Changing synaptic specificities in the nervous system of Caenorhabditis elegans: Differentiation of the DD motoneurons

During postembryonic development, the DD motoneurons in the nematode Caenorhabditis elegans completely reorganize their pattern of synapses. Ablation of a pair of embryonic precursors results in the absence of this entire class of motoneurons. In their absence animals exhibit two developmentally distinct locomotory defects. The transition period from one defect to the other is correlated with the synaptic reorganization of the DD mns. Mutations in a gene (unc-123) have been isolated that exhibit locomotory defects similar to those of the ablated adult animals. Genetic and cellular analyses of one of these alleles suggest that the unc-123 gene product may be involved in the reestablishment of functional synapses in these neurons. © 1993 John Wiley & Sons, Inc.     

Directional sensitivity of an identified wind-sensitive interneuron during the postembryonic development of the locust

Simultaneous extracellular recordings from both locust abdominal connectives show a differential activation of both bilateral homologues of an identified long projection interneuron (A4I1) in response to wind stimuli from different directions. Despite the previously shown extensive structural dynamics of sensory afferents and synaptic rearrangement of the direct afferent-to-interneuron connections during postembryonic development, a directional sensitivity is already present in first instar nymphs. Only quantitative changes in the strength of the directional response can be detected. Intracellular stainings of the A4I1 interneuron in first instar nymphs and adults show that general morphological features do not change during postembryonic development, in contrast to the presynaptic sensory afferents. This also holds for general morphological features of pleuroaxillary flight motoneurons. The output connections of A4I1 to these motoneurons and an unidentified intersegmental interneuron are already present in flightless nymphs.     

Dominant Unc-37 Mutations Suppress the Movement Defect of a Homeodomain Mutation in Unc-4, a Neural Specificity Gene in Caenorhabditis Elegans

The unc-4 gene of Caenorhabditis elegans encodes a homeodomain protein that defines synaptic input to ventral cord motor neurons. unc-4 mutants are unable to crawl backward because VA motor neurons are miswired with synaptic connections normally reserved for their sister cells, the VB motor neurons. These changes in connectivity are not accompanied by any visible effects upon neuronal morphology, which suggests that unc-4 regulates synaptic specificity but not axonal guidance or outgrowth. In an effort to identify other genes in the unc-4 pathway, we have devised a selection scheme for rare mutations that suppress the Unc-4 phenotype. We have isolated four, dominant, extragenic, allele-specific suppressors of unc-4(e2322ts), a temperature sensitive allele with a point mutation in the unc-4 homeodomain. Our data indicate that these suppressors are gain-of-function mutations in the previously identified unc-37 gene. We show that the loss-of-function mutation unc-37(e262) phenocopies the Unc-4 movement defect but does not prevent unc-4 expression or alter VA motor neuron morphology. These findings suggest that unc-37 functions with unc-4 to specify synaptic input to the VA motor neurons. We propose that unc-37 may be regulated by unc-4. Alternatively, unc-37 may encode a gene product that interacts with the unc-4 homeodomain.     

Dendritic Targeting in the Leg Neuropil of Drosophila: The Role of Midline Signalling Molecules in Generating a Myotopic Map

Neural maps are emergent, highly ordered structures that are essential for organizing and presenting synaptic information. Within the embryonic nervous system of Drosophila motoneuron dendrites are organized topographically as a myotopic map that reflects their pattern of innervation in the muscle field. Here we reveal that this fundamental organizational principle exists in adult Drosophila, where the dendrites of leg motoneurons also generate a myotopic map. A single postembryonic neuroblast sequentially generates different leg motoneuron subtypes, starting with those innervating proximal targets and medial neuropil regions and producing progeny that innervate distal muscle targets and lateral neuropil later in the lineage. Thus the cellular distinctions in peripheral targets and central dendritic domains, which make up the myotopic map, are linked to the birth-order of these motoneurons. Our developmental analysis of dendrite growth reveals that this myotopic map is generated by targeting. We demonstrate that the medio-lateral positioning of motoneuron dendrites in the leg neuropil is controlled by the midline signalling systems Slit-Robo and Netrin-Fra. These results reveal that dendritic targeting plays a major role in the formation of myotopic maps and suggests that the coordinate spatial control of both pre- and postsynaptic elements by global neuropilar signals may be an important mechanism for establishing the specificity of synaptic connections.     

Cuticle morphogenesis in crustacean embryonic and postembryonic stages

The crustacean cuticle is a chitin-based extracellular matrix, produced in general by epidermal cells and ectodermally derived epithelial cells of the digestive tract. Cuticle morphogenesis is an integrative part of embryonic and postembryonic development and it was studied in several groups of crustaceans, but mainly with a focus on one selected aspect of morphogenesis. Early studies were focused mainly on in vivo or histological observations of embryonic or larval molt cycles and more recently, some ultrastructural studies of the cuticle differentiation during development were performed. The aim of this paper is to review data on exoskeletal and gut cuticle formation during embryonic and postembryonic development in crustaceans, obtained in different developmental stages of different species and to bring together and discuss different aspects of cuticle morphogenesis, namely data on the morphology, ultrastructure, composition, connections to muscles and molt cycles in relation to cuticle differentiation. Based on the comparative evaluation of microscopic analyses of cuticle in crustacean embryonic and postembryonic stages, common principles of cuticle morphogenesis during development are discussed. Additional studies are suggested to further clarify this topic and to connect the new knowledge to related fields.     

Developmental attenuation of Manduca pre-ecdysis behavior involves neural changes upstream of motoneurons and relay interneurons

Each molt in the hawkmoth, Manduca sexta, culminates in the shedding of the old cuticle at ecdysis. Prior to each larval ecdysis, the old cuticle is loosened by pre-ecdysis behavior, which includes rhythmic, synchronous compressions in all abdominal segments. Prior to ecdysis to the pupal stage, pre-ecdysis behavior and its underlying motor pattern are markedly attenuated. A single pair of interneurons located in the terminal abdominal ganglion, the IN-402s, drives compression motoneuron activity during the pre-ecdysis motor pattern via monosynaptic excitatory connections. The present study tested the hypotheses that (1) changes in intrinsic properties (resting membrane potential, spike threshold, input resistance and excitability) of compression motoneurons, or (2) changes in the strength of synaptic connections from IN-402s to compression motoneurons, underlie the developmental attenuation of the pre-ecdysis motor pattern. Membrane potential was slightly more hyperpolarized in prepupal as compared to larval motoneurons, but no other findings supported the tested hypotheses. These results suggest that developmental weakening of the pre-ecdysis motor pattern results from changes upstream of the compression motoneurons and their synaptic connections from IN-402s. Accepted: 29 September 1999     

Elucidating the molecular mechanisms that underlie the target control of motoneuron death

Approximately half of the motoneurons generated during normal embryonic development undergo programmed cell death. Most of this death occurs during the time when synaptic connections are being formed between motoneurons and their target, skeletal muscle. Subsequent muscle activity stemming from this connection helps determine the final number of surviving motoneurons. These observations have given rise to the idea that motoneuron survival is dependent upon access to muscle derived trophic factors, presumably through intact neuromuscular synapses. However, it is not yet understood how the muscle regulates the supply of such trophic factors, or if there are additional mechanisms operating to control the fate of the innervating motoneuron. Recent observations have highlighted target independent mechanisms that also operate to support the survival of motoneurons, such as early trophic-independent periods of motoneuron death, trophic factors derived from Schwann cells and selection of motoneurons during pathfinding. Here we review recent investigations into motoneuron cell death when the molecular signalling between motoneurons and muscle has been genetically disrupted. From these studies, we suggest that in addition to trophic factors from muscle and/or Schwann cells, specific adhesive interactions between motoneurons and muscle are needed to regulate motoneuron survival. Such interactions, along with intact synaptic basal lamina, may help to regulate the supply and presentation of trophic factors to motoneurons.     

Postural interneurons in the abdominal nervous system of lobster

The nature of the synaptic relationship between 7 identified postural interneurons and 5 pairs of superficial motoneurons was examined by obtaining dual intracellular recordings from interneuron-motoneuron pairs in the lobster 2nd abdominal ganglion. For six different interneuron-motoneuron pairs EPSPs recorded from motoneurons occurred with a short (1 to 3 ms) fixed latency following each presynaptic spike recorded from the interneuron. This suggests that there is a monosynaptic relationship between these interneurons and motoneurons. Monosynaptic pathways accounted for 27% of all excitatory connections. Preliminary evidence indicates that the monosynaptic potentials are mediated by an excitatory chemical synapse since: all IPSPs occurred with latencies greater than 5 ms, there was no evidence for electrical coupling, and one of the interneurons produced facilitating PSPs. A majority of all monosynaptic connections were made by two of the flexion producing interneurons (FPIs), 201 and 301. The synaptic outputs of these FPIs were similar in that both made monosynaptic connections with a different bilaterally homologous pair of motoneurons. Both also produced larger EPSPs and more vigorous spiking in contralateral members of the bilateral motoneuron pairs. A previous study demonstrated that interneurons 201 and 301 are the only postural interneurons yet identified that express motor programs indistinguishable from command neurons. Taken together, these results suggest that certain intersegmental interneurons share properties with command neurons and driver neurons, and that there may not be a sharp morphological or functional distinction between these two cell types.     

Copulation in C. elegans males requires a nuclear hormone receptor

In Caenorhabditis elegans, uncoordinated (unc)-55 encodes a nuclear hormone receptor that is necessary for coordinated movement and male mating. An unc-55 reporter gene revealed a sexually dimorphic pattern: early in post-embryonic motor neurons in both sexes; and later in a subset of male-specific cells that included an interneuron and eight muscle cells. A behavioral analysis coupled with RNA interference (RNAi) revealed that males require UNC-55 to execute copulatory motor programs. Two mRNA isoforms (unc-55a and unc-55b) were detected throughout post-embryonic development in males, whereas only one, unc-55a, was detected in hermaphrodites. In unc-55 mutant males isoform a rescued the locomotion and mating defect, whereas isoform b rescued the mating defect only. Isoform b represents the first report of male-specific splicing in C. elegans. In addition, isoform b extended the number of days that transgenic unc-55 mutant males mated when compared to males rescued with isoform a, suggesting an anabolic role for the nuclear hormone receptor. The male-specific expression and splicing is part of a regulatory hierarchy that includes two key genes, male abnormal (mab)-5 and mab-9, required for the generation and differentiation of male-specific cells. We suggest that UNC-55 acts as an interface between genes involved in male tail pattern formation and those responsible for function.     

Motor pattern generation of the posterior cardiac plate-pyloric system in the stomatogastric ganglion of the mantis shrimp Squilla oratoria

Activity patterns of the constituent neurons of the posterior cardiac plate-pyloric system in the stomatogastric ganglion of the mantis shrimp Squilla oratoria were studied by recording spontaneous burst discharges intracellularly from neuronal somata. These neurons were identified electrophysiologically, and synaptic connections among them were qualitatively analysed. The posterior cardiac plate constrictor, pyloric constrictor, pyloric dilator and ventricular dilator motoneurons, and the pyloric interneuron were involved in the posterior cardiac plate-pyloric system. All the cell types could produce slow burst-forming potentials which led to repetitive spike discharges. These neurons generated sequentially patterned outputs. Most commonly, the posterior cardiac plate neuron activity was followed by the activity of pyloric constrictor neurons, and then by the activity of pyloric dilator/pyloric interneuron, and ventricular dilator neurons. The motoneurons and interneuron in the posterior cardiac plate-pyloric system were connected to each other either by electrical or by inhibitory chemical synapses, and thus constructed the neural circuit characterized by a wiring diagram which was structurally similar to the pyloric circuit of decapods. The circuitry in the stomatogastric ganglion was strongly conserved during evolution between stomatopods and decapods, despite significant changes in the peripheral structure of the foregut. There were more electrical synapses in stomatopods, and more reciprocal inhibitory synapses in decapods.Abbreviations EJP excitatory junctional potential - IPSP inhibitory postsynaptic potential - CoG commissural ganglion - CPG central pattern generator - ion inferior oesophageal nerve - OG oesophageal ganglion - pcp posterior cardiac plate - son superior oesophageal nerve - STG stomatogastric ganglion - stn stomatogastric nerve - PY pyloric constrictor - PD pyloric dilator - VD ventricular dilator - AB pyloric interneuron - lvn lateral ventricular nerves - tcpm transverse cardiac plate muscle     

Programmed cell death in the embryonic central nervous system of Drosophila melanogaster

Although programmed cell death (PCD) plays a crucial role throughout Drosophila CNS development, its pattern and incidence remain largely uninvestigated. We provide here a detailed analysis of the occurrence of PCD in the embryonic ventral nerve cord (VNC). We traced the spatio-temporal pattern of PCD and compared the appearance of, and total cell numbers in, thoracic and abdominal neuromeres of wild-type and PCD-deficient H99 mutant embryos. Furthermore, we have examined the clonal origin and fate of superfluous cells in H99 mutants by DiI labeling almost all neuroblasts, with special attention to segment-specific differences within the individually identified neuroblast lineages. Our data reveal that although PCD-deficient mutants appear morphologically well-structured, there is significant hyperplasia in the VNC. The majority of neuroblast lineages comprise superfluous cells, and a specific set of these lineages shows segment-specific characteristics. The superfluous cells can be specified as neurons with extended wild-type-like or abnormal axonal projections, but not as glia. The lineage data also provide indications towards the identities of neuroblasts that normally die in the late embryo and of those that become postembryonic and resume proliferation in the larva. Using cell-specific markers we were able to precisely identify some of the progeny cells, including the GW neuron, the U motoneurons and one of the RP motoneurons, all of which undergo segment-specific cell death. The data obtained in this analysis form the basis for further investigations into the mechanisms involved in the regulation of PCD and its role in segmental patterning in the embryonic CNS.     

The origin of postembryonic neuroblasts in the ventral nerve cord of Drosophila melanogaster

Embryonic and postembryonic neuroblasts in the thoracic ventral nerve cord of Drosophila melanogaster have the same origin. We have traced the development of threefold-labelled single precursor cells from the early gastrula stage to late larval stages. The technique allows in the same individual monitoring of progeny cells at embryonic stages (in vivo) and differentially staining embryonic and postembryonic progeny within the resulting neural clone at late postembryonic stages. The analysis reveals that postembryonic cells always appear together with embryonic cells in one clone. Furthermore, BrdU labelling suggests that the embryonic neuroblast itself rather than one of its progeny resumes proliferation as a postembryonic neuroblast. A second type of clone consists of embryonic progeny only.     

Voltage-gated calcium channels direct neuronal migration in Caenorhabditis elegans

Calcium signaling is known to be important for regulating the guidance of migrating neurons, yet the molecular mechanisms underlying this process are not well understood. We have found that two different voltage-gated calcium channels are important for the accurate guidance of postembryonic neuronal migrations in the nematode Caenorhabditis elegans. In mutants carrying loss-of-function alleles of the calcium channel gene unc-2, the touch receptor neuron AVM and the interneuron SDQR often migrated inappropriately, leading to misplacement of their cell bodies. However, the AVM neurons in unc-2 mutant animals extended axons in a wild-type pattern, suggesting that the UNC-2 calcium channel specifically directs migration of the neuronal cell body and is not required for axonal pathfinding. In contrast, mutations in egl-19, which affect a different voltage-gated calcium channel, affected the migration of the AVM and SDQR bodies, as well as the guidance of the AVM axon. Thus, cell migration and axonal pathfinding in the AVM neurons appear to involve distinct calcium channel subtypes. Mutants defective in the unc-43/CaM kinase gene showed a defect in SDQR and AVM positioning that resembled that of unc-2 mutants; thus, CaM kinase may function as an effector of the UNC-2-mediated calcium influx in guiding cell migration.     

Mutations in the Drosophila neuroglian cell adhesion molecule affect motor neuron pathfinding and peripheral nervous system patterning

We have identified and characterized three embryonic lethal mutations that alter or abolish expression of Drosophila Neuroglian and have used these mutations to analyze Neuroglian function during development. Neuroglian is a member of the immunoglobulin superfamily. It is expressed by a variety of cell types during embryonic development, including expression on motoneurons and the muscle cells that they innervate. Examination of the nervous systems of neuroglian mutant embryos reveals that motoneurons have altered pathfinding trajectories. Additionally, the sensory cell bodies of the peripheral nervous system display altered morphology and patterning. Using a temperature-sensitive mutation, the phenocritical period for Neuroglian function was determined to occur during late embryogenesis, an interval which coincides with the period during which neuromuscular connections and the peripheral nervous system pattern are established. © 1997 John Wiley & Sons, Inc. J Neurobiol 32: 325–340, 1997     

Hexamethonium sensitivity of the swim musculature of the pteropod mollusc, <Emphasis Type="Italic">Clione limacina</Emphasis>

Swimming in reduced electrophysiological preparations of the pteropod mollusc, Clione limacina, was blocked by bath application of hexamethonium even though pattern generator activity continued with this treatment. Neuromuscular recordings indicated that hexamethonium blocked synaptic input from Pd-3 and Pd-4 motoneurons to slow-twitch muscle cells, while connections from Pd-1A and Pd-2A motoneurons to fast-twitch muscle cells were variable in their response to hexamethonium—synaptic inputs were suppressed in most cases and occasionally blocked, but the latter only with high concentrations and long incubations. Acutely dissociated wing muscle cells showed a concentration-dependency in the percentage of contracted cells with bath application of acetylcholine, and this contractile activity was blocked in preparations that were first bathed in hexamethonium. Intracellular recordings from dissociated slow-twitch muscle cells showed conductance-increase depolarizations of approximately 20 mV following 1 s pressure ejections of 10⁻⁴ M acetylcholine from micropipettes placed immediately adjacent to the muscle cells. These responses were blocked when hexamethonium was bath applied prior to the pressure-applied acetylcholine. The results suggest the Pd-3/Pd-4 motoneuron to slow-twitch muscle cell junctions are cholinergic with nicotinic-like receptors, while the Pd-1A/Pd-2A to fast-twitch muscle cell connections are likely cholinergic, but with a different receptor type.     

A neural degeneration mutation that spares primary neurons in the zebrafish

We describe an embryonic lethal mutation in the zebrafish Brachydanio rerio that specifically affects the viability of most cells in the embryonic central nervous system (CNS). The mutation ned-1 (b39rl) was induced with gamma-irradiation and segregates as a single recessive allele closely linked to its centromere. It produces massive cell death in the CNS but a small set of specific neurons, including Rohon-Beard sensory neurons, large hindbrain interneurons, and primary motoneurons, survive embryogenesis and are functional. Synaptic connections between embryonic motoneurons and muscle cells appear physiologically normal, and the normally observed spontaneous flexions are present. Correlated with the presence of sensory neurons and interneurons, mutant embryos display reflexive movements in response to mechanical stimulation. Together, the surviving neurons, called primary neurons, form a class of cells that are prominent in size and arise early during development. Thus, this mutation may define a function that is differentially required by developmentally distinguishable sets of cells in the embryonic CNS.     

Regeneration of central and peripheral synaptic connections in the locomotor system of the pteropod molluscClione limacina

The neural network underlying rhythmic wing movements in the molluscClione limacina is well-studied. Two different groups of motoneurons innervate two distinct groups of wing muscles. The locomotor rhythm generated in the left and right pedal ganglia is synchronized by interneurons. When the axons of the locomotor motoneurons are crushed, numerous fine neurites sprout towards the denervated muscles and reach them in 8–15 days. At this stage motoneurons project to and synapse on not only correct but equally incorrect muscle targets. After 2 weeks of regeneration the number of incorrect neurites and synaptic connections begins to decrease and following 1.5–2 months all incorrect connections are eliminated, incorrect axons are withdrawn and the behavioral deficit is compensated. In this study the regeneration of interneurons and the growth profiles of inter- and motoneurons were also studiedin vitro. Two individually isolated pedal ganglia were co-cultured in three different configurations: a) the wing nerve stump from one ganglion was fixed against the commissural stump from another ganglion; b) the wing nerve stumps were fixed against each other; c) the commissural stumps were fixed against each other. Under the above experimental conditions we found that the interneurons were able to cross only the contact between two commissural stumps, and in this case found their original targets, restored correct connections and synchronized the rhythm in two pedal ganglia. In contrast, motoneurons were able to cross all types of contacts.