200 kD neurofilament protein and synapse elimination in the rat soleus muscle.

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.     

Protein and enzyme distribution in microsomal and myelin fractions from rat and jimpy mouse brain

4-Aminopyridine (4-AP) powerfully increases transmitter release from motor nerve terminals of rat and frog skeletal muscle in response to single nerve impulses. The drug also enhances transmitter release during repetitive nerve activity but, at D-tubocurarine-blocked endplates, only the first impulses cause increased transmitter release at stimulation frequencies at or above 50 Hz. At magnesium- and botulinum-poisoned endplates, 4-AP potentiates transmitter release at every stimulus during tetanic nerve stimulation and restores neuromuscular transmission. Spontaneous transmitter release in the rat is not affected by the drug, but at some frog endplates miniature endplate potential (mepp) frequency increases. The drug has no post-synaptic action, as evidenced by its lack of effect on amplitude or time course of mepps. Decreasing the temperature from 37 to 15 degrees C does not abolish the effect of 4-AP on neuromuscular transmission. In the presence of 4-AP, single nerve impulses produce repetitive spontaneous activity in the nerve terminal of the frog nerve-muscle preparation. Experiments on the mode of action of 4-AP suggest that the drug increases transmitter release by enhancing the influx of calcium ions during depolarization of the nerve terminal.     

Motor axon sprouting and site of synapse formation in intact innervated skeletal muscle of the frog

The pattern of motor innervation to the cutaneous-pectoris muscle of the frog is altered after injuring the motor nerve to the contralateral muscle in that muscle fibers innervated by a single motor nerve cell become polyneuronally innervated. This polyneuronal pattern of innervation was detected by single cell recordings of multiple end-plate potentials from muscle fibers. The present study shows that the anatomical basis for this electrophysiological observation is the formation of new additional synapses by intact motor neurons on already innervated muscle fibers at new sites. Light and electron microscopical examination of muscles stained with Zinc Iodide and Osmium revealed that the sources for the new axon terminals were the intact motor axons and nerve endings that gave rise to sprouts that formed synaptic connections with muscle fibers apparently not innervated by their parent axons. Furthermore, new synapses were formed at new synaptic sites. The increased incidence of polyneuronal innervation that was detected electrophysiologically was associated, in the same muscles, with a proportional increase in the average end-plate size estimated from the measurements of cholinesterase-stained sites. Additional evidence that synapses were formed at new sites was that the shape of the different components of multiple end-plate potentials in some polyneuronally innervated muscle fibers differed in their rise-times. No such recordings were observed in polyneuronally innervated muscle fibers of normal frogs.     

Motoneuron death and motor unit size during embryonic development of the rat

Chronic paralysis of rat embryos during the last 4 to 6 prenatal days causes a diminution in skeletal muscle fiber numbers but inhibits motoneuron death. Consequently, as paralyzed muscles develop, an increased number of motoneurons attempts to form synapses at a reduced number of synaptic sites. Paralyzed muscle fibers receive their synapses at a single endplate, as in control muscles, but these endplates are hyperinnervated, with about twice the normal number of inputs. Counts of axons, synaptic inputs, and muscle units showed that motoneurons normally contact a maximum number of muscle fibers shortly before birth, and this number remains stable for several days postnatal until it finally is reduced to the adult number. The average motor unit size in paralyzed embryos at the time of birth was the same as in controls. We suggest that it is not necessary to postulate the existence of competition between embryonic nerve terminals in order to explain regulation of the number of muscle fibers initially contacted by a motoneuron. Motoneuron death was not immediately affected by paralysis, but paralysis "rescued" all motoneurons whose death normally would have occurred 24 hr or more after the time when paralysis was initiated, regardless of when this was. This implies that the peak period for determination to die is during embryonic day 14, when myotube formation is just beginning and no recognizable endplate structures are present in muscles. When paralyzed, motoneurons normally destined to die are capable of forming a normal number of functional nerve-muscle contacts.     

Neuromuscular relationships in a muscle having segregated motor endplate zones. II. The response to partial denervation

The cranial belly of the anterior gracilis muscle of the rat has two discrete motor endplate zones. A proximal zone is innervated by short branches of the obturator nerve, and a distal zone is innervated by (usually) two longer branches. Each muscle fiber is innervated at a single motor endplate although a substantial number lie within both endplate zones. In addition, motor units are divided between the two zones. In order to dissociate the role of the denervated endplate from that of the denervated muscle fiber in the promotion of motoneuron sprouting, the distal endplate zone in this model was denervated and the response at the proximal zone was studied. Comparisons were made with partial denervation of the muscle by division of the L4 ventral ramus and with partial denervation of the distal endplate zone. Denervation of the distal endplate zone produced profuse terminal sprouting at the proximal zone whereas division of L4 predominantly produced nodal sprouting at both zones. Partial denervation of the distal zone resulted in nodal sprouts in that zone and again mainly terminal sprouts at the proximal zone. The repeated association of terminal sprouting with division of the motor axons supplying the distal zone together with the knowledge that motor units are distributed between the two zones led to the conclusion that the terminal sprouting was stimulated by the reduction in size of motor units rather than by the presence of denervated muscle fibers in the vicinity of the endplates.     

Physiological properties of isolated motor units in normal and bilaterally innervated Xenopus gastrocnemius muscles.

Physiological properties of isolated gastrocnemius motor units were measured in normal juvenile postmetamorphic Xenopus frogs and in a group of juvenile animals with a single bilaterally innervated hindlimb. The aim of this study was to evaluate changes to the organisation of the motor unit when the muscle is hyperinnervated. Animals with a single bilaterally innervated hindlimb have previously been shown to support up to twice the normal number of motoneurons projecting into a single hindlimb. Under these circumstances there was a lowering of average neuromuscular efficacy (as judged by motor unit twitch/tetanus ratio) in comparison with normal age-matched siblings. Motor units with neurons in the lateral motor column contralateral to the remaining hindlimb were indistinguishable from those originating ipsilaterally. There is a wide range of safety margins for neuromuscular transmission at the various terminals of individual frog motor units, and comparison of motor unit contraction times with twitch/tetanus ratios showed that under pressure of hyperinnervation, motoneurons tend to retain their safest terminals on muscle fibres with fast contraction times.     

Regulation of Motoneuronal Calcitonin Gene–related Peptide (CGRP) During Axonal Growth and Neuromuscular Synaptic Plasticity Induced by Botulinum Toxin in Rats

The aim of this study was to examine whether changes in rat motoneuronal calcitonin gene–related peptide (CGRP) can be correlated with axonal growth and plasticity of neuromuscular synapses. Nerve terminal outgrowth was induced by local paralysis with botulinum toxin. Normal adult soleus and tibialis anterior did not show detectable CGRP content at the motor endplates. Following botulinum toxin injection there was a progressive, transient and bimodal increase in CGRP in both motoneuron cell bodies which innervated poisoned muscles and their motor endplates. CGRP content was moderately increased 1 day after paralysis and, after an initial decline, reached a peak 20 days after injection. This was followed by a gradual decrease and a return to normal levels at the 200th day. CGRP changes in intoxicated endplates were less evident in the tibialis anterior than in the soleus muscle. The CGRP content in motoneurons was positively correlated with the degree of intramuscular nerve sprouting found by silver staining. In situ hybridization revealed an increase in CGRP mRNA in spinal cord motoneurons 20 days after toxin administration. We conclude that motoneurons regulate their CGRP in situations in which peripheral synapse remodelling and plasticity occur.     

Role for the skeletal muscle action potential in non-Hebbian long-term depression at the amphibian (Bufo marinus) neuromuscular junction

Retrograde signaling from skeletal muscle cells to motor nerve terminals is a recognized mechanism for modulating the strength of neuromuscular transmission. We recently described a form of long-term depression of transmitter release at the mature neuromuscular junction that is dependent on the production of nitric oxide, most likely by the muscle cell (Etherington and Everett 2004 J Physiol (Lond) 559:507-517). We now show that the depression is blocked by treating neuromuscular preparations with mu-conotoxin G111A, an antagonist of skeletal muscle voltage gated sodium channels, indicating that the depression requires postsynaptic action potential firing. Experiments on dually-innervated sartorius muscles revealed that propagation of action potentials generated by low-frequency stimulation of one nerve branch gives rise to nitric-oxide mediated depression at unstimulated nerve terminals located many millimetres away on the same muscle fiber. The non-Hebbian pattern of expression of the depression, as well as its reliance on postsynaptic action potential firing, distinguish it from forms of synaptic depression described at immature neuromuscular synapses and may provide a mechanism for coregulation of the strength of motoneurons innervating the same postsynaptic cell.     

Integrins at the neuromuscular junction are important for motoneuron survival

During development motoneurons depend on target contact for their survival. Following injury to the sciatic nerve in neonatal rats, a large proportion of motoneurons die. However, the same injury inflicted at 5 days of age results in no loss of motoneurons. This critical period of postnatal development coincides with the time during which there is a significant increase in the release of transmitter from the nerve terminals at the neuromuscular junction. We have proposed that the role of the target muscle cell during this period is to induce this up-regulation of transmitter release from motor nerve terminals. It has been shown that stretch-induced increase in transmitter release from frog motor nerve terminals is accomplished via an integrin-dependent mechanism. In this study we examined the role of integrins at the rat neuromuscular junction in motoneuron survival. We found that blocking integrin binding at the developing neuromuscular junction delayed the increase in choline acetyltransferase activity that normally takes place during the early postnatal period, and resulted in motoneuron death. Furthermore, the maturation of those motoneurons that survived was delayed so they remained susceptible to subsequent nerve injury. These results support the possibility that integrins, by their involvement in modulating transmitter release, can influence motoneuron survival.     

Nitric oxide synthase in rat neuromuscular junctions and in nerve terminals of torpedo electric organ: Its role as regulator of acetylcholine release

The distribution of nitric oxide synthase on peripheral motor system was studied using a specific antibody against the neuronal isoform of nitric oxide synthase (nNOS). The immunoreactivity for nNOS was detected on the sarcolemmal surface of muscle cells, in intramuscular axons and in neuromuscular synapses. At the neuromuscular junctions, ultrastructural immunolabeling demonstrated that nNOS immunoreactivity was localized mainly into the presynaptic nerve terminals as well as adjacent postsynaptic muscle membrane. Similar immunostaining pattern was present in frog muscles and Torpedo electric organs. After chronic muscle denervation, nNOS immunoreactity at endplate level decreased during the first week but it was upregulated after 30 days of denervation. In denervated endplates, nNOS immunoreactivity was localized in the terminal Schwann cells covering the degenerated neuromuscular junctions whereas nNOS was not detected in Schwann cells under normal conditions. In Torpedo synaptosomes, acetylcholine (ACh) release elicited by potassium depolarization was inhibited by NO donors such as sodium nitroprusside. In contrast, application of inhibitors of NOS activity, aminoguanidine (AMG) and Nω-Nitro-L-arginine methyl esther (L-NAME) increased acetylcholine release. These results indicate that nNOS is present at the motor nerve terminals in a variety of vertebrates and that it may be involved in the physiological modulation of ACh release and in the regulation of muscle response to nerve injury.     

Inverse relationship between transmitter release and terminal length in synapses on frog muscle fibers of uniform input resistance

Transmitter release at frog neuromuscular junctions is known to be related positively to nerve terminal length. However, the correlation is inexact, with a wide scatter of data. We have analyzed the endplate potentials (EPPs) of identified frog cutaneous pectoris muscle fibers, correlating release with terminal size and fiber input resistance (Rin). Transmitter release was assayed by quantal analysis of endplate activity in low Ca2+ Ringer solution and by measurement of the EPPs evoked in normal Ringer solution and curare. For fibers of approximately the same Rin, there is an inverse relationship between the level of transmitter release per unit length and total terminal length. Terminals with high levels of release per unit length tend to be shorter than do those which release relatively less transmitter per unit length. Furthermore, if the analysis is restricted similarly to fibers with nearly identical Rin, the total transmitter release of the largest endplates is usually less than that of the shorter terminals in the sample. These findings do not contradict the overall trend of greater release and longer terminals on larger muscle fibers (with lower Rin). Instead, they help explain the variability in measurement of release versus terminal length. The relationship that we find is consistent with the hypothesis that, in the cutaneous pectoris, terminals are induced to grow until an adequate safety factor for transmission is achieved.     

Motoneurons of twitch and nontwitch extraocular muscle fibers in the abducens, trochlear, and oculomotor nuclei of monkeys

Eye muscle fibers can be divided into two categories: nontwitch, multiply innervated muscle fibers (MIFs), and twitch, singly innervated muscle fibers (SIFs). We investigated the location of motoneurons supplying SIFs and MIFs in the six extraocular muscles of monkeys. Injections of retrograde tracers into eye muscles were placed either centrally, within the central SIF endplate zone; in an intermediate zone, outside the SIF endplate zone, targeting MIF endplates along the length of muscle fiber; or distally, into the myotendinous junction containing palisade endings. Central injections labeled large motoneurons within the abducens, trochlear or oculomotor nucleus, and smaller motoneurons lying mainly around the periphery of the motor nuclei. Intermediate injections labeled some large motoneurons within the motor nuclei but also labeled many peripheral motoneurons. Distal injections labeled small and medium-large peripheral neurons strongly and almost exclusively. The peripheral neurons labeled from the lateral rectus muscle surround the medial half of the abducens nucleus: from superior oblique, they form a cap over the dorsal trochlear nucleus; from inferior oblique and superior rectus, they are scattered bilaterally around the midline, between the oculomotor nucleus; from both medial and inferior rectus, they lie mainly in the C-group, on the dorsomedial border of oculomotor nucleus. In the medial rectus distal injections, a "C-group extension" extended up to the Edinger-Westphal nucleus and labeled dendrites within the supraoculomotor area. We conclude that large motoneurons within the motor nuclei innervate twitch fibers, whereas smaller motoneurons around the periphery innervate nontwitch, MIF fibers. The peripheral subgroups also contain medium-large neurons which may be associated with the palisade endings of global MIFs. The role of MIFs in eye movements is unclear, but the concept of a final common pathway must now be reconsidered.     

Innervation pattern of muscles of one-legged Xenopus laevis supplied by motoneurons from both sides of the spinal cord

In Xenopus tadpoles one limb bud was removed before innervation and motoneurons from both sides of the spinal cord were induced to innervate the remaining limb. When examined after metamorphosis the motor innervation of the limb had the following characteristics. In agreement with previous findings a large proportion of contralateral motoneurons survived (51-82% of the ipsilateral numbers) and sent axons to the limb. By acetylcholinesterase staining and intracellular recording from muscle fibers of the response to electrical stimulation of the two limb innervations, the neuromuscular junctions from contralateral motoneurons were indistinguishable from those from the ipsilateral side in their morphology, spacing along the fiber, and physiological properties. Many single muscle fibers shared innervation from both sides of the cord by symmetrically placed spinal nerves. By the same techniques junctions in one-legged frogs were morphologically indistinguishable from those in normal frogs, but the quantal content of transmitter release was increased by up to 63%. Recording twitch and tetanic tensions from individual motor units from the gastrocnemius muscle showed that the one-legged animals had many more and smaller motor units than do normal frogs. We confirm that the hind-limb musculature has the ability, normally unexpressed, to sustain, through the period of normal developmental cell death, up to twice the usual number of motoneurons. In maturity, motoneurons accommodate themselves to the limb muscles by making fewer than the normal number of synapses. The above suggests that developmental motoneuron death is not primarily a mechanism for adjusting the number of motoneurons to the size of the peripheral musculature and is likely to be related to mechanisms for securing specific neuromuscular connections.     

Long-term changes in neuromuscular synapses with altered sensory input to a crayfish motoneuron

Prolonged changes in crayfish motoneuron electrical activity result in adaptations in neuromuscular synapses which are consistent with findings at other synapses. In this study we establish that this long-term adaptation (LTA) of crayfish neuromuscular synapses to increased activation of the motoneuron does not require the activation of any other neurons. Selectively increasing the impulse activity of the relatively inactive fast closer excitor motoneuron (FCE) over a period of 7 days results in a 41% reduction in initial amplitude of the excitatory postsynaptic potential (EPSP), and a 42% decrease in synaptic fatigue. These changes in EPSP properties have been previously shown to be due to decreased initial transmitter release and greater sustained release of transmitter during prolonged stimulation. Chronic stimulation of sensory receptors known to produce subthreshold synaptic potentials in the central processes of the FCE elicits LTA of its neuromuscular synapses. The initial EPSP is decreased by 21%, and the synaptic fatigue is reduced by 17%. These results lead to the hypothesis that the primary event leading to LTA of neuromuscular synapses is depolarization of the motoneuron.     

Promotion of motoneuron survival and branching in rapsyn-deficient mice

Inhibition of programmed cell death of motoneurons during embryonic development requires the presence of their target muscle and coincides with the initial stages of synaptogenesis. To evaluate the role of synapse formation on motoneuron survival during embryonic development, we counted the number of motoneurons in rapsyn-deficient mice. Rapsyn is a 43 kDa protein needed for the formation of postsynaptic specialisations at vertebrate neuromuscular synapses. Here we show that the rapsyn-deficient mice have a significant increase in the number of motoneurons in the brachial lateral motor column during the period of naturally occurring programmed cell death compared to their wild-type littermates. In addition, we observed an increase in intramuscular axonal branching in the rapsyn-deficient diaphragms compared to their wild-type littermates at embryonic day 18.5. These results suggest that deficits in the formation of the postsynaptic specialisation at the neuromuscular synapse, brought about by the absence of rapsyn, are sufficient to induce increases in both axonal branching and the survival of the innervating motoneuron. Moreover, these results support the idea that skeletal muscle activity through effective synaptic transmission and intramuscular axonal branching are major mechanisms that regulate motoneuron survival during development.     

Morphology and ultrastructure of medial rectus subgroup motoneurons in the macaque monkey

There are two muscle fiber types in extraocular muscles: those receiving a single motor endplate, termed singly innervated fibers (SIFs), and those receiving multiple small terminals along their length, termed multiply innervated fibers (MIFs). In monkeys, these two fiber types receive input from different motoneuron pools: SIF motoneurons found within the extraocular motor nuclei, and MIF motoneurons found along their periphery. For the monkey medial rectus muscle, MIF motoneurons are found in the C‐group, while SIF motoneurons lie in the A‐ and B‐groups. We analyzed the somatodendritic morphology and ultrastructure of these three subgroups of macaque medial rectus motoneurons to better understand the structural determinants controlling the two muscle fiber types. The dendrites of A‐ and B‐group motoneurons lay within the oculomotor nucleus, but those of the C‐group motoneurons were located outside the nucleus, and extended into the preganglionic Edinger–Westphal nucleus. A‐ and B‐group motoneurons were very similar ultrastructurally. In contrast, C‐group motoneurons displayed significantly fewer synaptic contacts on their somata and proximal dendrites, and those contacts were smaller in size and lacked dense‐cored vesicles. However, the synaptic structure of C‐group distal dendrites was quite similar to that observed for A‐ and B‐group motoneurons. Our anatomical findings suggest that C‐group MIF motoneurons have different physiological properties than A‐ and B‐group SIF motoneurons, paralleling their different muscle fiber targets. Moreover, primate C‐group motoneurons have evolved a special relationship with the preganglionic Edinger–Westphal nucleus, suggesting these motoneurons play an important role in near triad convergence to support increased near work requirements. J. Comp. Neurol. 522:626–641, 2014. © 2013 Wiley Periodicals, Inc.     

Protein kinase C activity affects neurotransmitter release at polyinnervated neuromuscular synapses

By using intracellular recording, we studied how protein kinase C (PKC) activity affected transmitter release in singly and dually innervated endplates of the Levator auris longus muscle of 5-6-day-old rats during axonal competition in the postnatal synaptic elimination period. In dually innervated fibers, a second endplate potential (EPP) may appear after the first one when the stimulation intensity is increased. The nerve terminals that generate the lowest and the highest EPP amplitudes are designated "small-EPP generating ending" (SEGE) and "large-EPP generating ending" (LEGE), respectively. Blocking PKC with calphostin C, staurosporine, or chelerythrine results in an increased release from SEGE ( approximately 80%), whereas release from LEGE and from endings generating only one EPP (OEGE) is not significantly affected. Blocking PKC also leads to the recruitment of silent synapses (acetylcholine cannot be released before PKC inhibition). The mean number of functional axon terminals per synapse increases by approximately 47%, and these are now designated the "recruited-EPP generating endings" (REGE). This suggests that axonal PKC can modulate postnatal synaptic elimination by favoring the nerve terminal disconnection of certain weak axonal endings (REGE and SEGE). We conclude that a PKC-mediated mechanism should occupy a pivotal place in neonatal synapse elimination, because functional axonal withdrawal can indeed be turned back by PKC block.     

Kinetics of acetylcholine quanta release at the neuromuscular junction during high-frequency nerve stimulation

The effects of high‐frequency nerve stimulation (10–100 Hz) on the kinetics of evoked acetylcholine quanta secretion from frog motor nerve endings were studied. The amplitude and temporal parameters of uni‐ and multiquantal endplate currents were analysed to estimate the possible changes in the degree of synchrony of quantal release. The frog neuromuscular synapse is unusually long and we have placed special emphasis on evaluating the velocity of propagation of excitation along the nonmyelinated nerve ending as this might influence the synchrony of release from the whole terminal and hence affect the time course of postsynaptic currents. The data show that high‐frequency firing leads to the desynchronization of acetylcholine release from motor nerve endings governed by at least two independent factors, namely a reduction of nerve pulse propagation velocity in the nonmyelinated parts of the axon and a change of secretion kinetics at single active zones. A computer reconstruction of the multiquantal synaptic response was performed to estimate any contribution of each of the above factors to the total rate of release and amplitude and time characteristics of the endplate currents. The results indicate that modification of the kinetics of neurotransmitter quanta release during high‐frequency firing should be taken into account when mechanisms underlying the plasticity of chemical synapses are under investigation.     

Motor endplate disease affects neuromuscular junction maturation

Postnatal formation of the neuromuscular synapse requires complex interactions among nerve terminal, muscle fibres and terminal Schwann cells. In motor endplate disease (med) mice, neuromuscular transmission is severely impaired without alteration of axonal conduction and a lethal paralytic phenotype occurs during the postnatal period. The med phenotype appears at a crucial stage of the neuromuscular junction development, corresponding to the increase in terminal Schwann cell number, the elimination of the multiple innervations and the pre- and postsynaptic maturation. Here we investigated the early cellular and molecular consequences of the med mutation on neuromuscular junction development. We observed that cellular defects preceded overt clinical phenotype. The first detectable cellular effect of the mutation at the onset of the clinical phenotype was a drastic reduction in the number of terminal Schwann cells, in part due to an increase in glial apoptosis, and a delayed maturation of motor endplates. We also showed that, in terminally ill animals, mono-innervation was not achieved, synaptic vesicles had accumulated in the presynaptic compartment and, finally, the size of motor endplates was reduced. All together, our findings suggested that the clinical weakness in these mutant mice was likely to be related to postnatal structural abnormalities of the neuromuscular junction maturation.     

Morphological transformation of synaptic terminals of a phasic motoneuron by long-term tonic stimulation

In vivo stimulation of a relatively "silent" phasic crayfish motoneuron changes the ultrastructure of its synaptic terminals to a more tonic phenotype. The closer muscle of the crayfish claw is supplied by only 2 excitatory motoneurons, one of which is phasic and the other tonic. The ultrastructures of conditioned phasic, unconditioned phasic, and tonic motor terminals were compared. The terminals of the tonic motor axon were larger in cross-sectional area, had larger mitochondria, greater synaptic contact area, and were more varicose than unconditioned phasic terminals. Following long-term tonic stimulation of the phasic axon, its terminals became more varicose, mitochondrial cross-sectional area more than doubled, and synapses and mitochondria came into closer proximity, although mean terminal cross-sectional area did not change. Thus, the conditioned phasic terminals became more similar to those of the tonic motor axon. These changes in ultrastructure correlate with, and may be causally linked to, previously reported changes in neuromuscular synaptic physiology produced by in vivo tonic stimulation of this motoneuron. We conclude that the ongoing level of impulse activity can affect the ultrastructural differentiation of synaptic terminals and synapses of the phasic motoneuron.