Growth of inhibitory innervation in a lobster muscle

The fine structure of inhibitory innervation to a limb muscle was examined in larval, juvenile, and adult lobsters. The innervation is essentially similar in qualitative features among these different stages, although there are some marked quantitative changes associated with growth. From being localized to discrete regions in the larval muscle, the inhibitory innervation spreads to groups of muscle fibers in the early juvenile muscle and to single fibers in the late juvenile and adult muscles. Concurrently, its neuromuscular synapses enlarge in area, become perforated, and acquire more active sites of transmitter release. Inhibitory nerve terminals occur in close proximity to their excitatory counterparts in the muscles of larval and early juvenile stages, although in later stages this juxtaposition occurs preferentially in some muscle fibers but not others. The inhibitory innervation is, nevertheless, much more restricted in occurrence than is the excitatory innervation.     

Innervation and motor patterns of the abdominal superficial flexor muscles in larval lobsters

The pattern of innervation and motor program of the abdominal superficial flexor muscle was investigated electrophysiologically in larval lobsters (Homarus americanus). The muscle receives both excitatory and inhibitory innervation in the larval as well as in the embryonic stages. Individual muscle fibers receive a single inhibitory neuron (f5) and a maximum of three excitors. Based on spike heights these axons belong to either the small (f1 or f2) or large (f3, f4) motoneurons. While the small axons preferentially innervate the medial muscle fibers the large axons innervate medial as well as lateral fibers. This larval pattern of innervation resembles the pattern in the adult lobster. The resemblance extends to the firing patterns as well with both large and small excitors firing spontaneously. Furthermore, evoked activity in the larvae produces reciprocal (and occasionally cyclical) bursts of excitor and inhibitor neurons denoting abdominal extension and flexion and resembling the firing patterns in adults. Consequently motor programs employed in steering the pelagic larvae are reminiscent of the programs for maintaining posture in the benthic adult lobsters.     

Single-cell analysis of Drosophila larval neuromuscular synapses

The neuromuscular system of Drosophila has been widely used in studies on synaptic development. In the embryo, the cellular components of this model system are well established, with uniquely identified motoneurons displaying specific connectivity with distinct muscles. Such knowledge is essential to analyzing axon guidance and synaptic matching mechanisms with single-cell resolution. In contrast, to date the cellular identities of the larval neuromuscular synapses are hardly established. It is not known whether synaptic connections seen in the embryo persist, nor is it known how individual motor endings may differentiate through the larval stages. In this study, we combine single-cell dye labeling of individual synaptic boutons and counterstaining of the entire nervous system to characterize the synaptic partners and bouton differentiation of the 30 motoneuron axons from four nerve branches (ISN, SNa, SNb, and SNd). We also show the cell body locations of 4 larval motoneurons (RP3, RP5, V, and MN13-Ib) and the types of innervation they develop. Our observations support the following: (1) Only 1 motoneuron axon of a given bouton type innervates a single muscle, while up to 4 motoneuron axons of different bouton types can innervate the same muscle. (2) The type of boutons which each motoneuron axon forms is likely influenced by cell-autonomous factors. The data offer a basis for studying the properties of synaptic differentiation, maintenance, and plasticity with a high cellular resolution.     

Common and specific inhibition in leg muscles of decapods: sharpened distinctions

Crustaceans are characteristically parsimonious in their neuromuscular innervation. In extreme instances, a single efferent axon, excitatory or inhibitory, may innervate two or more muscles that have totally different actions. In particular, the inhibitory axons of the reptantian decapod leg have been reported, in various studies within four different infraorders, to innervate anywhere from one to all seven of the leg's distal muscles and to vary in number from two to four. These axons' often inexplicable combinations of target muscles have in many cases precluded interpretation of their behavioral significance. Recent findings reviewed in this paper suggest that in fact all reptants share the same three inhibitory axons: one is a universal common inhibitor, making synaptic connections within all leg muscles; the other two are specific (single-target) inhibitors of the opener and stretcher muscles, respectively (muscles which share a single excitatory axon as their sole source of activation even though they act on different joints). The literature suggests two distinct roles in the control of limb movement for these two classes of inhibitors.     

Developmental changes in transmitter sensitivity and synaptic transmission in embryonic chicken sympathetic neurons innervated in vitro.

Dispersed neurons from embryonic chicken sympathetic ganglia were innervated in vitro by explants of spinal cord containing the autonomic preganglionic nucleus or somatic motor nucleus. The maturation of postsynaptic acetylcholine (ACh) sensitivity and synaptic activity was evaluated from ACh and synaptically evoked currents in voltage-clamped neurons at several stages of innervation. All innervated cells are more sensitive to ACh than uninnervated neurons regardless of the source of cholinergic input. Similarly, medium conditioned by either dorsal or ventral explants mimics innervation by enhancing neuronal ACh sensitivity. This increase is due to changes in the rate of appearance of ACh receptors on the cell surface. There are also several changes in the nature of synaptic transmission with development in vitro, including an increased frequency of synaptic events and the appearance of larger amplitude synaptic currents. In addition, the mean amplitude of the unit synaptic current mode increases, as predicted from the observed changes in postsynaptic sensitivity. Although spontaneous synaptic current amplitude histograms with multimodal distributions are seen at all stages of development, histograms from early synapses are typically unimodal. Changes in the synaptic currents and ACh sensitivity between 1 and 4 days of innervation were paralleled by an increase in the number of synaptic events that evoked suprathreshold activity in the postsynaptic neurons. The early pre- and postsynaptic differentiation described here for interneuronal synapses formed in vitro may be responsible for increased efficacy of synaptic transmission during development in vivo.     

Neuromuscular relationships in the abdomen of the Californian shore crab Pachygrapsus crassipes

There are two pairs of muscles in each abdominal segment of the crab; one pair of flexors and one pair of extensors. In the early larval stages the muscles have short sarcomeres--a property of fast fibers--and high thin to thick filament ratios--a property of slow fibers. In the adult the abdominal muscles are intermediate and slow, since they have fibers with intermediate and long sarcomeres, high thin to thick filament ratios, low myofibrillar ATPase activity, and high NADH diaphorase activity. The different fiber types are regionally distributed within the flexor muscle. Microelectrode recordings from single flexor muscle fibers in the adult showed that most fibers are supplied by three excitatory motor axons, although some are supplied by as many as five efferents. One axon supplies all of the flexor muscle fibers in its own hemisegment, and the evoked junctional potentials exhibit depression. This feature together with the innervation patterns of the fibers are similar to those reported for the deep flexor muscles of crayfish and lobsters. Therefore, in the adult crab, the abdominal flexor muscles have some features in common with the slow superficial flexors of crayfish and other features in common with the fast deep flexor muscles.     

Spontaneous and evoked postsynaptic potentials in an embryonic neuromuscular system of the lobster,Homarus americanus

The embryonic motor innervation to the deep extensor abdominal muscles was studied in lobster eggs in which reflex twitches and tail flips could be evoked by mechanical stimulation in early embryos. Recordings from impaled fibers during early and later stages of embryonic development revealed spontaneous depolarizing and hyperpolarizing potentials, suggesting the presence of excitatory and inhibitory axons. Stimulation of the extensor motor innervation produced a variety of EPSPs and IPSPs. The depolarizing responses included small and large EPSPs and nonovershooting spikes. Although moderate facilitation of the EPSP was sometimes observed, defacilatation was observed in the majority of fibers of all stages. Spiking could not be evoked by motor axon stimulation in embryos of early stages. These findings indicate that from the outset the deep abdominal extensor neuromuscular system of the lobster is phasic in its response to nerve stimulation and is functional as part of the tail flip reflex at least six months before hatching.     

Myopodia (postsynaptic filopodia) participate in synaptic target recognition

Synaptic partner cells recognize one another by utilizing a variety of molecular cues. Prior to neuromuscular synapse formation, Drosophila embryonic muscles extend dynamic actin-based filopodia called "myopodia." In wild-type animals, myopodia are initially extended randomly from the muscle surface but become gradually restricted to the site of motoneuron innervation, a spatial redistribution we call "clustering." Previous experiments with prospero mutant embryos demonstrated that myopodia clustering does not occur in the absence of motoneuron outgrowth into the muscle field. However, whether myopodia clustering is due to a general signal from passing axons or is a result of the specific interactions between synaptic partners remained to be investigated. Here, we have examined the relationship of myopodia to the specific events of synaptic target recognition, the stable adhesion of synaptic partners. We manipulated the embryonic expression of alphaPS2 integrin and Toll, molecules known to affect synaptic development, to specifically alter synaptic targeting on identified muscles. Then, we used a vital single-cell labeling approach to visualize the behavior of myopodia in these animals. We demonstrate a strong positive correlation between myopodia activity and synaptic target recognition. The frequency of myopodia clustering is lowered in cases where synaptic targeting is disrupted. Myopodia clustering seems to result from the adherence of a subset of myopodia to the innervating growth cone while the rest are eliminated. The data suggest that postsynaptic cells play a dynamic role in the process of synaptic target recognition.     

Expression of several adhesive macromolecules (N-CAM, L1, J1, NILE, uvomorulin, laminin, fibronectin, and a heparan sulfate proteoglycan) in embryonic, adult, and denervated adult skeletal muscle

Levels of the neural cell adhesion molecule N-CAM in muscle are regulated in parallel with the susceptibility of muscle to innervation: N-CAM is abundant on the surface of early embryonic myotubes, declines in level as development proceeds, reappears when adult muscles are denervated or paralyzed, and is lost after reinnervation (Covault, J., and J. R. Sanes, 1985, Proc. Natl. Acad. Sci. USA, 82:4544-4548). Here we used immunocytochemical methods to compare this pattern of expression with those of several other molecules known to be involved in cellular adhesion. Laminin, fibronectin, and a basal lamina-associated heparan sulfate proteoglycan accumulate on embryonic myotubes after synapse formation, and their levels change little after denervation. L1, J1, nerve growth factor-inducible large external protein, uvomorulin, and a carbohydrate epitope (L2/HNK-1) shared by several adhesion molecules are undetectable on the surface of embryonic, perinatal, adult, or denervated adult muscle fibers. Thus, of the molecules tested, only N-CAM appears on the surface of muscle cells in parallel with the ability of the muscle cell surface to accept synapses. However, four antigens--N-CAM, J1, fibronectin, and a heparan sulfate proteoglycan--accumulate in interstitial spaces near denervated synaptic sites; regenerating axons traverse these spaces as they preferentially reinnervate original synaptic sites. Of particular interest is J1, antibodies to which block adhesion of central neurons to astrocytes (Kruse, J., G. Keihauer, A. Faissner, R. Timpl, and M. Schachner, 1985, Nature (Lond.), 316:146-148). J1 is associated with collagen and other fibrils in muscle and thus may be an extracellular matrix molecule employed in both the central and peripheral nervous systems.     

A retrograde signal is involved in activity-dependent remodeling at a C. elegans neuromuscular junction

We have characterized how perturbations of normal synaptic activity influence the morphology of cholinergic SAB motor neurons that innervate head muscle in C. elegans. Mutations disrupting components of the presynaptic release apparatus, acetylcholine (ACh) synthesis or ACh loading into synaptic vesicles each induced sprouting of SAB axonal processes. These sprouts usually arose in the middle of the normal innervation zone and terminated with a single presynaptic varicosity. Sprouting SAB neurons with a similar morphology were also observed upon reducing activity in muscle, either by using mutants lacking a functional nicotinic ACh receptor subunit or through muscle-specific expression of a gain-of-function potassium channel. Analysis of temperature-sensitive mutants in the choline acetyltransferase gene revealed that the sprouting response to inactivity was developmentally regulated; reduction of synaptic activity in early larval stages, but not in late larval stages, induced both sprouting and addition of varicosities. Our results indicate that activity levels regulate the structure of certain synaptic connections between nerve and muscle in C. elegans. One component of this regulatory machinery is a retrograde signal from the postsynaptic cell that mediates the formation of synaptic connections.     

The adult abdominal neuromuscular junction of Drosophila: a model for synaptic plasticity

During its life cycle, Drosophila makes two sets of neuromuscular junctions (NMJs), embryonic/larval and adult, which serve distinct stage-specific functions. During metamorphosis, the larval NMJs are restructured to give rise to their adult counterparts, a process that is integrated into the overall remodeling of the nervous system. The NMJs of the prothoracic muscles and the mesothoracic dorsal longitudinal (flight) muscles have been previously described. Given the diversity and complexity of adult muscle groups, we set out to examine the less complex abdominal muscles. The large bouton sizes of these NMJs are particularly advantageous for easy visualization. Specifically, we have characterized morphological attributes of the ventral abdominal NMJ and show that an embryonic motor neuron identity gene, dHb9, is expressed at these adult junctions. We quantified bouton numbers and size and examined the localization of synaptic markers. We have also examined the formation of boutons during metamorphosis and examined the localization of presynaptic markers at these stages. To test the usefulness of the ventral abdominal NMJs as a model system, we characterized the effects of altering electrical activity and the levels of the cell adhesion molecule, FasciclinII (FasII). We show that both manipulations affect NMJ formation and that the effects are specific as they can be rescued genetically. Our results indicate that both activity and FasII affect development at the adult abdominal NMJ in ways that are distinct from their larval and adult thoracic counterparts     

The zebrafish ennui behavioral mutation disrupts acetylcholine receptor localization and motor axon stability

The zebrafish ennui mutation was identified from a mutagenesis screen for defects in early behavior. Homozygous ennui embryos swam more slowly than wild-type siblings but normal swimming recovered during larval stages and homozygous mutants survived until adulthood. Electrophysiological recordings from motoneurons and muscles suggested that the motor output of the CNS following mechanosensory stimulation was normal in ennui, but the synaptic currents at the neuromuscular junction were significantly reduced. Analysis of acetylcholine receptors (AChRs) in ennui muscles showed a marked reduction in the size of synaptic clusters and their aberrant localization at the myotome segment borders of fast twitch muscle. Prepatterned, nerve-independent AChR clusters appeared normal in mutant embryos and dispersed upon outgrowth of motor axons onto the muscles. Genetic mosaic analysis showed that ennui is required cell autonomously in muscle fibers for normal synaptic localization of AChRs. Furthermore, exogenous agrin failed to induce AChR aggregation, suggesting that ennui is crucial for agrin function. Finally, motor axons branched more extensively in ennui fast twitch muscles especially in the region of the myotome borders. These results suggest that ennui is important for nerve-dependent AChR clustering and the stability of axon growth.     

Studies on induction of polydactyly in rats with cytosine arabinoside.

The development of the retino-tectal projection in Rana pipiens has been studied by the intraocular injection of small amounts of [³H]proline at late embryonic and at several larval stages. After survival periods varying from 1–24 hr the distribution of the radioactively labeled proteins in the axons of the retinal ganglion cells was studied autoradiographically. It is evident from the appearance of labeled proteins in the optic nerve and chiasm at late embryonic and early larval stages that there is a rapid phase of axonal transport at these stages and that some fraction of the materials transported in this phase are distributed to the tips of the growing axons.The first retinal fibers reach the contralateral optic tectum at embryonic Stage 22; at this stage they are confined to the rostrolateral portion of the tectum where the first tectal neurons are generated. At successively later stages the fibers appear to grow across the surface of the tectum in a general rostrolateral to caudomedial direction, reaching the dorsal part of the mid-tectum at larval Stage II and the lateral part of its caudal third by Stage V. However, it is not until relatively late larval stages (XVIII) that the fibers reach the caudomedial region of the tectum, and it is only at the time of metamorphosis (Stage XXV) that the retinal projection appears to cover the entire tectum.     

Elevated levels of polyneuronal innervation persist for as long as two years in reinnervated frog neuromuscular junctions

When the nerve to an adult frog sartorius muscle is crushed, and axons are allowed to regenerate, the level of polyneuronal innervation at reinnervated neuromuscular junctions is higher than normal. With time, much of this polyneuronal innervation is reduced by the process of synapse elimination (Werle and Herrera, 1988). Using intracellular recording, we estimated the level of polyneuronal innervation in adult frog (Rana pipiens) sartorius muscles 2 years (range: 1.7-2.4 years) after crushing the sartorius nerve. We found that 27% (S.E. = 1.4%) of the junctions in muscles 2 years after reinnervation were polyneuronally innervated, whereas only 10% (S.E. = 1.2%) of the junctions in normal frog muscles were polyneuronally innervated. Thus, the synapse elimination that occurs following reinnervation does not restore the normal level of polyneuronal innervation. Histological comparisons of junctional structure between muscles 2 years after reinnervation and normal muscles revealed substantial differences. Reinnervated junctions had a greater length of synaptic gutter apposed by nerve terminal processes, more axonal inputs, more empty synaptic gutter, more instances of single synaptic gutters innervated by more than one axon, and longer lengths of nerve terminal processes that connect synaptic gutters within a junction. On the basis of this physiological and anatomical evidence, we conclude that nerve injury causes persistent changes in the pattern of muscle innervation.     

Development of the embryo, larva and early juvenile of Nile tilapia Oreochromis niloticus (Pisces: Cichlidae). Developmental staging system

We described the developmental stages for the embryonic, larval and early juvenile periods of Nile tilapia Oreochromis niloticus to elucidate sequential events of craniofacial development. Craniofacial development of cichlids, especially differentiation and morphogenesis of the pharyngeal skeleton, progresses until about 30 days postfertilization (dpf). Because there is no comprehensive report describing the sequential processes of craniofacial development up to 30 dpf, we newly defined 32 stages using a numbered staging system. For embryonic development, we defined 18 stages (stages 1-18), which were grouped into seven periods named the zygote, cleavage, blastula, gastrula, segmentation, pharyngula and hatching periods. For larval development, we defined seven stages (stages 19-25), which were grouped into two periods, early larval and late larval. For juvenile development until 30 dpf, we defined seven stages (stages 26-32) in the early juvenile period. This developmental staging system for Nile tilapia O. niloticus will benefit researchers investigating skeletogenesis throughout tilapia ontogeny and will also facilitate comparative evolutionary developmental biology studies of haplochromine cichlids, which comprise the species flocks of Lakes Malawi and Victoria.     

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.     

Motor innervation of the supercontracting longitudinal ventro-lateral muscles of the blowfly larva

The gross motor innervation of the abdominal longitudinal ventro-lateral muscles of the larva of Calliphora erythrocephala is described. Two of these muscles, 6A and 7A, are innervated by the same two multiterminally-ending axons, and thus comprise a single motor unit. No difference is found between the axon diameters in the main nerve trunks, but there is a difference where the axons run over the muscle surface. Only the dorsal, inner, surface of the muscle is innervated. Electro-physiological results show two sizes of EPSP: the large fast EPSP presumably corresponds to the thicker axon and the small slow one to the thinner axon. Preliminary work indicates that it is not possible to distinguish between the two axons with electron microscopy; the presynaptic regions possess both ‘classical’ synaptic and ‘neurosecretory’ type vesicles, and have no glial cell covering.     

Morphological effects of ciliary neurotrophic factor treatment during neuromuscular synapse elimination

In adult skeletal muscles, exogenous ciliary neurotrophic factor (CNTF) induces axons and their nerve terminals to sprout. CNTF also regulates the amount of multiple innervation in developing skeletal muscles during synapse elimination, maintaining multiple innervation of muscle fibers. While CNTF may maintain multiple innervation by regulating developmental synapse elimination, it is also possible that CNTF induces the formation of new multiple innervation through a sprouting response. In this study I examined morphologically the effects of CNTF during synapse elimination in the extensor digitorum longus (EDL) muscle. Rat pups received injections of CNTF in one leg and vehicle in the other either early [postnatal day 7 (P7)-P13] or late (P14–P20) in development. The early treatment period corresponds to that time when the pattern of innervation in the EDL is converted from predominantly multiple to single innervation. The late treatment period is at the end of synapse elimination for the EDL but corresponds to the major period of synapse elimination in the levator ani (LA), allowing a comparison of effects on these two muscles from the same animals. On the day after the final injection, EDL muscles were dissected and stained with tetranitroblue tetrazolium and the resulting pattern of innervation was assessed. The present findings indicate that only the early CNTF treatment regulates the level of multiple innervation in the EDL. Moreover, the effect of early CNTF treatment was local, affecting multiple innervation only in the EDL from the CNTF-treated leg. CNTF injected during the late treatment period had no apparent effects on the EDL but had a potent effect on the pattern of innervation in the LA, significantly increasing the level of multiple innervation in this muscle. Thus, CNTF affected multiple innervation in these two muscles only if provided during the period when single innervation normally develops. There was no evidence to indicate that CNTF induced axons or their terminals to sprout during either treatment period. In conclusion, CNTF increases the level of multiple innervation, probably by regulating synapse elimination, and skeletal muscles themselves may be an important target site for CNTF action. Presumably, the sprouting response to CNTF found in adult muscle develops sometime after P21. © 1996 John Wiley & Sons, Inc.     

Compensating for delayed hatching across consecutive life‐history stages in an amphibian

Environmental conditions experienced early in the ontogeny can have a strong impact on individual fitness and performance later in life. Organisms may counteract the negative effects of poor developmental conditions by developing compensatory responses in growth and development. However, previous studies on compensatory responses have largely ignored the effects that poor embryonic conditions could have during the later life stages. In this study, we examined the effects of artificially delayed development in early life over two later life history transitions by investigating the compensatory growth of larval moor frogs Rana arvalis in response to temperature variation during embryonic development, and the associated costs during the larval ′and postmetamorphic stages. Low temperature during embryonic stage lead to delayed hatching at smaller size. The groups with delayed embryonic development showed strong compensatory growth during the larval stage, and reached similar metamorphic size than the controls in a shorter time. However, the most strongly delayed group was not able to fully catch up the total development time. These compensatory responses were found in the absence of photoperiod cues indicating that the delay in embryonic development was sufficient to initiate the compensatory response in larval growth and development. No apparent costs of compensatory growth were detected in terms of morphology or locomotor performance at the juvenile stage. We found that compensatory responses can be activated as early as at the embryonic stage and extend over several consecutive life history transitions, mitigating the effects of poor conditions experienced early in development. Potential short‐term costs in natural environments and the occurrence of long‐term costs, which prevent the generalisation of a faster larval life style, are discussed.     

Specificity and Plasticity of Thalamocortical Connections in Sema6A Mutant Mice

The establishment of connectivity between specific thalamic nuclei and cortical areas involves a dynamic interplay between the guidance of thalamocortical axons and the elaboration of cortical areas in response to appropriate innervation. We show here that Sema6A mutants provide a unique model to test current ideas on the interactions between subcortical and cortical guidance mechanisms and cortical regionalization. In these mutants, axons from the dorsal lateral geniculate nucleus (dLGN) are misrouted in the ventral telencephalon. This leads to invasion of presumptive visual cortex by somatosensory thalamic axons at embryonic stages. Remarkably, the misrouted dLGN axons are able to find their way to the visual cortex via alternate routes at postnatal stages and reestablish a normal pattern of thalamocortical connectivity. These findings emphasize the importance and specificity of cortical cues in establishing thalamocortical connectivity and the spectacular capacity of the early postnatal cortex for remapping initial sensory representations.