Inhibition of axonal morphogenesis by nonlethal, submicromolar concentrations of methylmercury.

We investigated the effects of sublethal concentrations of the neurotoxicant methylmercury (MeHg) on the developmental progression of cultured neurons to the stage of axonal morphogenesis. Chick (E8) forebrain neurons in vitro develop axons by a stereotyped developmental sequence nearly identical to that of widely used rat hippocampal neurons, but at much less cost and difficulty. In this chick forebrain system, 40% of neurons develop long axons after 2 days in culture, and 80% have axons after 4 days. A single, 2-h exposure to 0.5 or 0.25 microM MeHg reduced the number of neurons developing axons to approximately half that of controls without causing significant cell death for at least 2 days after treatment. Although MeHg caused an immediate depolymerization of neuronal microtubules, after 1 day of recovery the microtubule array of MeHg-treated neurons was indistinguishable by immunofluorescent assay from that of untreated cells at equivalent development stages. Thus, the inhibition of axonal development by submicromolar concentrations of MeHg did not appear to be the direct effect of microtubule disassembly. Chelation of Ca(2+) during MeHg exposure appeared to exert a small immediate protective effect, as previously reported, but was itself toxic within 1 day after chelation. We suggest that this inhibition of axonal morphogenesis by acute, sublethal concentrations of MeHg may play a role in the developmental syndrome caused by environmental exposure to MeHg.

Axonal outgrowth of cultured neurons is not limited by growth cone competition.

We have examined the question of scarcity-driven competition for outgrowth among growth cones of a single neuron. We measured spontaneous neurite elongation rates from 85 hours of videotape of the arbors of 31 chick sensory neurons in culture. These rate measurements were analyzed in ten minute periods that allowed cell bodies to be classified as to the number of their growth cones and the elongation to be analyzed as a series of discrete events. Comparing periods in which neurons maintained simple bipolar morphology we find no temporal competition between the two growth cones. That is, periods of above-average growth by one growth cone are not compensated by below-average growth during the same period by its sibling growth cone. Analyzing all outgrowth from a neuron based on its number of growth cones shows that net elongation rate from a single cell body is a linear function of the number of growth cones from 1 to 11. These observations suggest that growth cones behave independently and are not limited by availability of structural precursors. A surplus pool of structural precursors available for normal growth is also indicated by the high capacity for growth from single neurites when experimentally stimulated by mechanical tension. In addition, towing one or more neurites at above average rates does not cause any decline in simultaneous growth cone-mediated outgrowth from a single neuron compared to the 2-3 hour period prior to experimentally induced elongation. This high capacity for growth combined with the often observed, intermittant growth behavior of individual growth cones suggests that neurite outgrowth is intrinsically limited primarily by poor growth cone 'performance,' not scarcity-driven competition. We postulate that growth cones are poor 'tractors,' exerting too little tension to exploit the available capacity for axonal elongation.

Cytomechanics of neurite outgrowth from chick brain neurons.

Mechanical tension is a direct and immediate stimulus for neurite initiation and elongation from peripheral neurons. We report here that the relationship between tension and neurite outgrowth is equally initimate for embryonic chick forebrain neurons. Culture of forebrain neurons was unusually simple and reliable, and some of these cells undergo early events of axonal-dendritic polarity. Neurite outgrowth can be initiated de novo by experimental application of tension to the cell margin of forebrain neurons placed into culture 8-12 hours earlier, prior to spontaneous neurite outgrowth. Experimentally induced neurite elongation from these neurons shows the same robust linear relationship between elongation rate and magnitude of applied tension as peripheral neurons, i.e. both show a fluid-like growth response to tension. Although forebrain and sensory neurons manifest a similar distribution of growth sensitivity to tension (growth rate/unit tension), chick forebrain neurons initiated and elongated neurites at substantially lower net tensions than peripheral neurons. This is because, unlike peripheral neurons, there is no minimum threshold tension required for elongation in forebrain neurons; all positive tensions stimulate neurite outgrowth. Consistent with this observation, chick forebrain neurons showed weak retractile behavior in response to slackening compared to sensory neurons. Neurites that were slackened showed only transient elastic behavior and never actively produced tension, as do chick sensory neurons after slackening. We conclude that tension is an important regulator of both peripheral and central neuronal growth, but that elastic behavior is much weaker for forebrain neurons than peripheral neurons from the same developing organism. These data have significance for the understanding of the morphogenetic events of brain development.

Rac is required for growth cone function but not neurite assembly.

Recent work has suggested that rac1 and other members of the rho family of small GTP-binding proteins play an important role in the formation of neural processes. We have explored the mechanism of this effect by comparing the spontaneous, growth cone-mediated growth and experimental tension-induced growth of axons in normal PC12 cells and in mutant cells expressing a dominant negative form of rac. PC12 that have been primed by exposure to NGF, but not naive PC12 cells, initiate a microtubule-rich process de novo in response to tension applied to cell body. As in chick sensory neurons, neurite elongation rate is proportional to applied tension above a threshold. Addition of cyclic AMP, which has been shown to rapidly augment NGF-induced neurite outgrowth in PC12, causes a rapid increase in the rate of neurite elongation at a given tension level. Expression of a dominant negative form of rac1 inhibits spontaneous, growth cone-mediated neurite elongation in response to NGF, but does not substantially affect tension-induced neurite elongation. That is, rac-deficient cells show a normal linear relationship between applied tension and elongation rate and the elongations contain a normal density of axial microtubules by immunofluorescent assay. Thus, rac1 is apparently required for the mechanisms that normally generate tension in an elongating neurite, but if this tension is provided from an outside source, then axonal elongation can proceed normally in rac1-deficient cells. We conclude that rac1 is required for the adhesive and motile function of growth cones rather than the assembly of neurites per se.

The psychoanalytic group situation.

This article explores the theoretical and clinical underpinnings of a specific form of group-centered psychotherapy. In this modality, the nature of the phenomena developing in the clinical situation are related to the prevailing levels of the therapeutic regression occurring in the group. It is suggested that the most regressive phenomena tend to be experienced as groupwide phenomena with individual members assuming the position of part objects, whereas lesser degrees of regression center on the experience of whole-object relations. Five dimensions of the therapeutic situation are explored: regression, the group as a setting, the nature of anxiety, the creation of objects, and symbolization. This model allows for a differentiated exploration of therapist-centered and peer-centered transferences. A session is discussed in detail.

Osmotic dilution stimulates axonal outgrowth by making axons more sensitive to tension.

Mechanical tension is a potent stimulator of axonal growth rate, which is also stimulated by osmotic dilution. We wished to determine the relationship, if any, between osmotic stimulation and tensile regulation of axonal growth. We used calibrated glass needles to apply constant force to elongate axons of cultured chick sensory neurons. We find that a neurite being pulled at a constant force will grow 50-300% faster following a 50% dilution of inorganic ions in the culture medium. That is, osmotic dilution appears to cause axons to increase their sensitivity to applied tensions. Experimental interventions suggest that this effect is not mediated by dilution of extracellular calcium, or to osmotic stimulation of adenylate cyclase, or to osmotic stimulation of mechanosensitive ion channels. Rather, experiments measuring the static tension normally borne by neurites suggest a direct mechanical effect on the cytoskeletal proteins of the neurite shaft. Our results are consistent with a formal thermodynamic model for axonal growth in which removing a compressive load on axonal microtubules promotes their assembly, thus promoting axonal elongation.

[The epidemiology and cost of AIDS in France]. / Epidémiologie et coût du SIDA en France.

Throughout the world the AIDS epidemic continues and even seems to accelerate in regions previously thought to be untouched. The number of cases has increased by 60% in one year. With its 15,000 seropositive persons and 35,000 cases of declared AIDS, France is the most affected of all European countries. Sexual contamination remains predominant with a relative increase of heterosexual transmissions. However, particularly in the South, transmission is prevalent in intra-venous drug addicts. In all cases worldwide it is the socially deprived, marginal and fragile populations which are the most vulnerable. Various previsions are possible in France, depending on the prophylactic measures applied. In any event, the number of seropositive subjects should be levelling out at the end of this decade, and the prevalence of the disease should stabilized at between 15,000 to 20,000 patients. At present, the cost of AIDS exceeds 5.5 billion francs.

Cytomechanics of axonal development.

Mechanical tension is a robust regulator of axonal development of cultured neurons. We review work from our laboratory, using calibrated glass needles to measure or apply tension to chick sensory neurons, chick forebrain neurons, and rat PC12 cells. We survey direct evidence for two different regimes of tension effects on neurons, a fluid-like growth regime, and a nongrowth, elastic regime. Above a minimum tension threshold, we observe growth effects of tension regulating four phases of axonal development: 1. Initiation of process outgrowth from the cell body; 2. Growth cone-mediated elongation of the axon; 3. Elongation of the axon after synaptogenesis, which normally accommodates the skeletal growth of vertebrates; and 4. Axonal elimination by retraction. Significantly, the quantitative relationship between the force and the growth response is surprisingly similar to the simple relationship characteristic of Newtonian fluid mechanical elements: elongation rate is directly proportional to tension (above the threshold), and this robust linear relationship extends from physiological growth rates to far-above-physiological rates. Thus, tension apparently integrates the complex biochemistry of axonal elongation, including cytoskeletal and membrane dynamics, to produce a simple "force input/growth output" relationship. In addition to this fluid-like growth response, peripheral neurons show elastic behaviors at low tensions (below the threshold tension for growth), as do most cell types. Thus, neurites could exert small static forces without diminution for long periods. In addition, axons of peripheral neurons can actively generate modest tensions, presumably similar to muscle contraction, at tensions near zero. The elastic and force-generating capability of neural axons has recently been proposed to play a major role in the morphogenesis of the brain.

[Effect of ranitidine on blood alcohol and glucose levels after ingestion of alcohol]. / Effet de la ranitidine sur l'alcoolémie et la glycémie après ingestion d'alcool.

Are alcohol and glucose blood levels modified in fasting subjects taking ranitidine? This experience tries to simulate normal life conditions. Nine men, volunteer, aged from 24 to 29 years old, without any digestive symptoms, ate a standard lunch after five hours of fasting, took 0.35 g of alcohol per kg. Ethanol blood levels, glycemia and blood levels of insulin and glucagon were taken at regular intervals every 10 to 15 minutes during all the experiment (120 minutes). After the initial experiment, all subjects took 150 mg of ranitidine p.o. b.i.d. during seven days. Afterward they were submitted to the same protocol. Between both experiments no differences were found on blood levels of ethanol. Peak concentration, decreasing rate, and biodisponibility (estimated by area under the curve) did not change. There was a tendency to have a faster decrease in glucose blood level (p < 0.05). This study does not show any significant modification of ethanol metabolism after taking ranitidine p.o.; those results are differing from data already found with studies using cimetidine.

A cytomechanical investigation of neurite growth on different culture surfaces.

We have examined the relationship between tension, an intrinsic stimulator of axonal elongation, and the culture substrate, an extrinsic regulator of axonal elongation. Chick sensory neurons were cultured on three substrata: (a) plain tissue culture plastic; (b) plastic treated with collagen type IV; and (c) plastic treated with laminin. Calibrated glass needles were used to increase the tension loads on growing neurites. We found that growth cones on all substrata failed to detach when subjected to two to threefold and in some cases 5-10-fold greater tensions than their self-imposed rest tension. We conclude that adhesion to the substrate does not limit the tension exerted by growth cones. These data argue against a "tug-of-war" model for substrate-mediated guidance of growth cones. Neurite elongation was experimentally induced by towing neurites with a force-calibrated glass needle. On all substrata, towed elongation rate was proportional to applied tension above a threshold tension. The proportionality between elongation rate and tension can be regarded as the growth sensitivity of the neurite to tension, i.e., its growth rate per unit tension. On this basis, towed growth on all substrata can be described by the simple linear equation: elongation rate = sensitivity x (applied tension - tension threshold) The numerical values of tension thresholds and neurite sensitivities varied widely among different neurites. On all substrata, thresholds varied from near zero to greater than 200 mudynes, with some tendency for thresholds to cluster between 100 and 150 mudynes. Similarly, the tension sensitivity of neurites varied between 0.5 and 5.0 microns/h/mudyne. The lack of significant differences among sensitivity or threshold values on the various substrata suggest to use that the substratum does not affect the internal "set points" of the neurite for its response to tension. The growth cone of chick sensory neurons is known to pull on its neurite. The simplest cytomechanical model would assume that both growth cone-mediated elongation and towed growth are identical as far as tension input and elongation rate are concerned. We used the equation above and mean values for thresholds and sensitivity from towing experiments to predict the mean growth cone-mediated elongation rate based on mean rest tensions. These predictions are consistent with the observed mean values.

Tensile regulation of axonal elongation and initiation.

Neurites of chick sensory neurons in culture were attached by their growth cones to glass needles of known compliance and were subjected to increasing tensions as steps of constant force; each step lasted 30-60 min and was 25-50 mu dyn greater than the previous step. After correcting for elastic stretching, neurite elongation rate increased in proportion to tension magnitude greater than a tension threshold. The value of the tension threshold required for growth varied between 25 and 560 mu dyn, with most between 50 and 150 mu dyn. The growth sensitivity of neurites to tension was surprisingly high: an increase in tension of 1 mu dyn increased the elongation rate an average of about 1.5 microns/hr. The linear relationship between growth rate and tension provides a simple control mechanism for axons to accommodate tissue expansion in growing animals that consistently maintains a moderate rest tension on axons. Styrene microspheres treated with polyethyleneimine were used to label the surface of neurites in order to determine the site and pattern of surface addition during the experimental "towed growth" regime. New membrane is added interstitially throughout the neurite, but different regions of neurite vary widely in the amount of new membrane added. This contrasts with membrane addition specifically at the distal end in growth-cone-mediated growth. The different sites for membrane addition in growth mediated by towing and by the growth cone indicate that the membrane addition process is sensitive to the mode of growth. We confirmed the finding of Bray (1984) that neurites can be initiated de novo by application of tension to the cell margin of chick sensory neurons.(ABSTRACT TRUNCATED AT 250 WORDS)

On the cytomechanics and fluid dynamics of growth cone motility.

Following a brief review of the controversy concerning the physical mechanism of growth cone advance, we present cytomechanical data to support a version of the classic model of growth cone motility. In this model, the growth cone is pulled forward by filopodial tension. Observations of growth cone behavior and axonal guidance suggest that this model should include fluid flow mechanisms as well as the original solid, elastic mechanism. Recent data are reviewed on the similarity of the fluid behavior of cytoplasm and of suspensions of cytoskeletal filaments. The thixotropic behavior of cytoplasm is used to develop a model for lamellipodial protrusion caused by filopodial tension.

Growth cone behavior and production of traction force.

The growth cone must push its substrate rearward via some traction force in order to propel itself forward. To determine which growth cone behaviors produce traction force, we observed chick sensory growth cones under conditions in which force production was accommodated by movement of obstacles in the environment, namely, neurites of other sensory neurons or glass fibers. The movements of these obstacles occurred via three, different, stereotyped growth cone behaviors: (a) filopodial contractions, (b) smooth rearward movement on the dorsal surface of the growth cone, and (c) interactions with ruffling lamellipodia. More than 70% of the obstacle movements were caused by filopodial contractions in which the obstacle attached at the extreme distal end of a filopodium and moved only as the filopodium changed its extension. Filopodial contractions were characterized by frequent changes of obstacle velocity and direction. Contraction of a single filopodium is estimated to exert 50-90 microdyn of force, which can account for the pull exerted by chick sensory growth cones. Importantly, all five cases of growth cones growing over the top of obstacle neurites (i.e., geometry that mimics the usual growth cone/substrate interaction), were of the filopodial contraction type. Some 25% of obstacle movements occurred by a smooth backward movement along the top surface of growth cones. Both the appearance and rate of movements were similar to that reported for retrograde flow of cortical actin near the dorsal growth cone surface. Although these retrograde flow movements also exerted enough force to account for growth cone pulling, we did not observe such movements on ventral growth cone surfaces. Occasionally obstacles were moved by interaction with ruffling lamellipodia. However, we obtained no evidence for attachment of the obstacles to ruffling lamellipodia or for directed obstacle movements by this mechanism. These data suggest that chick sensory growth cones move forward by contractile activity of filopodia, i.e., isometric contraction on a rigid substrate. Our data argue against retrograde flow of actin producing traction force.

Extracellular matrix allows PC12 neurite elongation in the absence of microtubules.

Several groups have shown that PC12 will extend microtubule-containing neurites on extracellular matrix (ECM) with no lag period in the absence of nerve growth factor. This is in contrast to nerve growth factor (NGF)-induced neurite outgrowth that occurs with a lag period of several days. During this lag period, increased synthesis or activation of assembly-promoting microtubule-associated proteins (MAPs) occurs and is apparently required for neurite extension. We investigated the growth and microtubule (MT) content of PC12 neurites grown on ECM in the presence or absence of inhibitors of neurite outgrowth. On ECM, neurites of cells with or without prior exposure to NGF contain a normal density of MTs, but frequently contain unusual loops of MTs in their termini that may indicate increased MT assembly. On ECM, neurites extend from PC12 cells in the presence of 10 microM LiCl at significantly higher frequency than on polylysine. On other substrates, LiCl inhibits neurite outgrowth, apparently by inhibiting phosphorylation of particular MAPs (Burstein, D. E., P. J. Seeley, and L. A. Greene. 1985. J. Cell Biol. 101:862-870). Although 35-45% of 60 Li(+)-neurites examined were found to contain a normal array of MTs, 25-30% were found to have a MT density approximately 15% of normal. The remaining 30% of these neurites were found to be nearly devoid of MTs, containing only occasional, ambiguous, short tubular elements. We also found that neurites would extend on ECM in the presence of the microtubule depolymerizing drug, nocodazole. At 0.1 micrograms/ml nocodazole, cells on ECM produce neurites that contain a normal density of MTs. This is in contrast to the lack of neurite outgrowth and retraction of extant neurites that this dose produces in cells grown on polylysine. At 0.2 microgram/ml nocodazole, neurites again grew out in substantial number and four of five neurites examined ultrastructurally were found to be completely devoid of microtubules. We interpret these results by postulating that growth on ECM relieves the need for MTs to serve as compressive supports for neurite tension (Dennerll, T. J., H. C. Joshi, U. L. Steel, R. E. Buxbaum, and S. R. Heidemann. 1988. J. Cell Biol. 107:665). Because compression destabilizes MTs and favors disassembly, this would tend to increase MT assembly relative to other conditions, as we found. Additionally, if MTs are not needed as compressive supports, neurites could grow out in their absence, as we also observed.

The cytomechanics of axonal elongation and retraction.

Neurites of PC12 and chick dorsal root ganglion neurons behave as viscoelastic solids in response to applied forces. This passive behavior can be modeled with three mechanical elements; a relatively stiff, undamped spring in series with a Voight element composed of a less stiff spring in parallel with a dashpot. In response to applied tensions greater than 100 microdynes, PC12 cells show lengthening behavior distinct from and in addition to the passive viscoelastic response. We interpret this as "towed growth" (Bray, D. 1984. Dev. Biol. 102:379-389) because the neurites can become twice as long without obvious thinning of the neurite and because in two cases neurite tensions fell below original rest tensions, a result that cannot be obtained with passive viscoelastic elements. The rate of towed growth showed a linear dependence of growth rate with applied tensions in 8 of 12 PC12 neurites exposed to applied tension greater than 100 microdynes. Both PC12 and chick sensory neurons showed evidence of retraction when neurite tensions were suddenly diminished. This response was measured as tension recovery after slackening in chick sensory neurites. In 62% of the cases, tension recovery exceeded and sometimes doubled the preexperimental steady-state tension. Our data indicate that this response is active tension generation by the neurite shaft. We conclude that neurite length is regulated by axial tension in both elongation and retraction. Our data suggest a three-way controller: above some tension set point, the neurite is stimulated to elongate. Below some different, lower tension threshold the neurite is stimulated to retract. Between these two tension thresholds, the neurite responds passively as a viscoelastic solid.

Direct evidence that growth cones pull.

There is controversy over whether axonal elongation is the result of a pulling growth cone and the role of tension in axonal elongation. Earlier in this decade, the consensus was that axons or neurites elongated from tension generated by forward motility of the growth cone. It was presumed that contractile filopodia were the source of the tension moving the growth cone. But this view was challenged by experiments showing that neurites elongate, albeit abnormally, in the presence of cytochalasin, which inhibits growth-cone and filopodial movements. Additionally, high resolution, video-enhanced observations of growth-cone activity argued against filopodial shortening as a source of tension, suggesting instead that an extrusion of cytoplasm rather than a pulling process, is the key event in neurite elongation. Studies of slow axonal transport, however, indicate that much slower cytoskeletal pushing underlies axonal elongation. We report here direct measurements of neurite force as a function of growth-cone advance which show that they are linearly related and accompanied by apparent neurite growth. No increase in force occurs in neurites whose growth cone fails to advance.

[Professional eczema of trawlermen by contact with bryozoaires in the "baie de scine" (first French cases 1975-1977) (author's transl)]. / Eczéma professionnel du marin pêcheur par contact de bryozoaires en Baie de Seine (premiers cas Français 1975-1977).

Since 1972 in the "Baie de Seine" trawlermen have been affected with a particular eczema of hands and forearms. It is caused by repeated contact with a bryozoaire, Alcyonidium gelatinosum and patch-tests are positive. A survey of 120 trawlermen in Le Havre has shown 13 cases.

[Alcyonidium gelatinosum (L.) (Bryozoa) and cutaneous hypersensitivity reactions. Preliminary results of an experimental study]. / Alcyonidium gelatinosum (L.) (Bryozoaire) et réactions cutanées d'hypersensibilité. Résultats préliminaires d'une étude expérimentale.

The appearance of acute eczematous dermatitis in fishermen from the estuary of the Seine, about which the responsibility of Alcyonidium gelatinosum (L.) is still under discussion, led us to estimate the ability of this Bryozoa to induce cutaneous responses of delayed hypersensitivity. Intradermal tests carried out in guinea-pigs after subcutaneous, respiratory or percutaneous sensitization, gave evidence of a clear allergenic ability of this alcyonelline. The deposit of the biological product on normal skin during four consecutive days is enough to induce strong responses of delayed hypersensitivity. This experimental study needs further investigations in which the possible existence of anaphylactic reactions should be confirmed.