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Uptake of the thymidine analogue 5-bromo-2-deoxyuridine into non-neuronal cells of the insect central nervous system has been examined following a controlled lesioning of the glial elements. The pattern of BUdR labelling along the penultimate abdominal connective was examined over a period of 17 days. Cell proliferation occurred in and immediately around the site of damage in both perineurial and subperineurial glial cells but at different times post-lesion for the two regions. Proliferation in the perineurial zone was maximal at 6-8 days post-lesion but continued for at least 17 days. Subperineurial proliferation was less dramatic and peaked between days 8-11 post-lesion. In both areas division appears to be confined to the reactive glial cells. These results are discussed in the context of past research on this system, particularly with regard to the restoration of the blood-brain barrier.

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Glial cells from an adult insect, Periplaneta americana, have been grown in neurone-free cultures. No growth occurred from freshly-excised fragments of abdominal nervous connectives. Vigorous growth was obtained, however, from explants of connectives induced to proliferate by prior exposure to a toxin, ethidium bromide, applied selectively to glial cells in vivo. Glial growth in vitro is dependent upon the initiation of early stages of repair in vivo: this supports the idea that haemocytes which invade the lesion zone immediately after damage are involved in directing proliferation of perineurial and sub-perineurial glia. In contrast, both glial and neuronal cells grew in vitro from explanted abdominal ganglia without prior glial lesioning, indicating that different factors may determine cellular regeneration in this domain. The morphology of the proliferating cells was influenced by the substrate; extensive glial migration was restricted to areas of close contact between cell and substrate surface.

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Selective disruption of the neuroglia in penultimate abdominal connectives of the cockroach nerve is followed by a rapid accumulation of cells in the perineurial layer of the lesion. Subsequently, there is an abrupt, secondary, rise in cell numbers in the undamaged perineurial tissues, anterior to the lesion and adjacent to the 4th abdominal ganglia. By 7 days the increased cell numbers are again effectively confined to the original lesion zone. The initial rise in cell numbers is postulated to result from an invasion by blood-borne haemocytes and the subsequent increase, in undamaged perineurial tissues, from the mobilization of endogenous reactive cells. Recruitment of the endogenous cells is inhibited if the haemocytes are excluded from the lesion. There is a slower mobilization of sub-perineurial cells, which, again, is inhibited following exclusion of haemocytes from the lesion zone. It is postulated that the recruitment of the endogenous reactive cells is initiated by the invading haemocytes which transform to granule-containing cells and release diffusible morphogenic and/or mitogenic factors.

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As in other repairing systems, glial regeneration in insect central nervous connectives, following selective chemical lesioning, involves both exogenous and endogenous elements. Our current evidence, including that obtained with monoclonal antibodies, indicates that the reactive, granule-containing cells are derived from a sub-population of circulating haemocytes which, within 24 h, invade, and are restricted to, the lesion zone. The granule-containing cells are involved in the initial repair of the perineurial region. They also contribute to the first stage in the restoration of the blood-brain barrier and are responsible for recruiting reactive endogenous glia, apparently from the vicinity of the anterior abdominal ganglion. The granule-containing cells transform into or are replaced by functional glia between 3 and 5 days after selective glial disruption, coincident with the appearance in the lesion zone of dividing reactive cells.

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The repair of cockroach central nervous connectives, following selective glial disruption, involves an initial invasion of the lesion by a novel cell class. The available evidence, including that obtained using monoclonal antibodies, shows that these cells arise from circulating haemocytes. These invasive exogenous cells are restricted to the lesion zone. They are not only involved in initial repair of the peripheral glial elements, but may also be responsible for initiating recruitment and division of endogenous reactive cells. There is a clear anterior polarity in this recruitment, with significantly higher numbers of cells appearing anterior to, and then within, the lesion area. Characteristically, recognizable exogenous cells decline in number after 3 days, although there is no overall reduction in cell numbers within the lesion at this stage, nor has significant cell division begun. This suggests that the haemocyte-derived cells transform into, or are replaced by, functional perineurial glia, between 3 and 5 days, coincident with the restoration of the blood-brain barrier and the onset of endogenous cell division. Glial repair in the insect CNS can thus be divided into three phases which show striking similarities to the repair sequence in vertebrate brain. These include: an initial invasion of the lesion by exogenous cells, subsequent glial proliferation and then longer term fluxes in cell numbers and distribution.

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Using antibodies specific for haemocytes, we have shown that these blood cells penetrate the abdominal nervous connectives of the cockroach following selective disruption of the glia using the DNA-intercalating drug, ethidium bromide, as a glial toxin. Within 4 days post-lesion, the labelled cells formed a mosaic beneath the neural lamella and penetrated deeply among the disrupted subperineurial glia. These observations confirm that exogenous cells are involved in glial repair and support a previous hypothesis that they play critical roles in both structural repair and the recruitment of endogenous reactive cells.

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Octopamine caused only a slight reduction in the potential across the perineurial glia of the cockroach, had no effect upon sodium-induced changes in potential, but did reduce potassium-induced changes (at 10(-7) M and above). The effect of 10(-7) M octopamine was accompanied by a rise in resistance, was mimicked by 10(-7) M synephrine and blocked by 10(-6) M phentolamine. Transperineurial potassium permeability was reduced by 10(-6) M octopamine. It is concluded that octopamine receptors mediate a reduction in potassium conductance of the basolateral membrane of these glia, and a reduction in the net potassium permeability of the barrier.

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Surgical lesioning of central nervous connectives in the cockroach (Periplaneta americana (L.], although causing only local glial damage, resulted in complex and prolonged cellular changes. An early response to mechanical disruption was the appearance of granule-containing cells within the damaged perineurium, among adjacent, undamaged, perineurial cells, and between glial processes deep within the connectives. These cells, which were strikingly similar to hemocytes, were clearly involved in phagocytic activity and persisted in the damaged regions for more than a month after lesioning. There was only a slow restoration of organized perineurial glia and re-establishment of the blood-brain barrier, as indicated by the exclusion of an extracellular tracer, ionic lanthanum. These observations contrast with the speedy, ordered repair of the neuroglia observed following selective glial disruption and suggest that undamaged axons and/or the extracellular matrix exert a profound influence on the mechanisms of glial repair.

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In vivo application of ethidium bromide to cockroach central nervous connectives caused extensive disruption of the neuroglia within 24 hr. Axonal conduction persisted following treatment with the glial toxin. A consistent feature of glial damage and repair was the prominent involvement of granule-containing cells. These cells (which were never seen in control cords) shared a number of cytological features with hemocytes that were seen adhering to and penetrating the neural lamella, in the early stages of glial damage. The granule-containing cells appear to serve dual functions: phagocytosis and structural repair. After 48 hr, granule-containing cells, or their processes, formed layers at the periphery of the connectives. By 4 to 6 days after treatment, the peripheral cells had assumed the morphological characteristics of normal perineurial cells and by 28 days were indistinguishable, ultrastructurally, from those of the perineurium of normal, untreated animals. These structural changes paralleled the re-establishment of the normal permeability properties of the blood-brain interface revealed by the exclusion of an extracellular tracer, ionic lanthanum, and electrophysiological observations.

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The effects of lesions on cockroach giant interneurones (GIs) were studied to investigate the transient, Ca-dependent, action potentials which have been reported to appear near the tips of regenerating GIs (Meiri, Spira & Parnas, 1981). We were unable to repeat these observations. Ligation of connectives, before cutting, delayed the degenerative changes in segments of GIs that were separated from their cell bodies. The short-term injury response (which was normally observed on cutting while recording close to the site of lesion) was not recorded when the axon was ligated. The effect of cutting GIs at different distances from their cell bodies was also investigated. There was no apparent relation between the response of GIs to a lesion and the site of the lesion. Sprouting of GIs was observed both at the tip of the axon and also at sites some distance from the lesion. No changes of the cell body or its dendritic tree in the terminal ganglion were seen but sprouting was observed in other ganglia.

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The efflux of radiocations (22Na, 2K and 45Ca) and of radiochloride occur as two-stage processes from intact cockroach nerve cords. It is suggested that the initial, fast fraction of efflux comes mainly from the superficial connective tissue layer, the neural lamella, and the clefts between the underlying layer of neuroglia, the perineurium. This is deduced from the lack of effect of a metabolic inhibitor and sodium-transport inhibitors on the fast component of 22Na efflux (which contrast with their effects both on the size an the half-time of the slow component) and from the typically extracellular ratios between the fast components of substantial increase in the fast fractions of 22Na and 45Ca efflux but only a small increase in 36Cl efflux: effects which would be expected if the addition to the fast fraction consisted of ions maintained in Donnan equilibrium with fixed anionic sites within the extracellular system. The presence of such anionic sites is also indicated by lanthanum-binding in the extracellular matrix and by the previous histochemical demonstration of hyaluronic acid in the matrix by Ashhurst and Costin. It is suggested that the anionic glycosaminoglycans provide an extracellular cation reservoir which could serve a role in short-term ionic homeostasis of the brain microenvironment.

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Field observations on the ocean skater, Halobates robustus Barber showed that the flotillas of adults, unlike those of juveniles, occurred very close to the shore at specific sites from which they were not easily dislodged.The adult flotillas consisted of small all-male groups or aggregations of males and copulating pairs. The proportion of free males in copulating flotillas decreased with increasing group size. There was no correlation between the sizes of the paired males and females.Single adult females, which were not observed in the copulating flotillas, were usually teneral with undeveloped eggs. In contrast, the copulating females contained numerous well-developed eggs. The pairs remained in prolonged genital contact.The behaviour of free males in copulating flotillas was characterized by frequent, non-random encounters with copulating pairs. Experiments with marked individuals established that male take-overs can occur within flotillas.It is concluded that the copulating flotillas are initiated by the males and not only provide protection from predators, as previously shown, but also act as arenas for mate finding and selection. It is presumed that prolonged copulation reduces sperm competition between contending males.

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Extracellular ionic homeostasis in an insect central nervous system involves a peripheral intercellular diffusion barrier, an extracellular matrix and neuroglial cation transport. The peripheral location of the barrier in the superficial neuroglia is confirmed by intracellular recording from glial cells identified by peroxidase injection. This barrier protects the underlying neurones from large changes in ionic composition of the blood-plasma, but renders them more susceptible to fluctuations in ion composition resulting from neuronal signalling within the very restricted extracellular system. Because of the peripheral intercellular barrier, sodium movements between the blood and the extracellular fluid are largely transcellular and are mediated by ion pumps on the perineurial and underlying glial membranes. It is suggested that the homeostatic role of the neuroglial ion pumps is augmented by an anion matrix which functions as an extracellular sodium reservoir. It is proposed that during depletion of extracellular sodium, this cation is released by the matrix to maintain the sodium activity in the fluid at the axon surfaces.

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Intracellular recordings demonstrated a transfer of impulses between the paired giant axons of Sabella, apparently along narrow axonal processes contained within the paired commissures which link the nerve cords in each segment of the body. This transfer appears not to be achieved by chemical transmission, as has been previously supposed. This is indicated by the spread of depolarizing and hyperpolarizing voltage changes between the giant axons, the lack of effects of changes in the concentrations of external divalent cations on impulse transmission and by the effects of hyperpolarization in reducing the amplitude of the depolarizing potential which precedes the action potentials in the follower axon. The ten-to-one attenuation of electronic potentials between the giant axons argues against the possibility of an exclusively passive spread of potential along the axonal processes which link the axons. Observation of impulse traffic within the nerve cord commissures indicates, on the other hand, that transmission is achieved by conduction of action potentials along the axonal processes which link the giant axons. At least four pairs of intact commissures are necessary for inter-axonal transmission, the overall density of current injected at multiple sites on the follower axon being, it is presumed, sufficient to overcome the reduction in safety factor imposed by the geometry of the system in the region where axonal processes join the giant axons. The segmental transmission between the giant axons ensures effective synchronization of impulse traffic initiated in any region of the body and, thus, co-ordination of muscular contraction, during rapid withdrawal responses of the worm.

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The following three aspects of the biology of the marine insect Halobates robustus were studied, during a two week observation period, at several sites in the Galapagos Islands: distribution, aggregation behaviour and rhythmicity of locomotory activity. H. robustus occurred in highest numbers on the water surface at shores fringed with mangroves. The aggregations of H. robustus varied according to their location and density. Copulating adults formed dense, floating aggregations, which tended to be close to rocks or mangroves. Late instar nymphs were less aggregated and, in lagoons (where there was some shelter from direct tidal forces), were furthest from the shore. In two types of habitat (mangrove-fringed, sandy shores and rocky shores) the aggregations of H. robustus showed a pronounced ability to maintain a floating station in relation to the surrounding environment, irrespective of tidal movements (in one case at 34 m from the nearest fixed objects). Evidence of the ability of the aggregations to maintain station on the water surface was also obtained by comparing the movements of H. robustus with those of floating polystyrene particles, which move passively with wind and tide. Laboratory observations and experiments indicated no clear periodicity in locomotory movements throughout a 24 h period. However, the frequency of encounters between individuals showed two daily peaks, post-dawn and pre-dusk, with fewer encounters during the day and only occasional encounters during the night. By shifting the light-dark cycle it was demonstrated that the daily bimodal rhythm of encounters is triggered by dawn and, since it is not maintained in constant light or dark, an 'hour-glass' mechanism is suggested. The contributions of single adults, of copulating pairs and of immature stages to the overall pattern of activity were also determined. Immature stages did not affect the overall rate of encounters significantly and the interactions between single and copulating pairs of adults appear to have been responsible for the bimodal pattern.

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Freeze-fractured axonal membrane surfaces from the connectives of Mytilus edulis show an increment in particle frequency of 52% (fixed tissues) or 68% (unfixed tissues) after long-term adaptation to low salinity. Particle size distribution was unaffected by osmotic adaptation, but was significantly different in fixed and unfixed material. The possibility that these structural changes reflect the known increase in sodium pump frequency in this osmoconforming tissue is considered.

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On a marine saltmarsh, the intertidal mite, Bdella interrupta Evans, is exposed to periods of regular tidal submergence which alternate with periods of tidal emergence. The mite shows well defined day and night peaks of locomotory activity on the soil surface. During periods of tidal submergence the activity peaks showed an apparent periodicity, of around 12.5 h, characteristic of a tidal rhythm. In the presence of non-submerging tides the periodicity of the activity peaks changed to around 11.5 h. It is suggested that this shorter periodicity results either from free-running of the circatidal clock in the absence of entraining tides or from a sequence of transients that are phaseshifting towards a stable relationship with a second zeitgeber. It is further suggested that the alternation between a ca. 12.5 h and a ca. 11.5 h rhythm ensures that an activity peak is maintained in daylight and, also, reduces the possibility of inundation during the first critical floodings during sequences of rising spring tides.

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The giant axons in Mercierella are overlaid by narrow glial processes which provide an incomplete covering of the axonal surface. Where more complete covering occurs the intercellular clefts are not sealed by tight junctional complexes. Ionic lanthanum penetrates to the surfaces of axons from sea-water-adapted animals (in normal saline and during initial exposure to hyposmotic saline) and, also, to the surface of hyposmotically adapted axons. A relatively free intercellular access to the axon surfaces is also indicated by the rapid electrical responses of sea-water-adapted axons to hyposmotic dilution and of hyposmotically adapted axons to sodium-deficient saline. The giant axon possesses an unusual ultrastructural specialization: hemidesmosome-like structures (associated with the axon membrane) which are connected to a network of neurofilaments within the axon. Theoretical considerations suggest that these structures could enable the axons to withstand appreciable excesses in intracellular hydrostatic pressure resulting from osmotic imbalance during hyposmotic stress.

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The giant axon of this extreme euryhaline osmoconformer possess an unusual ability to produce action potentials of large amplitude over a wide range of ionic dilution when constant osmotic concentration is maintained by the addition of mannitol to the bathing medium. Ionic dilution under these circumstances causes a decline in the overshoot of the action potential (resulting largely from reduction in [Na+]o) and an appreciable axonal hyperpolarization (primarily as a result of decrease in [K+]O). This hyperpolarization tends to compensate for the reduction in the extent of the overshoot and so maintains the amplitude of the sodium-mediated action potentials during isosmotic dilution of the bathing medium. The axonal hyperpolarization also appears to reduce sodium inactivation so as to maintain a rapid rate of rise of the action potential despite drastic reduction in the ionic concentration of the bathing medium. Prolonged exposure to reduced ionic concentrations appears to induce a ouabain sensitive reduction in intracellular sodium concentration which increases the sodium gradient across the axon membrane during isosmotic dilution of the external medium.

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