The role of target size in neuronal survival.

A loss of about half of the trochlear motor neurons occurs during the course of normal development in duck and quail embryos. The role of the size of the target muscle in controlling the number of surviving motor neurons was examined by making motor neurons innervate targets either larger or smaller in size than their normal target. In one experiment the smaller trochlear motor neuron pool of the quail embryo was forced to innervate the larger superior oblique muscle of the duck embryo. This was accomplished by grafting the midbrain of a quail embryo in the place of the midbrain of a duck embryo. Results indicated that no additional quail trochlear motor neurons were rescued in spite of a considerable increase in target size. In another experiment the larger trochlear motor neuron pool of the duck embryo was made to innervate the smaller superior oblique muscle of the quail embryo. This resulted in loss of some additional neurons; however, the number of surviving motor neurons was not proportionate to the reduction in target size. These experiments failed to provide support for the hypothesis that the size of the target muscle controls the number of surviving motor neurons. Although contact with target is necessary for survival of neurons, factors other than the number or size of target cells are involved in the control of motor neuron numbers during development.     

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.     

Influence of altered afferent input on the number of trochlear motor neurons during development.

A loss of about half of the trochlear motor neurons occurs during the course of normal development. The present investigation was undertaken to examine the role of afferent input in regulating the number of surviving or dying trochlear motor neurons. A majority of the afferent input to the trochlear nucleus comes from the vestibular nuclei of the hindbrain via the medial longitudinal fasciculus. Portions of the hindbrain were lesioned in duck embryos on embryonic day 3, considerably prior to the time motor neurons send their axons out and cell death begins. The effectiveness of hindbrain lesion was verified by electron microscopical examination of synapses. There was a significant decrease in the number of synapses on trochlear motor neurons following hindbrain lesion. Cell counts made after the period of cell death indicated a significant decrease in the final number of surviving trochlear motor neurons. Cell counts made prior to the onset of cell death indicated that there was a drastic reduction in the initial number of trochlear motor neurons produced in hindbrain lesion embryos. In spite of a significant reduction in the initial number of neurons, the percentage loss of neurons was about the same as during normal development. Since trochlear motor neurons are generated prior to the formation of afferent synapses on them, it is unlikely that the reduction in the number of motor neurons initially produced is due to reduced afferent synaptic input. Since the percentage of cell loss in hindbrain lesion and normal embryos is about the same, it seems that the magnitude of cell death is genetically programmed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of altered afferent input on the number of trochlear motor neurons during development

A loss of about half of the trochlear motor neurons occurs during the course of normal development. The present investigation was undertaken to examine the role of afferent input in regulating the number of surviving or dying trochlear motor neurons. A majority of the afferent input to the trochlear nucleus comes from the vestibular nuclei of the hindbrain via the medial longitudinal fasciculus. Portions of the hindbrain were lesioned in duck embryos on embryonic day 3, considerably prior to the time motor neurons send their axons out and cell death begins. The effectiveness of hindbrain lesion was verified by electron microscopical examination of synapses. There was a significant decrease in the number of synapses on trochlear motor neurons following hindbrain lesion. Cell counts made after the period of cell death indicated a significant decrease in the final number of surviving trochlear motor neurons. Cell counts made prior to the onset of cell death indicated that there was a drastic reduction in the initial number of trochlear motor neurons produced in hindbrain lesion embryos. In spite of a significant reduction in the initial number of neurons, the percentage loss of neurons was about the same as during normal development. Since trochlear motor neurons are generated prior to the formation of afferent synapses on them, it is unlikely that the reduction in the number of motor neurons initially produced is due to reduced afferent synaptic input. Since the percentage of cell loss in hindbrain lesion and normal embryos is about the same, it seems that the magnitude of cell death is genetically programmed. These observations suggest that the afferent synaptic input to the trochlear motor neurons may not be as important as previously thought in regulating cell number during development. They also suggest that some afferent input, of nonsynaptic type, available at very early stages of development may specify the initial number of trochlear motor neurons produced and that a fixed percentage of that number is programmed to die during development. It is suggested that this early influence may be provided by the embryonic medial longitudinal fasciculus.     

Role of innervation on the embryonic development of skeletal muscle

Summary The extent to which the motor innervation regulates the embryonic development of skeletal muscle was investigated by comparing changes in normal, aneural, and paralyzed superior oblique muscle of the duck embryo. The muscle was made aneural by permanently destroying the trochlear motor neurons with electrocautery on day 7 i.e., three days prior to innervation. Embryos were paralyzed by daily application of -bungarotoxin onto the chorioallantoic membrane from day 10 onwards. The differentiation of myoblasts and myotubes in the aneural muscle was severely affected and did not progress to the myofiber stage. A mass of dead cells in the aneural muscle was replaced by connective tissue. Although the differentiation of myoblasts and myotubes was also retarded in the paralyzed muscle, numerous muscle cells progressed to the myofiber stage. Neuromuscular junctions of normal ultrastructure were seen in all paralyzed muscles. Degeneration of some cells in the paralyzed muscle occurred but there was no evidence of a massive wave of cell death similar to that observed in the aneural muscle. These observations suggest that both the trophic factors from the nerve and the nerve-evoked muscle activity are essential for the execution of the developmental program of the muscle. Trophic factors may play a larger role in differentiation, and maintenance of the muscle than muscle activity.Supported by a grant from the Muscular dystrophy Association and a grant from NIHWe are grateful to Beth McBride and Greg Oblak for their technical assistance     

Glial cell-line derived neurotrophic factor-dependent fusimotor neuron survival during development

Glial cell-line derived neurotrophic factor (GDNF) is a potent survival factor for motor neurons. Previous studies have shown that some motor neurons depend upon GDNF during development but this GDNF-dependent motor neuron subpopulation has not been characterized. We examined GDNF expression patterns in muscle and the impact of altered GDNF expression on the development of subtypes of motor neurons. In GDNF hemizygous mice, motor neuron innervation to muscle spindle stretch receptors (fusimotor neuron innervation) was decreased, whereas in transgenic mice that overexpress GDNF in muscle, fusimotor innervation to muscle spindles was increased. Facial motor neurons, which do not contain fusimotor neurons, were not changed in number when GDNF was over expressed by facial muscles during their development. Taken together, these data indicate that fusimotor neurons depend upon GDNF for survival during development. Since the fraction of cervical and lumbar motor neurons lost in GDNF-deficient mice at birth closely approximates the size of the fusimotor neuron pool, these data suggest that motor neuron loss in GDNF-deficient mice may be primarily of fusimotor neuron origin.     

Serial homologies of the motor neurons of the dorsal intersegmental muscles of the cockroach,Periplaneta americana (L.)

The types and locations of serially homologous motor neurons of the dorsal muscles in the cockroach Periplaneta americana remain rather constant regardless of the various adaptations of their muscles or the fusion of ganglia. However, the size and number of neurons do vary according to the development of the muscles they innervate. Neurons in four distinctive locations, two ipsisegmental and two antesegmental, innervate the dorsal longitudinal (DL) muscles in most segments. One of the ipsisegmental neurons (DLC) is common to all of the DL muscles of a segment and probably has a modulatory function. The dorsal oblique (DO) muscles of most segments have neurons in two antesegmental positions. One of these, an antesegmental, contralateral neuron, innervates both DO and DL muscles in each segment and is also probably modulatory. One neuron (DOC) of the prothoracic ganglion is the principal exception to the constancy of these serially homologous neurons. This neuron appears to be homologous to the DLC neurons of other segments but innervates the DO rather than the DL muscles.     

Appearance of high-molecular-weight acetylcholinesterase in aneural muscle developing in vivo

Acetylcholinesterase was studied in the superior oblique muscle of the duck embryo during the course of in vivo development. Normally developing, paralyzed, and uninnervated muscles were studied using velocity sedimentation for separation of various forms and biochemical determination of enzyme activity, and light and electron microscopy for histochemical and cytochemical localization of enzyme. Results indicate that neither muscle activity nor contact by the motor neurons is essential for the appearance of high-molecular-weight form of acetylcholinesterase on muscle cells developing in vivo. Acetylcholinesterase activity per muscle was considerably lower in the paralyzed and aneural muscles than the normal muscle. The absolute loss of acetylcholinesterase parallels loss of muscle protein in paralyzed and aneural muscles and may be secondary. Paralysis or absence of innervation had no significant effect on the specific activity of acetylcholinesterase.     

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.     

Mechanisms and molecules in motor neuron specification and axon pathfinding.

The vertebrate nervous system performs the most complex functions of any organ system. This feat is mediated by dedicated assemblies of neurons that must be precisely connected to one another and to peripheral tissues during embryonic development. Motor neurons, which innervate muscle and regulate autonomic functions, form an integral part of this neural circuitry. The first part of this review describes the remarkable progress in our understanding of motor neuron differentiation, which is arguably the best understood model of neuronal differentiation to date. During development, the coordinate actions of inductive signals from adjacent non-neural tissues initiate the differentiation of distinct motor neuron subclasses, with specific projection patterns, at stereotypical locations within the neural tube. Underlying this specialisation is the expression of specific homeodomain proteins, which act combinatorially to confer motor neurons with both their generic and subtype-specific properties. Ensuring that specific motor neuron subtypes innervate the correct target structure, however, requires precise motor axon guidance mechanisms. The second half of this review focuses on how distinct motor neuron subtypes pursue highly specific projection patterns by responding differentially to spatially discrete attractive and repulsive molecular cues. The tight link between motor neuron specification and axon pathfinding appears to be established by the dominant role of homeodomain proteins in dictating the ways that navigating motor axons interpret the plethora of guidance cues impinging on growth cones.     

Sexual dimorphisms in the vocal control system of a teleost fish: morphology of physiologically identified neurons

In one species of vocalizing (sonic) fish, the midshipman (Porichthys notatus), there are two classes of sexually mature males--Types I and II--distinguished by a number of traits including body size, gonad size, and reproductive tactic. The larger Type-I males (unlike Type-II males and females) build nests, guard eggs, and generate several types of vocalizations. Sound production by Type-I males is paralleled by a proportionate increase of 600% in their sonic muscle mass. The motor volley from ventral occipital roots innervating the sonic muscles establishes their contraction rate and, in turn, the fundamental frequency of emitted sounds. Electrical stimulation of the midbrain in every male and female elicited a rhythmic sonic discharge as recorded in the occipital roots; however, the fundamental frequency was slightly, but significantly, higher (20%) in Type-I males. Intracellular recording from identified motoneurons and presumed presynaptic "pacemaker" neurons showed their synaptic and action potentials had the same frequency as that of the nerve volley in every male and female. Reconstructions of physiologically identified motoneurons and pacemaker neurons following intracellular horseradish-peroxidase (HRP) filling showed their somata and dendrites to be 100-300% larger in Type-I males. These data unambiguously show that the size of a target muscle is correlated with the size of both the respective motoneurons and their presynaptic afferent neurons. As discussed, this implies that the dramatic increase in neuron size in the sonic motor system of Type-I males is causally dependent upon expansion of the sonic muscle. It is further likely that the more modest sex difference in the rhythmic central discharge is established by the intrinsic membrane properties of sonic neurons. These results also corroborate, at a number of behavioral, morphological, and neurophysiological levels, that the sonic motor system of "sneak spawning" Type-II males is similar to that of females. Thus, unlike the vocalizing Type-I males, sexual differentiation of the reproductive system in Type-II males is not linked to concomitant changes in the neurophysiological and morphological features of the sonic motor circuit.     

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.     

Neural control of embryonic acetylcholine receptor and skeletal muscle

The manner by which motor neurons exert control over the distribution and number of acetylcholine receptors, and muscle development was investigated in the superior oblique muscle of white Peking duck embryos. Clusters of receptors in the normally developing muscle first appeared on day 10 of incubation as determined with I125 alpha-bungarotoxin autoradiography. The initial appearance of receptor clusters coincided with the arrival of motor nerve fibers in the muscle. Clusters of receptors also appeared in normal fashion in muscles made aneural by destruction of motor neurons on day 7. But after day 14 these clusters had disappeared and no new clusters were seen thereafter in the aneural muscle. Receptor clusters persisted throughout development in muscle in which neuromuscular transmission was blocked with either curare or botulinum toxin and in muscles denervated on day 10.5, i.e., shortly after the initial nerve-muscle contact but prior to the onset of muscle activity. A progressive increase in the total number of receptors and in the total amount of protein occurred during the course of normal development. However, the specific activity of the receptor protein declined sharply following innervation on day 10. The total number of receptors and the specific activity of the receptor was affected depending on whether the motor neurons were destroyed before or after innervation and following chronic blockade of neuromuscular transmission. The half-life of the receptor protein was similar in normal, aneural, and paralyzed muscles (26, 25, 26 h, respectively). Measurements of total protein indicated that essentially no muscle growth occurred in the complete absence of innervation. Paralyzed muscles continued to develop but at a slower pace.     

Elimination of multiple innervation in the developing avian superior oblique muscle (1)

Motor endplates in the developing avian superior oblique muscle first appear on day 18 of incubation. Most of the endplates from this time through hatching (day 27) are innervated by multiple fibers. Each endplate in the post-hatching period is innervated by only one fiber. Time of elimination of multineuronal innervation does not correlate with the time of trochlear neuron loss; the former occurs much later in development. Removal of multiple innervation is therefore, not the cause of the naturally occurring neuron loss.     

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.     

Reinnervation of murine muscle following fetal sciatic nerve transection

A technique is reproted that permits transection of the sciatic nerve of mouse fetuses without interfering with fetal viability. Sciaticotomy was performed on Swiss Webster mice at day 17 of gestation; the contralateral side served as a control. Six weeks later the extensor digitorum longus (EDL) muscles on both sides were injected with horseradish peroxidase (HRP). Examination of the lumbar spinal cord revealed that while a substantial number of motor neurons in the region of the spinal cord giving rise to the sciatic nerve died, the EDL muscle did become reinnervated. The size of the EDL motor neuron pool on the denervated-reinnervated side was ～43% of that seen on the control side. While the control EDL motor neuron pool was located in lumbar segments L3–L5, the location of the pool to the denervated-reinnervated EDL was shifted cranially to L2–L4. Denervated-reinnervated EDL muscles were analyzed immunohistochemically to study the effect of fetal denervation on the neuronal cell adhesion molecule (N-CAM) expression. At 2 weeks postnatal, N-CAM immunoreactivity in control muscle was segregated to the motor end plate region, while fetally denervated muscle continued to express N-CAM along the length of the sarcolemma. Thus fetally denervated muscle does not develop the same pattern of N-CAM expression as normal, innervated muscle. By 6 weeks of age, the denervated-reinnervated muscle showed the same level and distribution of N-CAM immunoreactivity as did age-matched control muscle, indicating that most, if not all, of its myofibers had been reinnervated.     

Reinnervation of murine muscle following fetal sciatic nerve transection.

A technique is reported that permits transection of the sciatic nerve of mouse fetuses without interfering with fetal viability. Sciaticotomy was performed on Swiss Webster mice at day 17 of gestation; the contralateral side served as control. Six weeks later the extensor digitorum longus (EDL) muscles on both sides were injected with horseradish peroxidase (HRP). Examination of the lumbar spinal cord revealed that while a substantial number of motor neurons in the region of the spinal cord giving rise to the sciatic nerve died, the EDL muscle did become reinnervated. The size of the EDL motor neuron pool on the denervated-reinnervated side was approximately 43% of that seen on the control side. While the control EDL motor neuron pool was located in lumbar segments L3-L5, the location of the pool to the denervated-reinnervated EDL was shifted cranially to L2-L4. Denervated-reinnervated EDL muscles were analyzed immunohistochemically to study the effect of fetal denervation on the neuronal cell adhesion molecule (N-CAM) expression. At 2 weeks postnatal, N-CAM immunoreactivity in control muscle was segregated to the motor end-plate region, while fetally denervated muscle continued to express N-CAM along the length of the sarcolemma. Thus fetally denervated muscle does not develop the same pattern of N-CAM expression as normal, innervated muscle. By 6 weeks of age, the denervated-reinnervated muscle showed the same level and distribution of N-CAM immunoreactivity as did age-matched control muscle, indicating that most, if not all, of its myofibers had been reinnervated.