A screen of cell-surface molecules identifies leucine-rich repeat proteins as key mediators of synaptic target selection

In Drosophila embryos and larvae, a small number of identified motor neurons innervate body wall muscles in a highly stereotyped pattern. Although genetic screens have identified many proteins that are required for axon guidance and synaptogenesis in this system, little is known about the mechanisms by which muscle fibers are defined as targets for specific motor axons. To identify potential target labels, we screened 410 genes encoding cell-surface and secreted proteins, searching for those whose overexpression on all muscle fibers causes motor axons to make targeting errors. Thirty such genes were identified, and a number of these were members of a large gene family encoding proteins whose extracellular domains contain leucine-rich repeat (LRR) sequences, which are protein interaction modules. By manipulating gene expression in muscle 12, we showed that four LRR proteins participate in the selection of this muscle as the appropriate synaptic target for the RP5 motor neuron.     

Quantitative differences among EMG activities of muscles innervated by subpopulations of hypoglossal and upper spinal motoneurons during non-REM sleep - REM sleep transitions: a window on neural processes in the sleeping brain

In the rat, a species widely used to study the neural mechanisms of sleep and motor control, lingual electromyographic activity (EMG) is minimal during non-rapid eye movement (non-REM) sleep and then phasic twitches gradually increase after the onset of REM sleep. To better characterize the central neural processes underlying this pattern, we quantified EMG of muscles innervated by distinct subpopulations of hypoglossal motoneurons and nuchal (N) EMG during transitions from non-REM sleep to REM sleep. In 8 chronically instrumented rats, we recorded cortical EEG, EMG at sites near the base of the tongue where genioglossal and intrinsic muscle fibers predominate (GG-I), EMG of the geniohyoid (GH) muscle, and N EMG. Sleep-wake states were identified and EMGs quantified relative to their mean levels in wakefulness in successive 10 s epochs. During non-REM sleep, the average EMG levels differed among the three muscles, with the order being N>GH>GG-I. During REM sleep, due to different magnitudes of phasic twitches, the order was reversed to GG-I>GH>N. GG-I and GH exhibited a gradual increase of twitching that peaked at 70-120 s after the onset of REM sleep and then declined if the REM sleep episode lasted longer. We propose that a common phasic excitatory generator impinges on motoneuron pools that innervate different muscles, but twitching magnitudes are different due to different levels of tonic motoneuronal hyperpolarization. We also propose that REM sleep episodes of average durations are terminated by intense activity of the central generator of phasic events, whereas long REM sleep episodes end as a result of a gradual waning of the tonic disfacilitatory and inhibitory processes.     

Innervation and control of tension in abdominal muscles of Blaberus discoidalis and Gromphadorhina portentosa

In Blaberus discoidalis and Gromphadorhina portentosa, the distribution of motor axons to the muscles which control movements of the spiracular valves at both respiratory and non-respiratory spiracles is identical. Both fast and slowly contracting heads of the opener muscles are innervated by an excitatory motor axon. Physiological properties of the opener excitor axon correlate with valve function. The slowly contracting head of the opener muscle is, in addition, innervated by a common inhibitor which also occasionally innervates closer muscle fibers. Activation of the common inhibitor terminates contraction of slowly contracting opener muscle fibres and initiates a rapid relaxation of these fibres.     

Plasticity of skeletal muscle: regenerating fibers adapt more rapidly than surviving fibers

The properties of mammalian skeletal muscle demonstrate a high degree of structural and functional plasticity as evidenced by their adaptability to an atypical site after cross-transplantation and to atypical innervation after cross-innervation. We tested the hypothesis that, regardless of fiber type, skeletal muscles composed of regenerating fibers adapt more readily than muscles composed of surviving fibers when placed in an atypical site with atypical innervation. Fast muscles of rats were autografted into the site of slow muscles or vice versa with the donor muscle innervated by the motor nerve to the recipient site. Surviving fibers in donor muscles were obtained by grafting with vasculature intact (vascularized muscle graft), and regenerating fibers were obtained by grafting with vasculature severed (free muscle graft). Our hypothesis was supported because 60 days after grafting, transposed muscles with surviving fibers demonstrated only a slight change from the contractile properties and fiber typing of donor muscles, whereas transplanted muscles with regenerating fibers demonstrated almost complete change to those of the muscle formerly in the atypical site.     

Crustacean Neuromuscular Mechanisms

Properties of crustacean muscle fibers and neuromuscular synapsesare discussed, with particular reference to the problems offast and slow contraction, synaptic diversity, and peripheralinhibition. Electrical and mechanical responses of crustacean muscle fibersare variable, and govern to a large extent the muscle's performance.Fast and slow contractions are often mediated by distinct "phasic"and "tonic" muscle fibers, as in abdominal muscles, in whichsuch fibers are segregated into two parallel sets of muscles.In leg muscles the fibers are often heterogeneous in propertiesand innervation. In doubly-motor-innervated muscles of crabsthe axons producing fast and slow contractions preferentiallyinnervate rapidly and slowly contracting fibers, respectively. Crustacean neuromuscular synapses vary greatly in electricalbehavior and in ultrastructural characteristics. Some motoraxons possess both facilitating and nonfacilitating synapses.The proportion of the different types of synapse associatedwith a motor axon probably determines in large measure the propertiesof the postsynaptic potentials evoked by that axon. Pre-synaptic and post-synaptic inhibition both occur, sometimesin the same muscle. The latter type is more common. Pre-synapticinhibition is thought to be mediated by the action of an inhibitorytransmitter-substance on receptors of the motor nerve terminals.     

Histological study of motor innervation to long nuclear chain intrafusal fibers in the muscle spindle of the cat

Several muscle spindles of the cat tenuissimus muscle were cut in serial, 1-micron thick transverse sections and stained with toluidine blue in search for long nuclear chain intrafusal muscle fibers. Five complete poles of the long chain fibers were located. Each fiber pole displayed one plate-type motor ending situated in the extracapsular fiber region. The endings were supplied by myelinated motor axons that originated from intramuscular nerve fascicles containing motor axons to extrafusal muscle fibers. One of the endings was innervated by a collateral from a motor axon that supplied an extrafusal end-plate. Ultrastructurally, the long chain endings resembled extrafusal end-plates. They were more complex, in terms of prominence of sole-plate and degree of post-junctional folding, than any other intrafusal ending present in the spindles. The motor endings of the long chain fibers were assumed to be the terminals of static (fast) skeletofusimotor axons, which preferentially innervate the longest nuclear chain fibers of cat muscle spindles.     

Transplantation of Xenopus laevis Tissues to Determine the Ability of Motor Neurons to Acquire a Novel Target

The evolutionary origin of novelties is a central problem in biology. At a cellular level this requires, for example, molecularly resolving how brainstem motor neurons change their innervation target from muscle fibers (branchial motor neurons) to neural crest-derived ganglia (visceral motor neurons) or ear-derived hair cells (inner ear and lateral line efferent neurons). Transplantation of various tissues into the path of motor neuron axons could determine the ability of any motor neuron to innervate a novel target. Several tissues that receive direct, indirect, or no motor innervation were transplanted into the path of different motor neuron populations in Xenopus laevis embryos. Ears, somites, hearts, and lungs were transplanted to the orbit, replacing the eye. Jaw and eye muscle were transplanted to the trunk, replacing a somite. Applications of lipophilic dyes and immunohistochemistry to reveal motor neuron axon terminals were used. The ear, but not somite-derived muscle, heart, or liver, received motor neuron axons via the oculomotor or trochlear nerves. Somite-derived muscle tissue was innervated, likely by the hypoglossal nerve, when replacing the ear. In contrast to our previous report on ear innervation by spinal motor neurons, none of the tissues (eye or jaw muscle) was innervated when transplanted to the trunk. Taken together, these results suggest that there is some plasticity inherent to motor innervation, but not every motor neuron can become an efferent to any target that normally receives motor input. The only tissue among our samples that can be innervated by all motor neurons tested is the ear. We suggest some possible, testable molecular suggestions for this apparent uniqueness.     

Differential effects of depolarization on the growth of crayfish tonic and phasic motor axons in culture

Previous studies have demonstrated neuron-specific differences in the inhibitory effects of depolarization upon neurite outgrowth. We examined whether there is a relationship between the normal impulse activity level of an axon and the effect of depolarization upon its growth. Inactive phasic motor axons and active tonic motor axons grow from crayfish abdominal nerve cord explants in culture. Depolarization of these axons with high K⁺ solutions produced greater inhibition of advancing growth cones from the phasic axons than from the tonic axons. During the period 20–40 min after the beginning of depolarization, tonic axon growth cones continued to advance, whereas phasic axon growth cones retracted. During chronic depolarization, all of the phasic axons retracted during the first day and approximately half of the phasic axons had degenerated after 4 days of depolarization. The majority of tonic axons continue to grow after 3 days of depolarization, and all of the tonic axon growth survived the 4 days of depolarization. The different responses of the growing phasic and tonic axons to depolarization appear to be Ca²⁺ dependent. The inhibitory effects of depolarization upon phasic axon growth were reduced by the Ca²⁺ channel blockers La³⁺ and Mg²⁺. Application of a Ca²⁺ ionophore, A23187, produces greater inhibition of phasic axon growth than tonic axon growth. This study demonstrates that depolarization produces greater inhibition of growth from inactive motor axons than from active motor axons. This is likely due to differences in Ca²⁺ regulation and/or sensitivity to intracellular Ca²⁺. © 1997 John Wiley & Sons, Inc. J Neurobiol 33: 85–97, 1997     

Muscle type-specific myosin isoforms in crustacean muscles

Differential expression of multiple myosin heavy chain (MyHC) genes largely determines the diversity of critical physiological, histochemical, and enzymatic properties characteristic of skeletal muscle. Hypotheses to explain myofiber diversity range from intrinsic control of expression based on myoblast lineage to extrinsic control by innervation, hormones, and usage. The unique innervation and specialized function of crayfish (Procambarus clarkii) appendicular and abdominal musculature provide a model to test these hypotheses. The leg opener and superficial abdominal extensor muscles are innervated by tonic excitatory motoneurons. High resolution SDS-PAGE revealed that these two muscles express the same MyHC profile. In contrast, the deep abdominal extensor muscles, innervated by phasic motoneurons, express MyHC profiles different from the tonic profiles. The claw closer muscles are dually innervated by tonic and phasic motoneurons and a mixed phenotype was observed, albeit biased toward the phasic profile seen in the closer muscle. These results indicate that multiple MyHC isoforms are present in the crayfish and that differential expression is associated with diversity of muscle type and function.     

Induced neuroma formation and target muscle perturbation in the giant fiber pathway of the Drosophila temperature-sensitive mutant shibire

Summary The temperature-sensitive mutation shibire (shi) in Drosophila melanogaster is thought to disrupt membrane recycling processes, including endocytotic vesicle pinch-off. This mutation can perturb the development of nerves and muscles of the adult escape response. After exposure to a heat pulse (6 h at 30° C) at 20 h of pupal development, adults have abnormal flight muscles. Wing depressor muscles (DLM) are reduced in number from the normal six to one or two fibers, and are composed of enlarged fibers that appear to represent fiber fusion; large spaces devoid of muscle fibers suggested fiber deletion. The normal five motor axons are present in the peripheral nerve PDMN near the ganglion. However, while some motor axons pass dorsally to the extant fibers, other motor axons lacking end targets pass into an abnormal posterior branch and terminate in a neuroma, i.e., a tangle of axons and glia without muscle target tissue. Hemisynapses are common in axons of the proximal PDMN and within the neuroma, but they are rarely seen in control (no heat pulse) shi or wild-type flies. All surviving muscle fibers are innervated; no muscle tissue exists without innervation. Fibrillar fine structure and neuromuscular synapses appear normal. Fused fibers have dual innervation, suggesting correct and specific matching of target tissue and motor axons. Motor axons lacking target fibers do not innervate erroneous targets but instead terminate in the neuroma. These results suggest developmental constraints and rules, which may contribute to the orderly, stereotyped development in the normal flight system. The nature of the anomalies inducible in the flight motor system in shi flies implies that membrane recycling events at about 20 h of pupal development are critical to the formation of the normal adult nerve-muscle pattern for DLM flight muscles.     

Sensitivity of transformed (phasic to tonic) motor neurons to the neuromodulator 5-HT

Long-term adaptation resulting in a 'tonic-like' state can be induced in phasic motor neurons of the crayfish, Procambarus clarkii, by daily low-frequency stimulation [Lnenicka, G.A., Atwood, H.L., 1985b. Long-term facilitation and long-term adaptation at synapses of a crayfish phasic motoneuron. J. Neurobiol. 16, 97-110]. To test the hypothesis that motor neurons undergoing adaptation show increased responses to the neuromodulator serotonin (5-HT), phasic motor neurons innervating the deep abdominal extensor muscles of crayfish were stimulated at 2.5 Hz, 2 h/day, for 7 days. One day after cessation of conditioning, contralateral control and conditioned motor neurons of the same segment were stimulated at 1 Hz and the induced excitatory post-synaptic potentials (EPSPs) were recorded from DEL(1) muscle fibers innervated by each motor neuron type. Recordings were made in saline without and with 100 nM 5-HT. EPSP amplitudes were increased by 5-HT exposure in all cases. Conditioned muscles exposed to 5-HT showed a 2-fold higher percentage of increase in EPSP amplitude than did control muscles. Thus, the conditioned motor neurons behaved like intrinsically tonic motoneurons in their response to 5-HT. While these results show that long-term adaptation (LTA) extends to 5-HT neuromodulation, no phenotype switch could be detected in the postsynaptic muscle. Protein isoform profiles, including the myosin heavy chains, do not change after 1 week of conditioning their innervating motor neurons.     

The oculomotor system of Daphnia magna

Summary The highly mobile cyclopic compound eye of Daphnia magna is rotated by six muscles arranged as three bilateral pairs. The three muscles on each side of the head share a common origin on the carapace and insert dorsally, laterally and ventrally on the eye. The dorsal and ventral muscles are each composed of two muscle fibers and the lateral muscle is composed of from two to five fibers, with three the most common number. Individual muscle fibers are spindle-shaped mononucleated cells with organized bundles of myofilaments. Lateral eye-muscle fibers are thinner than those of the other muscles but are otherwise similar in ultrastructure. Two motor neurons innervate each dorsal and each ventral muscle and one motor neuron innervates each lateral muscle. The cell bodies of the motor neurons are situated dorsally in the supraesophageal ganglion (SEG) and are ipsilateral to the muscles they innervate. The dendritic fields of the dorsal-muscle motor neurons are ipsilateral to their cell bodies; those of the ventral-muscle motor neurons are bilateral though predominantly contralateral. The central projections of the lateral-muscle motor neurons are unknown. In the dorsal and ventral muscles one motor axon synapses principally with one muscle fiber; in each lateral muscle the single motor axon branches to, and forms synapses with, all the fibers. The neuromuscular junctions, characterized by pre- and postsynaptic densities and clear vesicles, are similar in all the eye muscles.     

Neurite outgrowth on cryostat sections of innervated and denervated skeletal muscle

To localize factors that guide axons reinnervating skeletal muscle, we cultured ciliary ganglion neurons on cryostat sections of innervated and denervated adult muscle. Neurons extended neurites on sections of muscle (and several other tissues), generally in close apposition to sectioned cell surfaces. Average neurite length was greater on sections of denervated than on sections of innervated muscle, supporting the existence of functionally important differences between innervated and denervated muscle fiber surfaces. Furthermore, outgrowth was greater on sections of denervated muscle cut from endplate-rich regions than on sections from endplate-free regions, suggesting that a neurite outgrowth-promoting factor is concentrated near synapses. Finally, 80% of the neurites that contacted original synaptic sites (which are known to be preferentially reinnervated by regenerating axons in vivo) terminated precisely at those contacts, thereby demonstrating a specific response to components concentrated at endplates. Together, these results support the hypothesis that denervated muscles use cell surface (membrane and matrix) molecules to inform regenerating axons of their state of innervation and proximity to synaptic sites.     

Regeneration from crayfish phasic and tonic motor axons in vitro

An explant culture system is described that allows examination of axonal growth from the tonically and phasically active motoneurons of the abdominal nerve cord of the crayfish. In this preparation, growth occurs from the cut end of the axon while the remainder of the motoneuron is undisturbed. In vitro growth from the branches of the third roots, which contain the axons from the tonic and phasic motoneurons of abdominal ganglia one through four, was verified as axonal by retrograde labeling of axons and neuronal somata within the nerve cord. Growth from the axons of phasic and tonic cells was observed as early as 24 h after plating and continued for an additional 7–10 days. The morphology and growth rates of the motor terminals differed between the tonic and phasic axons. The phasic axons grew significantly faster and branched more often than did the tonic motor axons. These differences in growth may be related to differences in motoneuron size or, may result from differences in electrical activity. Tonic motoneurons show spontaneous impulse activity for up to 6 days in culture, whereas phasic motoneurons show no spontaneous impulse activity. In addition, the differences in growth may be related to the morphological differences in tonic and phasic motor terminals observed in situ. © 1993 John Wiley & Sons, Inc.     

The role of sensory input in motor neuron sprouting control

Primary sensory neurons project to motor neurons directly or through interneurons and affect their activity. In our previous paper we showed that intramuscular sprouting can be affected by changing the sensory synaptic input to motor neurons. In this work, motor axon sprouting within a peripheral nerve (extramuscular sprouting) was induced by nerve injury at such a distance from muscle so as not to allow nerve-muscle trophic interactions. Two different procedures were carried out: (1) sciatic nerve crush and (2) sciatic nerve crush with homosegmental ipsilateral L3-L5 dorsal rhizotomy. The number of regenerating motor axons innervating extensor digitorum longus muscle was determined by in vivo muscle tension recordings and an index of their individual conduction rate was obtained by in vitro intracellular recordings of excitatory postsynaptic end-plate potentials in muscle fibers. The main findings were: (1) there are more regenerated axons distally from the lesion than parent axons proximally to the lesion (sprouting at the lesion); (2) sprouting at the lesion was negatively affected by homosegmental ipsilateral dorsal rhizotomy; (3) the number of motor axons innervating extensor digitorum longus muscle extrafusal fibers counted proximally to the lesion increased following nerve injury and regeneration but this did not occur when sensory input was lost. A transient innervation of extrafusal fibers by &#110 motor neurons may explain the increase of motor axons counted proximally to the lesion.     

Structural and functional heterogeneity in an insect muscle.

Singing muscles of the katydid, Neoconocephalus robustus (Insecta, Tettigoniidae) are neurogenic, yet perform at contraction-relaxation frequencies as high as 212 Hz (Josephson and Halverson, '71). The mechanical and electrical responses of different bands of one of these muscles (the dorsal longitudinal muscle, DLM) has been examined with respect to ultrastructural features of each part which may be related to muscle performance. The DLM is composed of three bands and is innervated by four motoneurones. The cell bodies of three of these motoneurones occur ipsilaterally in the prothroracic ganglion; the cell body of the other motoneurone is contralateral in the mesothoracic ganglion. Three of the motoneurones (as yet unidentified fast axons) initiate extraordinarily fast twitches (rise time equal 7.3 msec, half duration equals 14.3 msec, 25 C), the fourth (an unidentified slower axon) evokes twitches which are considerably slower (rise time equals 18.9 msec, half duration equals 5.10 msec). Whereas the ventral and medial bands of the muscle are innervated only by fast axons (some fibers of the medial band are doubly innervated), the dorsal band is innervated by both a fast axon and the slower axon. A few fibers of the dorsal band are doubly innervated. The structure of fibers from the ventral and medial bands is very similar, with short sarcomeres (4.0 and 4.3 mum, respectively) and thin strap-like myofibrils delineated by well-developed sarcoplasmic reticulum (SR). Twenty-four percent of the volume of ventral band fibers is SR and the diffusion distance from SR to the center of the adjacent myofibril averages 0.083 mum. Twenty percent of the medial band fiber volume is SR, with a diffusion distance of 0.118 mum. Ventral and medial band fibers contain about 40% mitochondria, and 33% myofibrils. The dorsal band fibers have longer sarcomeres (9.5 mum), and only 10% of the fiber volume is SR. The muscle fibrils of the dorsal band are larger and consequently the diffusion distance is greater (0.227 mum) than in the ventral and medial bands. Mitochondria comprise 23% of the volume of dorsal band fibers. Most dorsal band mitochondria are aggregated into distinct clumps. Although some dorsal band fibers are innervated by a fast axon and some by the slower axon, the dorsal band fibers are structurally homogeneous, suggesting that neurotrophic effects are not important in maintaining the structure of dorsal band fibers. The mechanical-electrical performance and ultrastructure of the ventral and medial bands suggest their roll as fast, metabolically active but weak muscles, used in singing; the dorsal band as a slower but stronger muscle, perhaps involved in postural movements of the wing during singing.     

Influence of peripheral targets on the expression of calcitonin gene-related peptide immunoreactivity in rat cranial motoneurones

Calcitonin gene-related peptide-like immunoreactivity (CGRP-ir) is displayed by motoneurons that innervate striated muscle but is absent from preganglionic parasympathetic motoneurons. One hypothesis to explain this is that CGRP gene expression in motoneurons is, in part, dependent on influences from the innervated organ. To test this hypothesis, we cross-anastomosed the right hypoglossal and cervical vagal nerves of rats so that the vagal motoneurons grew to innervate the musculature of the tongue. Following a recovery period of 17 to 52 weeks, the distribution of CGRP-ir in the dorsal motor vagal nucleus was determined in both cross-anastomosed animals and self-anastomosed control animals. Successful reinnervation of the tongue musculature by vagal motoneurons was demonstrated by showing that electrical stimulation of the central vagus/peripheral hypoglossal nerve produced a twitch of the tongue muscles. Motoneurones of the dorsal motor vagal nucleus, which now innervated the tongue were found to express CGRP-ir, which was evident from the double labeling of neurons with both horseradish peroxidase and CGRP-ir. Motoneurones of the dorsal motor vagal nucleus contralateral to the cross-anastomosis remained CGRP negative. Similarly, motoneurons of the dorsal motor vagal nucleus in control animals where the vagus nerve was self-anastomosed remained CGRP negative, showing that an induction of CGRP expression is not a result of nerve section itself. We suggest that a signal from the striated muscle transported retrogradely via the motor axon regulates expression of CGRP-ir in motoneurons. © 1995 John Wiley & Sons, Inc.     

The role of sensory input in motor neuron sprouting control

Primary sensory neurons project to motor neurons directly or through interneurons and affect their activity. In our previous paper we showed that intramuscular sprouting can be affected by changing the sensory synaptic input to motor neurons. In this work, motor axon sprouting within a peripheral nerve (extramuscular sprouting) was induced by nerve injury at such a distance from muscle so as not to allow nerve-muscle trophic interactions. Two different procedures were carried out: (1) sciatic nerve crush and (2) sciatic nerve crush with homosegmental ipsilateral L3-L5 dorsal rhizotomy. The number of regenerating motor axons innervating extensor digitorum longus muscle was determined by in vivo muscle tension recordings and an index of their individual conduction rate was obtained by in vitro intracellular recordings of excitatory postsynaptic end-plate potentials in muscle fibers. The main findings were: (1) there are more regenerated axons distally from the lesion than parent axons proximally to the lesion (sprouting at the lesion); (2) sprouting at the lesion was negatively affected by homosegmental ipsilateral dorsal rhizotomy; (3) the number of motor axons innervating extensor digitorum longus muscle extrafusal fibers counted proximally to the lesion increased following nerve injury and regeneration but this did not occur when sensory input was lost. A transient innervation of extrafusal fibers by gamma motor neurons may explain the increase of motor axons counted proximally to the lesion.     

Independent development of sensory and motor innervation patterns in embryonic chick hindlimbs

Previous studies suggest that sensory axon outgrowth is guided by motoneurons, which are specified to innervate particular target muscles. Here we present evidence that questions this conclusion. We have used a new approach to assess the pathfinding abilities of bona fide sensory neurons, first by eliminating motoneurons after neural crest cells have coalesced into dorsal root ganglia (DRG) and second by challenging sensory neurons to innervate muscles in a novel environment created by shifting a limb bud rostrally. The resulting sensory innervation patterns mapped with the lipophilic dyes DiI and DiA showed that sensory axons projected robustly to muscles in the absence of motoneurons, if motoneurons were eliminated after DRG formation. Moreover, sensory neurons projected appropriately to their usual target muscles under these conditions. In contrast, following limb shifts, muscle sensory innervation was often derived from inappropriate segments. In this novel environment, sensory neurons tended to make more "mistakes" than motoneurons. Whereas motoneurons tended to innervate their embryologically correct muscles, sensory innervation was more widespread and was generally from more rostral segments than normal. Similar results were obtained when motoneurons were eliminated in embryos with limb shifts. These findings show that sensory neurons are capable of navigating through their usual terrain without guidance from motor axons. However, unlike motor axons, sensory axons do not appear to actively seek out appropriate target muscles when confronted with a novel terrain. These findings suggest that sensory neuron identity with regard to pathway and target choice may be unspecified or quite plastic at the time of initial axon outgrowth.     

THE FINE STRUCTURE OF MOTOR ENDPLATE MORPHOGENESIS

The fine structure of the developing neuromuscular junction of rat intercostal muscle has been studied from 16 days in utero to 10 days postpartum. At 16 days, neuromuscular relations consist of close membrane apposition between clusters of axons and groups of myotubes. Focal electron-opaque membrane specializations more intimately connect axon and myotube membranes to each other. What relation these focal contacts bear to future motor endplates is undetermined. The presence of a group of axons lying within a depression in a myotube wall and local thickening of myotube membranes with some overlying basal lamina indicates primitive motor endplate differentiation. At 18 days, large myotubes surrounded by new generations of small muscle cells occur in groups. Clusters of terminal axon sprouts mutually innervate large myotubes and adjacent small muscle cells within the groups. Nerve is separated from muscle plasma membranes by synaptic gaps partially filled by basal lamina. The plasma membranes of large myotubes, where innervated, simulate postsynaptic membranes. At birth, intercostal muscle is composed of separate myofibers. Soleplate nuclei arise coincident with the peripheral migration of myofiber nuclei. A possible source of soleplate nuclei from lateral fusion of small cells' neighboring areas of innervation is suspected but not proven. Adjacent large and small myofibers are mutually innervated by terminal axon networks contained within single Schwann cells. Primary and secondary synaptic clefts are rudimentary. By 10 days, some differentiating motor endplates simulate endplates of mature muscle. Processes of Schwann cells cover primary synaptic clefts. Axon sprouts lie within the primary clefts and are separated from each other. Specific neural control over individual myofibers may occur after neural processes are segregated in this manner.