas of uniform size averaging 0.6 micron2. All of the individual synapses had a presynaptic "dense body" at which synaptic vesicles clustered. If these structures represent release points for transmitter quanta, the initial high quantal content would have an ultrastructural basis. The mitochondial content of the nerve terminals, the synaptic vesicle population, and the specialized subsynaptic sarcoplasm were all much reduced in comparison with tonic axon synaptic regions in this and other crustaceans. The latter features may be correlated with the relatively infrequent use of this axon by the animal, and with rapid fatigue.     

Stimulation-induced changes at crayfish (Procambarus clarkii) neuromuscular terminals

Summary The fine structure of neuromuscular terminals of the single excitor axon was examined in the limb stretcher muscle of the crayfish Procambarus clarkii. A morphometric comparsion of the neuromuscular terminals of the left and right limbs of a control crayfish showed them to be similiar in qualitative as well as quantitative features. The excitor axon to the stretcher muscle of the right side was stimulated, by backfiring its branches in the adjacent opener muscle, at 20 Hz for 4–5 h per day over 4–5 days. The stretcher muscle on the left side was not stimulated and served as a control. Morphometric analysis of stimulated terminals revealed an increase in the number of dense bars and synaptic vesicles compared to their non-stimulated, contralateral counterparts. Since dense bars are regarded as active sites of transmitter release, changes in their number provide a morphological basis for synaptic plasticity.     

Activity and synapse elimination at the neuromuscular junction

The neuromuscular junction undergoes a loss of synaptic connections during early development. This loss converts the innervation of each muscle fiber from polyneuronal to single. During this change the number of motor neurons remains constant but the number of muscle fibers innervated by each motor neuron is reduced. Evidence indicates that a local competition among the inputs on each muscle fiber determines which inputs are eliminated. The role of synapse elimination in the development of neuromuscular circuits, other than ensuring a single innervation of each fiber, is unclear. Most evidence suggests that the elimination plays little or no role in correcting for errant connections. Rather, it seems that connections are initially highly specific, in terms of both which motor neurons connect to which muscles and which neurons connect to which particular fibers within these muscles. A number of attempts have been made to determine the importance of neuromuscular activity during early development for this rearrangement of synaptic connections. Experiments reducing neuromuscular activity by muscle tenotomy, deafferentation and spinal cord section, block of nerve impulse conduction with tetrodotoxin, and the use of postsynaptic and presynaptic blocking agents have all shown that normal activity is required for normal synapse elimination. Most experiments in which complete muscle paralysis has been achieved show that activity may be essential for the occurrence of synapse elimination. Furthermore, experiments in which neuromuscular activity has been augmented by external stimulation show that synapse elimination is accelerated. A plausible hypothesis to explain the activity dependence of neuromuscular synapse elimination is that a neuromuscular trophic agent is produced by the muscle fibers and that this production is controlled by muscle-fiber activity. The terminals on each fiber compete for the substance produced by that fiber. Inactive fibers produce large quantities of this substance; on the other hand, muscle activity suppresses the level of synthesis of this agent to the point where only a single synaptic terminal can be maintained. Inactive muscle fibers would be expected to be able to maintain more nerve terminals. The attractiveness of this scheme is that it provides a simple feedback mechanism to ensure that each fiber retains a single effective input.     

Synaptic plasticity in a regenerated crayfish phasic motoneuron.

Crustacean neuromuscular systems provide many advantages for the study of synaptic transmission and plasticity. The present study examines aspects of synaptic transmission in the phasic, fast closer excitor (FCE) motoneuron of regenerated crayfish claws. Excitatory postsynaptic potentials (EPSPs) fatigued rapidly and showed poor long-term facilitation (LTF) in the smallest of regenerating claws. EPSPs in larger regenerating claws fatigued less and showed pronounced facilitation. These observations were not the same as those previously made during primary development of this motoneuron (Lnenicka and Atwood, 1985a, J. Neuroscience 5:459-467). Hence, regeneration is not the recapitulation of primary development. In situ stimulation of the FCE is known to lead to long-lasting adaptation of synaptic performance. This adaptation is age dependent; it is expressed in young but not old animals. In the regenerated FCE of old animals, we observed a novel form of long-lasting adaptation to imposed activity: EPSPs showed large initial EPSPs and did not exhibit resistance to fatigue during maintained stimulation. This indicates that aged motoneurons can express adaptive changes to increased activity following axonal regeneration, but that the adaptive changes are the opposite to what is observed in nonregenerated motoneurons.     

Role of Activity in Determining Properties of the Neuromuscular System in Crustaceans

SYNOPSIS. Crustacean muscle fibers, like those of higher vertebrates,are diversified in physiology, morphology, and biochemical attributes.However, unlike motor units of mammals, those of crustaceansusually do not contain fibers of uniform type. Motor neuronactivity acts as a unifying force for the motor units of mammalianmuscles, but its role in determining properties of crustaceanmotor units is less well defined. In certain crustacean muscles,differential activity of sensory-motor systems is importantfor establishing muscle fiber properties during early development.In freshwater crayfish, neuromuscular junctions of a phasicmotor neuron are altered physiologically and morphologicallyby chronic stimulation; the adapted junctions release less transmitterper impulse and are more fatigue-resistant than naive junctions.The muscle fibers may also adapt to chronic stimulation, butless dramatically and at a slower rate. The adaptive responsesof the neuromuscular junction can be achieved through manipulationof sensory input and with little increase in motor impulse activity.This suggests that altered protein synthesis is triggered centrallyby synaptic input to the motor neuron. In general, present evidencesuggests that long-term adaptation of neuromuscular junctionsand muscle fibers of crustaceans can occur in response to alteredactivity in the nervous system, in spite of the fact that certainmuscle fiber properties appear to be genetically predetermined.Some aspects of matching between neuromuscular junction andmuscle fiber appear to be determined in response to growth ofthe muscle fiber; other features are activity-dependent; andsome may result from expression of inherent neuronal properties.     

Sexual differentiation of identified motor terminals in Drosophila larvae

In Drosophila, we have found that some of the motor terminals in wandering third-instar larvae are sexually differentiated. In three out of the four body-wall muscle fibers that we examined, we found female terminals that produced a larger synaptic response than their male counterparts. The single motor terminal that innervates muscle fiber 5 produces an EPSP that is 69% larger in females than in males. This is due to greater release of transmitter from female than male synaptic terminals because the amplitude of spontaneous miniature EPSPs was similar in male and female muscle fibers. This sexual difference exists throughout the third-instar: it is seen in both early (foraging) and late (wandering) third-instar larvae. The sexual differentiation appears to be neuron specific and not muscle specific because the same axon produces Is terminals on muscle fibers 2 and 4, and both terminals produce larger EPSCs in females than males. Whereas, the Ib terminals innervating muscle fibers 2 and 4 are not sexually differentiated. The differences in transmitter release are not due to differences in the size of the motor terminals. For the terminal on muscle fiber 5 and the Is terminal on muscle fiber 4, there were no differences in terminal length, the number of branches, or the number of synaptic boutons in males compared to females. These sexual differences in neuromuscular synaptic physiology may be related to male-female differences in locomotion.     

Ultrastructure of synapses with different transmitter-releasing characteristics on motor axon terminals of a crab,Hyas areneas

Synaptic terminals on branches of an excitatory motor axon in a spider crab (Hyas areneas) were examined by electron microscopy to determine whether differences in size, structure, and number of synapses could be correlated with differences in transmitter release. Terminals releasing relatively large amounts of transmitter during low frequencies of nerve impulses ("high-output" terminals) had larger synapses, more prominent presynaptic dense bodies (active zones), and fewer synapses per unit length than terminals releasing relatively small amounts of transmitter ("low-output" terminals). Neither the difference in synaptic area, nor the quantitative differences in the active zones, were sufficient in themselves to explain the difference in synaptic efficacy, and it is postulated that a non-linear relationship may exist between structural features of the synapse and release of transmitter by a nerve impulse, and that differences other than those apparent from the ultrastructure could be involved. Greater facilitation at low-output terminals with high frequencies of nerve impulses may be due to greater reserves of "immediately available" transmitter, and to recruitment or activation of more individual synaptic contacts.     

Synaptic plasticity in a regenerated crayfish phasic motoneuron

Crustacean neuromuscular systems provide many advantages for the study of synaptic transmission and plasticity. The present study examines aspects of synaptic transmission in the phasic, fast closer excitor (FCE) motoneuron of regenerated crayfish claws. Excitatory postsynaptic potentials (EPSPs) fatigued rapidly and showed poor long-term facilitation (LTF) in the smallest of regenerating claws. EPSPs in larger regenerating claws fatigued less and showed pronounced facilitation. These observations were not the same as those previously made during primary development of this motoneuron (Lnenicka and Atwood, 1985a, J. Neuroscience 5:459–467). Hence, regeneration is not the recapitulation of primary development. In situ stimulation of the FCE is known to lead to long-lasting adaptation of synaptic performance. This adaptation is age dependent; it is expressed in young but not old animals. In the regenerated FCE of old animals, we observed a novel form of long-lasting adaptation to imposed activity: EPSPs showed large initial EPSPs and did not exhibit resistance to fatigue during maintained stimulation. This indicates that aged motoneurons can express adaptive changes to increased activity following axonal regeneration, but that the adaptive changes are the opposite to what is observed in nonregenerated motoneurons. © 1992 John Wiley & Sons, Inc.     

Essential roles of the acetylcholine receptor gamma-subunit in neuromuscular synaptic patterning

Formation of the vertebrate neuromuscular junction (NMJ) takes place in a stereotypic pattern in which nerves terminate at select sarcolemmal sites often localized to the central region of the muscle fibers. Several lines of evidence indicate that the muscle fibers may initiate postsynaptic differentiation independent of the ingrowing nerves. For example, nascent acetylcholine receptors (AChRs) are pre-patterned at select regions of the muscle during the initial stage of neuromuscular synaptogenesis. It is not clear how these pre-patterned AChR clusters are assembled, and to what extent they contribute to pre- and post-synaptic differentiation during development. Here, we show that genetic deletion of the AChR gamma-subunit gene in mice leads to an absence of pre-patterned AChR clusters during initial stages of neuromuscular synaptogenesis. The absence of pre-patterned AChR clusters was associated with excessive nerve branching, increased motoneuron survival, as well as aberrant distribution of acetylcholinesterase (AChE) and rapsyn. However, clustering of muscle specific kinase (MuSK) proceeded normally in the gamma-null muscles. AChR clusters emerged at later stages owing to the expression of the AChR epsilon-subunit, but these delayed AChR clusters were broadly distributed and appeared at lower level compared with the wild-type muscles. Interestingly, despite the abnormal pattern, synaptic vesicle proteins were progressively accumulated at individual nerve terminals, and neuromuscular synapses were ultimately established in gamma-null muscles. These results demonstrate that the gamma-subunit is required for the formation of pre-patterned AChR clusters, which in turn play an essential role in determining the subsequent pattern of neuromuscular synaptogenesis.     

Regeneration of an identifiable motoneuron in the crayfish. II. Patterns of reconnection and synaptic strength established in the presence of an extra nerve

The regeneration of neuromuscular connections to the superficial flexor muscle system in the crayfish has been studied under a variety of experimental manipulations. These have provided insight into the factors that can influence the regeneration program of neurons. In this work the regeneration of the largest excitor motoneuron was studied under two different conditions: (1) when the original neuron and a transplanted neuron were growing simultaneously into a denervated target, and (2) when a transplanted neuron was growing into a target that had its original nerve supply intact. In condition 1 both the transplanted and the original neuron formed normal patterns of connectivity and synaptic strength in comparable periods of time. In condition 2 the rate of growth of the transplanted neuron is significantly reduced and does not extend into the lateral fibers of the muscle. It is concluded that the regeneration program of this neuron is not affected by the presence of other neurons growing at the same time into a denervated muscle. Since regeneration is seriously affected if growth occurs into a fully innervated target area, it is suggested that lack of growth stimuli from the target or competitive interactions between established and growing synaptic terminals could influence the regeneration program of this neuron.