Target cell contact suppresses neurite outgrowth from soma–soma paired Lymnaea neurons

Neurite extension from developing and/or regenerating neurons is terminated on contact with their specific synaptic partner cells. However, a direct relationship between the effects of target cell contact on neurite outgrowth suppression and synapse formation has not yet been demonstrated. To determine whether physical/synaptic contacts affect neurite extension from cultured cells, we utilized soma–soma synapses between the identified Lymnaea neurons. A presynaptic cell (right pedal dorsal 1, RPeD1) was paired either with its postsynaptic partner cells (visceral dorsal 4, VD4, and Visceral dorsal 2, VD2) or with a non‐target cell (visceral dorsal 1, VD1), and the interactions between their neurite outgrowth patterns and synapse formation were examined. Specifically, when cultured in brain conditioned medium (CM, contains growth‐promoting factors), RPeD1, VD4, and VD2 exhibited robust neurite outgrowth within 12–24 h of their isolation. Synapses, similar to those seen in vivo, developed between the neurites of these cells. RPeD1 did not, however, synapse with its non–target cell VD1, despite extensive neuritic overlap between the cells. When placed in a soma–soma configuration (somata juxtaposed against each other), appropriate synapses developed between the somata of RPeD1 and VD4 (inhibitory) and between RPeD1 and VD2 (excitatory). Interestingly, pairing RPeD1 with either of its synaptic partner (VD4 or VD2) resulted in a complete suppression of neurite outgrowth from both pre‐ and postsynaptic neurons, even though the cells were cultured in CM. A single cell in the same dish, however, extended elaborate neurites. Similarly, a postsynaptic cell (VD4) contact suppressed the rate of neurite extension from a previously sprouted RPeD1. This suppression of the presynaptic growth cone motility was also target cell contact specific. The neurite suppression from soma–soma paired cells was transient, and neuronal sprouting began after a delay of 48–72 h. In contrast, when paired with VD1, both RPeD1 and this non‐target cell exhibited robust neurite outgrowth. We demonstrate that this neurite suppression from soma–soma paired cells was target cell contact/synapse specific and Ca²⁺ dependent. Specifically, soma–soma pairing in CM containing either lower external Ca²⁺ concentration (50% of its control level) or Cd²⁺ resulted in robust neurite outgrowth from both cells; however, the incidence of synapse formation between the paired cells was significantly reduced. Taken together, our data show that contact (physical and/or synaptic) between synaptic partners strongly influence neurite outgrowth patterns of both pre‐ and postsynaptic neurons in a time‐dependent and cell‐specific manner. Moreover, our data also suggest that neurite outgrowth and synapse formation are differentially regulated by external Ca²⁺ concentration. © 2000 John Wiley & Sons, Inc. J Neurobiol 42: 357–369, 2000     

Differential localization of ErbB receptor ensembles influences their signaling in hippocampal neurons

Our studies indicate that ErbB complexes participate in both survival and synaptic plasticity signals of hippocampal neurons but in a manner that depends on the subcellular localization of the receptor ensembles. Using dissociated hippocampal cultures, we found that neurons, rather than glial cells, are the primary targets of ErbB receptor ligands such as epidermal growth factor and heregulin. Further investigation demonstrated that ErbB receptors distribute differentially in hippocampal neurons with the epidermal growth factor receptor confined to neural cell bodies and the p185(c-neu) and ErbB4 receptors distributed to both neural soma and neurites. Activation of ErbB receptor and downstream signaling molecules were observed in neurites only after heregulin stimulation. The receptor complex which mediated neurite located signals was the p185(c-neu)/ErbB4 heterodimer. Colocalization of p185(c-neu), but not epidermal growth factor receptor, with postsynaptic density protein 95 suggests that the heregulin signaling contributes to synapse specific activities. However, the epidermal growth factor receptor complex mediates physiological survival signals, as neuronal survival was enhanced by epidermal growth factor, rather than heregulin. Collectively, these studies indicate that different ErbB ensembles localize to different locations on the neuron to mediate distinct signals and functions.     

Glutamate regulates neurite outgrowth of cultured descending brain neurons from larval lamprey

In larval lamprey, descending brain neurons, which regenerate their axons following spinal cord injury, were isolated and examined in cell culture to identify some of the factors that regulate neurite outgrowth. Focal application of 5 mM or 25 mM L-glutamate to single growth cones inhibited outgrowth of the treated neurite, but other neurites from the same neuron were not inhibited, an effect that has not been well studied for neurons in other systems. Glutamate-induced inhibition of neurite outgrowth was abolished by 10 mM kynurenic acid. Application of high potassium media to growth cones inhibited neurite outgrowth, an effect that was blocked by 2 mM cobalt or 100 microM cadmium, suggesting that calcium influx via voltage-gated channels contributes to glutamate-induced regulation of neurite outgrowth. Application of glutamate to growth cones in the presence of 2 microM omega-conotoxin MVIIC (CTX) still inhibited neurite outgrowth, while CTX blocked high potassium-induced inhibition of neurite outgrowth. Thus, CTX blocked virtually all of the calcium influx resulting from depolarization. To our knowledge, this is the first direct demonstration that calcium influx via ligand-gated ion channels can contribute to regulation of neurite outgrowth. Finally, focal application of glutamate to the cell bodies of descending brain neurons inhibited outgrowth of multiple neurites from the same neuron, and this is the first demonstration that multiple neurites can be regulated in this fashion. Signaling mechanisms involving intracellular calcium, similar to those shown here, may be important for regulating axonal regeneration following spinal cord injury in the lamprey.     

Extensive fusion of mitochondria in spinal cord motor neurons

The relative roles played by trafficking, fission and fusion in the dynamics of mitochondria in neurons have not been fully elucidated. In the present study, a slow widespread redistribution of mitochondria within cultured spinal cord motor neurons was observed as a result of extensive organelle fusion. Mitochondria were labeled with a photoconvertible fluorescent protein (mitoKaede) that is red-shifted following brief irradiation with blue light. The behavior of these selectively labeled mitochondria was followed by live fluorescence imaging. Marking mitochondria within the cell soma revealed a complete mixing, within 18 hours, of these organelles with mitochondria coming from the surrounding neurites. Fusion of juxtaposed mitochondria was directly observed in neuritic processes at least 200 microns from the cell body. Within 24 hours, photoconverted mitoKaede was dispersed to all of the mitochondria in the portion of neurite under observation. When time lapse imaging over minutes was combined with long-term observation of marked mitochondria, moving organelles that traversed the field of view did not initially contain photoconverted protein, but after several hours organelles in motion contained both fluorescent proteins, coincident with widespread fusion of all of the mitochondria within the length of neurite under observation. These observations suggest that there is a widespread exchange of mitochondrial components throughout a neuron as a result of organelle fusion.     

Effect of the weaver (wv) mutation on cerebellar neuron differentiation. I. Qualitative observations of neuron behavior in culture

A mutant gene dose-dependent inhibition of cerebellar granule cell neuron survival and neurite growth in dissociated cultures of cerebellum from 7-day-old heterozygous (+/wv) and homozygous (wv/wv) weaver mutant mice (M. Willinger, D. M. Margolis, and R. L. Sidman. (1981), J. Supramol. Struc. 17, 79-86) has previously been observed. In the present phase-contrast study time-lapse microcinematography was performed between 10 and 80 hr in culture to determine which properties of neurite growth and neuron migration are affected by weaver gene expression. Neurite growth in +/+ cultures is rapid and discontinuous. Neurites are thin and cylindrical. Membrane movement occurs only at the growth cone. Growth cone contact with cell aggregates or glial somas results in the cessation of cone advancement and the induction of translocation of the neuronal soma toward the astrocyte. In cultures of +/wv and wv/wv cerebellar cells, abnormal neurite growth is characterized by frequent neurite retractions and reinitiations. Neuronal somas and neurite shafts are motile during elongation. Homozygous neurites and cones are pleomorphic. Normal, +/wv, and wv/wv neurons undergo nuclear translocation. Like +/+ neurons, +/wv neurons migrate in response to growth cone-cell soma contact. In contrast, homozygous soma frequently reverse direction and migrate independently of cell contact. Granule cell death occurs with increasing frequency with increasing gene dosage. Neurons are unusually active prior to the rapid onset of cell death. In summary, the weaver mutation impairs granule cell differentiation by affecting neurite maintenance, membrane motility, and neuron morphology. The loss of viability appears to be independent of, or secondary to, these targets of gene action.     

Formation of calyx-like contacts preferentially on appropriate target neurons in culture

The availability of culture systems for both Edinger Westphal and ciliary ganglion neurons has made it possible to examine the interactions in culture between two populations of vertebrate neurons that synapse in vivo. In the chick, Edinger Westphal neurons provide the sole presynaptic input to the ciliary ganglion and, through this projection, are responsible for the control of lens curvature (accommodation), iris constriction, and possibly smooth muscle function in the choroid layer of the eye. When embryonic chick Edinger Westphal and ciliary ganglion neurons were combined in culture and stained for enkephalin-like immunoreactivity to visualize Edinger Westphal terminals, stained calyx-like contacts were observed that resemble the calyciform terminals formed between Edinger Westphal processes and ciliary neurons in the ciliary ganglion in vivo. Although stained calyx-like contacts could also be found in Edinger Westphal-alone and ciliary ganglion-alone cultures, many more were observed when the two cell types were cultured together. The increase depended specifically on the ciliary ganglion neurons since substitution of either dorsal root ganglion or sympathetic ganglion neurons for them in the cocultures did not increase the number of calyx-like contacts staining positive for enkephalin over those present in cultures of Edinger Westphal neurons alone. When Edinger Westphal neurons were grown simultaneously with dorsal root and ciliary ganglion neurons, calyx-like contacts with enkephalin-like immunoreactivity were found to terminate preferentially on the latter. These findings suggest that vertebrate neurons can form morphologically specific contacts preferentially on appropriate target cells in culture in the absence of many of the potential cues present in the intact tissue.     

Compositional analysis of growing axons from rat sympathetic neurons

We describe culture systems for neurons of an adrenergic autonomic ganglion which: (a) permit cultivation of neurons without supporting cells, (b) permit separate harvest of somal and axonal material, and (c) permit direct access to the neuronal surface. The antimetabolites used to suppress supporting cell growth did not have demonstrable effects on neuronal polypeptide synthesis. Rapid neurite outgrowth, which characterized these cultures, was prevented by colchicine or cycloheximide and resumed promptly after their withdrawal. Axons separated from cell bodies showed no incorporation of label from leucine or fucose, but did exhibit incorporation of glucosamine. The major polypeptides present in this neuron, as demonstrated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis, are described. No major differences in polypeptide content were observed when soma and axons were compared. Likewise, there were no differences detected in polypeptides synthesized by neurons in suspension or neurons actively extending processes. Analysis of the polypeptides within the neurites after labeling with amino acids indicated transport at a number of different rates; certain of these polypeptides corresponded in size and transport characteristics to polypeptides observed in the rabbit optic nerve after labeling of retinal ganglion cells. Tubulin and actin have been definitively identified in this cell type (18); we found proteins similar in size and proportionate amounts to be among the rapidly transported soluble polypeptides. The prominent polypeptides observed after several methods of surface labeling are described.     

Organization of motor neurons to a multiply innervated insect muscle

Nine excitatory motor neurons have been identified as innervating the locust metathoracic flexor tibiae. The anatomical organization of the flexor motor neurons within the ganglion was examined with both light and electron microscopy. Flexor motor neurons were physiologically identified prior to intracellular staining with Procion or cobalt. Some of the cobalt-stained neurons were then silver intensified. The reliability of soma location and variability of neurite branching were examined. While the position of a soma could vary within its cluster by up to one radius, the anterior, posterior, and lateral soma clusters bore a consistent relationship to each other. The density of neurite branching varied greatly for any particular flexor. The ultrastructure of the tract containing the flexor neurites revealed the individual neurites to be glial wrapped, while the tract itself was isolated from the neuropil by additional glia. The hypothesis that subsets of the flexor motor neuron pool are recruited for different behaviors is discussed in light of the last two findings.     

Interactive properties of human glioblastoma cells with brain neurons in culture and neuronal modulation of glial laminin organization

The harmonious development of the central nervous system depends on the interactions of the neuronal and glial cells. Extracellular matrix elements play important roles in these interactions, especially laminin produced by astrocytes, which has been shown to be a good substrate for neuron growth and axonal guidance. Glioblastomas are the most common subtypes of primary brain tumors and may be astrocytes in origin. As normal laminin-producing glial cells are the preferential substrate for neurons, and glial tumors have been shown to produce laminin, we questioned whether glioblastoma retained the same normal glial-neuron interactive properties with respect to neuronal growth and differentiation. Then, rat neurons were co-cultured onto rat normal astrocytes or onto three human glioblastoma cell lines obtained from neurosurgery. The co-culture confirmed that human glioblastoma cells as well as astrocytes maintained the ability to support neuritogenesis, but non-neural normal or tumoral cells failed to do so. However, glioblastoma cells did not distinguish embryonic from post-natal neurons in relation to neurite pattern in the co-cultures, as normal astrocytes did. Further, the laminin organization on both normal and tumoral glial cells was altered from a filamentous arrangement to a mixed punctuate/filamentous pattern when in co-culture with neurons. Together, these results suggest that glioblastoma cells could identify neuronal cells as partners, to support their growth and induce complex neurites, but they lost the normal glia property to distinguish neuronal age. In addition, our results show for the first time that neurons modulate the organization of astrocytes and glioblastoma laminin on the extracellular matrix.     

Enhanced Neurite Outgrowth of Human Model (NT2) Neurons by Small-Molecule Inhibitors of Rho/ROCK Signaling

Axonal injury in the adult human central nervous system often results in loss of sensation and motor functions. Promoting regeneration of severed axons requires the inactivation of growth inhibitory influences from the tissue environment and stimulation of the neuron intrinsic growth potential. Especially glial cell derived factors, such as chondroitin sulfate proteoglycans, Nogo-A, myelin-associated glycoprotein, and myelin in general, prevent axon regeneration. Most of the glial growth inhibiting factors converge onto the Rho/ROCK signaling pathway in neurons. Although conditions in the injured nervous system are clearly different from those during neurite outgrowth in vitro, here we use a chemical approach to manipulate Rho/ROCK signalling with small-molecule agents to encourage neurite outgrowth in cell culture. The development of therapeutic treatments requires drug testing not only on neurons of experimental animals, but also on human neurons. Using human NT2 model neurons, we demonstrate that the pain reliever Ibuprofen decreases RhoA (Ras homolog gene family, member A GTPase) activation and promotes neurite growth. Inhibition of the downstream effector Rho kinase by the drug Y-27632 results in a strong increase in neurite outgrowth. Conversely, activation of the Rho pathway by lysophosphatidic acid results in growth cone collapse and eventually to neurite retraction. Finally, we show that blocking of Rho kinase, but not RhoA results in an increase in neurons bearing neurites. Due to its anti-inflammatory and neurite growth promoting action, the use of a pharmacological treatment of damaged neural tissue with Ibuprofen should be explored.     

The morphology and distribution of 'Kolmer-Agduhr cells', a class of cerebrospinal-fluid-contacting neurons revealed in the frog embryo spinal cord by GABA immunocytochemistry

An immunocytochemical method that localizes GABA in glutaraldehyde-fixed tissue has been applied to the study of the Xenopus embryo spinal cord. This procedure stained an anatomical class of neuron, which had somata forming two more or less continuous rows, one on either side of the central canal, in the ventral part of the spinal cord. The total number of stained neurons in the stage 37-38 embryo spinal cord was about 300. The medial surface on the soma protruded into the central canal and had a brush border which electron microscope studies showed to consist of many microvilli or stereocilia and one or two cilia. The external end of the neuron soma had an ipsilateral ascending axon. The axon of many of these neurons had a growth cone which was also clearly stained. We propose calling these neurons 'Kolmer-Agduhr cells' after W. Kolmer and E. Agduhr who described them in the spinal cords of many vertebrate classes. Their early embryonic origin, GABA-like immunoreactivity, axonal projections and distribution as a whole population have not previously been known.     

Regeneration of an identifiable motoneuron in the crayfish. II. Patterns of reconnection and synaptic strength established in the presence of an extra nerve

The regeneration of neuromuscular connections to the superficial flexor muscle system in the crayfish has been studied under a variety of experimental manipulations. These have provided insight into the factors that can influence the regeneration program of neurons. In this work the regeneration of the largest excitor motoneuron was studied under two different conditions: (1) when the original neuron and a transplanted neuron were growing simultaneously into a denervated target, and (2) when a transplanted neuron was growing into a target that had its original nerve supply intact. In condition 1 both the transplanted and the original neuron formed normal patterns of connectivity and synaptic strength in comparable periods of time. In condition 2 the rate of growth of the transplanted neuron is significantly reduced and does not extend into the lateral fibers of the muscle. It is concluded that the regeneration program of this neuron is not affected by the presence of other neurons growing at the same time into a denervated muscle. Since regeneration is seriously affected if growth occurs into a fully innervated target area, it is suggested that lack of growth stimuli from the target or competitive interactions between established and growing synaptic terminals could influence the regeneration program of this neuron.     

Defective interfering particles modulate VSV infection of dissociated neuron cultures.

Infection of dissociated neuron cultures of mice with VSV and its defective particle DI-T was studied using fluorescent light microscopy as well as transmission and scanning electron microscopy. When cultures are infected with wild virus, VSV replicates selectively in neurons, producing cell death within 24-48 hr. Sensory and immature neurons express viral antigen most rapidly. Viral antigen and viral budding sites are detected along the neuron soma and dendrites. When large amounts of DI-T particles are added to the wild virus inoculum, viral growth is completely suppressed in mature neurons, the cell killing effects of VSV are considerably delayed and co-infected cultures survive for 5-16 days. Viral antigen accumulates in cytoplasmic inclusions and on the membrane of neuron cell somas and dendrites in the virtual absence of viral assembly. Identical modulation of VSV infection in mature neuron cultures is obtained when DI-T particles are added before or after the wild virus, but ultraviolet inactivation of DIs completely abolishes their protective effect. Immature neurons or Vero cells cannot be protected from acute cytopathic changes by an equivalent amount of DI particles. Thus DIs interfere with replication and assembly of the wild virus and attenuate cell killing effects in mature neurons in vitro.     

Growth cone-growth cone interactions in cultures of rat sympathetic neurons

Growth cones of sympathetic neurons from the superior cervical ganglia of neonatal rats were studied using video-microscopy to determine events following contact between growth cones and other cell surfaces, including other growth cones and neurites. A variety of behaviors were observed to occur upon contact between growth cones. Most commonly, one growth cone would collapse and subsequently retract upon establishing filopodial contact with the growth cone of another sympathetic neuron. Contacts resulting in collapse and retraction were often accompanied by a rapid and transient burst of lamellipodial activity along the neurite 30-50 microns proximal to the retracting growth cone. In no instances did interactions between growth cones and either fibroblasts or red blood cells result in the growth cone collapsing, suggesting that a specific recognition event was involved. On several occasions, growth cones were seen to track other growth cones, although fasciculation was rare. In some cases, there was no obvious response between contacting growth cones. Growth cone-growth cone contact was almost four times more likely to result in collapse and retraction than was growth cone-neurite contact (45% vs 12%, respectively). These observations suggest that the superior cervical ganglion may be composed of neurons with different cell surface determinants and that these determinants are more concentrated on the surface of growth cones than on neurites. These results further suggest that contact-mediated inhibition of growth cone locomotion may play an important role in growth cone guidance.     

Neurite regeneration of long-term cultured adult insect neurosecretory cells identified as DUM neurons

A distinctive group of neurons having cell bodies located along the midline of the dorsal surface of the sixth abdominal (A6) ganglion of the adult cockroach Periplaneta americana has been characterized by direct anterogradc cobalt chloride staining. These neurons identified as dorsal unpaired median (DUM) neurons, present a T-shaped morphology. The soma gives rise to a single primary neurite running anteriorly in the ganglion before dividing into two lateral neurites which run into the left and the right side of the ganglion. A characteristic dendritic arborization arises from the lateral neurites within the ganglion. This major branching pattern is mainly located at the periphery of the A6 ganglion and forms a symmetrical complicated network. A new culture procedure of these same adult DUM neurons has been developed from the dissociation of the median parts of the A6 ganglia. In our experimental conditions, we show that cultured adult DUM neurons can survive for several weeks, and regenerate a single primary neuritc dividing into two symmetrical lateral neurites with a number of fine processes radiating from the endings. This corresponds to the typical DUM neuron morphology revealed in situ on the same preparation using the cobalt chloride staining technique. This culture system developed for the first time on A6 ganglia adult DUM neurons will allow a better understanding of the physiological intracellular mechanisms involved in the neurosecretory functions of DUM neurons, which are currently unknown.     

Assessment of TTX-s and TTX-r Action Potential Conduction along Neurites of NGF and GDNF Cultured Porcine DRG Somata

Nine isoforms of voltage-gated sodium channels (NaV) have been characterized and in excitable tissues they are responsible for the initiation and conduction of action potentials. For primary afferent neurons residing in dorsal root ganglia (DRG), individual neurons may express multiple NaV isoforms extending the neuron’s functional capabilities. Since expression of NaV isoforms can be differentially regulated by neurotrophic factors we have examined the functional consequences of exposure to either nerve growth factor (NGF) or glial cell line-derived neurotrophic factor (GDNF) on action potential conduction in outgrowing cultured porcine neurites of DRG neurons. Calcium signals were recorded using the exogenous intensity based calcium indicator Fluo-8®, AM. In 94 neurons, calcium signals were conducted along neurites in response to electrical stimulation of the soma. At an image acquisition rate of 25 Hz it was possible to discern calcium transients in response to individual electrical stimuli. The peak amplitude of electrically-evoked calcium signals was limited by the ability of the neuron to follow the stimulus frequency. The stimulus frequency required to evoke a half-maximal calcium response was approximately 3 Hz at room temperature. In 13 of 14 (93%) NGF-responsive neurites, TTX-r NaV isoforms alone were sufficient to support propagated signals. In contrast, calcium signals mediated by TTX-r NaVs were evident in only 4 of 11 (36%) neurites from somata cultured in GDNF. This establishes a basis for assessing action potential signaling using calcium imaging techniques in individual cultured neurites and suggests that, in the pig, afferent nociceptor classes relying on the functional properties of TTX-r NaV isoforms, such as cold-nociceptors, most probably derive from NGF-responsive DRG neurons.     

Extracellular matrix molecules in development and regeneration of the leech CNS.

As neurons grow to their targets their processes elongate, branch and form specialized endings into which are inserted appropriate ion channels. Our aim has been to analyse the role of the extracellular matrix molecules laminin and tenascin in inducing growth and in determining the form and physiological properties of growing neurites. A preparation in which development and regeneration can be followed at the cellular and molecular level in the animal and in tissue culture is the central nervous system (CNS) of the leech. In leech extracellular matrix (ECM) both laminin and tenascin are present; the molecules are structurally similar but not identical to their vertebrate counterparts. Tenascin extracted from leech ECM shows a typical hexabrachial structure whereas laminin shows a typical cruciform structure in rotary shadowed preparations. Leech laminin purified by means of a monoclonal antibody is a molecule of about 1000 kDa, with a polypeptide composition of 340, 200, 180 and 160 kDa. Substrates that contain tenascin or laminin produce rapid and reliable outgrowth of neurites by identified cells. A remarkable finding is that the outgrowth pattern produced by an individual neuron depends in part on its identity, in part on the substrate upon which it is placed. For example, a Retzius cell grows in a quite different configuration and far more rapidly on laminin substrate than does another type of neuron containing the same transmitter (serotonin); and the pattern of outgrowth of the Retzius cell is different on laminin and on the plant lectin Con A (concanavalin A). Thus Con A induces the growth of processes that are shorter, thicker, more curved and contain fewer calcium channels than those grown on laminin. To determine whether laminin can also influence neurite outgrowth in the animal, immunocytological techniques have been used to follow its distribution in the extracellular matrix of normal, developing and regenerating leech CNS. In adult leeches neuronal processes in the CNS are not in contact with laminin which is confined to the surrounding extracellular matrix. In embryos however, laminin staining appears between ganglionic primordia along the pathways that neurons will follow. Similarly, after injury to the adult CNS, laminin accumulates at the very sites at which sprouting and regeneration begin. How the laminin becomes redistributed to appear in the region of injury has not yet been established. Together these findings suggest a key role for laminin and for other extracellular matrix molecules.(ABSTRACT TRUNCATED AT 250 WORDS)     

Variations in location and size of identified flight motoneurons in the silk moth,Bombyx mori L. (Lepidoptera : Bombycidae)

Neurons are commonly identified by some specific features. However, recent studies showed variations in identified neurons, which casts doubt on the reliability of neuron identification. This paper tests the anatomical approach that groups of neurons, which look roughly the same in different preparations, really do contain the same neurons; it also tests the reliability of motor neuron identification by cell body size and position of flight motor neurons in the silk moth, Bombyx mori (Lepidoptera : Bombycidae).Soma size and position of 9 motor neurons, which innervate the mesothoracic dorsal longitudinal muscles (DLMs), were quantitatively measured in cobalt back-filled preparations. The neurons were classified into 5 subgroups by soma size and position, and muscle innervation, although neurons in the same subgroup could not be individually identified. The soma size was essentially constant for individual neuron subgroups, but the position varied somewhat. Two subgroups were generally distributed at one position in the ganglion, but others had 2 separate soma areas, and different animals showed different distributions in these 2 areas. These results show that DLM motor neurons can be identified by the soma size and position only when the variation of soma position is examined in advance.     

Formation of contacts between mast cells and sympathetic neurons in vitro

Summary Functional interactions between mast cells and peripheral nerves may occur at sites of association seen in vivo. To study the interactions, we developed a tissue culture model of murine sympathetic neurons co-cultured with rat basophilic leukaemia (RBL-2H3) cells (homologues of mucosal mast cells) or rat peritoneal mast cells. In co-cultures of up to 3 days, light microscopy identified neurite contacts with peritoneal mast cells or RBL-2H3 cells, but not with glial cells or fibroblasts. Electron microscopy confirmed membrane-membrane contact between neurites and RBL-2H3 cells. Time-lapse analysis of interactions between neurons and RBL-2H3 cells showed that 60–100% of the cells in a given field acquired neurite contact within 17 h. In matching control studies, there was no increase in the frequency of neurite contact with cells of the rat plasmacytoma line (YB2/0): these were not selected as targets, and contacts were broken if formed. Time-lapse records of the derivation of neurites from their path suggested a neurotropic effect of mast cells, with neurite contact ensuing when the intervening distance was less than 36±4 m. Once formed, contacts were invariably maintained throughout the period of examination (up to 72 h), in contrast to YB2/0 or fibroblast contacts. We conclude that neurons selectively form and maintain connections with cells representative of rat connective tissue-type and mucosal mast cells in vitro. Similar interactions in vivo could promote nerve/mast cell contacts, which may allow bidirectional communication between the nervous and immune systems.     

Neural maintenance roles for the matrix receptor dystroglycan and the nuclear anchorage complex in Caenorhabditis elegans

Recent studies in Caenorhabditis elegans have revealed specific neural maintenance mechanisms that protect soma and neurites against mispositioning due to displacement stresses, such as muscle contraction. We report that C. elegans dystroglycan (DG) DGN-1 functions to maintain the position of lumbar neurons during late embryonic and larval development. In the absence of DGN-1 the cell bodies of multiple lumbar neuron classes are frequently displaced anterior of their normal positions. Early but not later embryonic panneural expression of DGN-1 rescues positional maintenance, suggesting that dystroglycan is required for establishment of a critical maintenance pathway that persists throughout later developmental stages. Lumbar neural maintenance requires only a membrane-tethered N-terminal domain of DGN-1 and may involve a novel extracellular partner for dystroglycan. A genetic screen for similar lumbar maintenance mutants revealed a role for the nesprin/SYNE family protein ANC-1 as well as for the extracellular protein DIG-1, previously implicated in lumbar neuron maintenance. The involvement of ANC-1 reveals a previously unknown role for nucleus-cytoskeleton interactions in neural maintenance. Genetic analysis indicates that lumbar neuron position is maintained in late embryos by parallel DGN-1/DIG-1 and ANC-1-dependent pathways, and in larvae by separate DGN-1 and ANC-1 pathways. The effect of muscle paralysis on late embryonic- or larval-stage maintenance defects in mutants indicates that lumbar neurons are subject to both muscle contraction-dependent and contraction-independent displacement stresses, and that different maintenance pathways may protect against specific types of displacement stress.