Adult insect glial culture: Activation, substrate effects and proliferation

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.     

Glial repair in an insect

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.     

Neural repair in an insect: cell recruitment and deployment following selective glial disruption

Summary In Periplaneta americana, SEM of abdominal nervous connectives revealed a rapid accumulation of haemocytes on the surface of the neural lamella within 24 h of selective disruption of the underlying neuroglia by ethidium bromide. After 4 days the neural lamella was effectively clear of adhering haemocytes, but showed characteristic blisters, which, it is postulated, represented the points of entry of the cells from the haemocoel into the underlying tissues. A notable subsequent feature was a substantial increase in the number of cells within repairing connectives. Initially, there was a marked asymmetry in their distribution, with significantly higher numbers of cells anterior to, and within, the lesion area. It seems likely that this polarity resulted from differential cell division within the connectives. The initial asymmetry disappeared after seven days. However, increased perineurial cell numbers were maintained in the lesion area after one month and were still apparent two months after selective glial disruption. There was no equivalent increase in cell numbers in the lesion zone of cultured cords or, in vivo, after injection of the DNA-scission drug, bleomycin, treatments which preclude haemocyte involvement. It is suggested that in the absence of haemocytes and with suppression of proliferation by endogenous cells, repair is achieved by redeployment or growth of adjacent, undamaged glia.     

Neural repair in an insect central nervous system: cell kinetics and proliferation after selective glial disruption

Summary Autoradiographs of tritiated thymidine uptake and subsequent light- and electron-microscopical examination revealed an onset of perineurial glial cell proliferation 3 days after injury to the CNS. The number of cells labelled increased rapidly until 7 days post-lesioning. At 2 weeks, the labelled cells equalled the number of nuclei present in the perineurium. No label was seen in the subperineurial cells, possibly because of the inability of the label to penetrate into a region where localised division is taking place.Prior to the onset of thymidine uptake, the damaged nerve cord was invaded by an exogenous reactive cell. The number of these cells increased rapidly in the first 48 h, then decreased as a negative exponential, very few remaining after 7 days. We suggest that this cell type must either return to the haemocoel or transform into a functional glial cell class.The repair of the insect central nervous system can be divided into three phases which show striking similarities to vertebrate repair sequences. These include: initial invasion of the lesion by exogenous cells, subsequent proliferation of glial cells, the longer term flux of cell numbers, their distribution and the time scale of events. This suggests that the insect CNS might provide a system for examining common cellular mechanisms and events.     

Haemocyte involvement in the repair of the insect central nervous system after selective glial disruption

Summary Injection of physiologically inert particles (fluorescent microspheres) has a profound effect on neural repair of central nervous connectives of the cockroach Periplaneta americana following selective glial disruption. The injected particles, which do not gain direct access to the central nervous tissues, are taken up by a relatively small proportion (< 10%) of the haemocytes. This interference with haemocyte function virtually abolishes the appearance of the granule-containing cells (which are prominently involved in normal glial repair) and produces abnormal reorganization of the superficial glial elements. These results are interpreted as evidence that the granule-containing cells are derived from haemocytes which are critically involved in glial repair.     

Neurotransmitters as early signals for central nervous system development

During brain ontogenesis, the temporal and spatial generation of the different types of neuronal and glial cells from precursors occurs as a sequence of successive progenitor stages whose proliferation, survival and cell-fate choice are controlled by environmental and cellular regulatory molecules. Neurotransmitters belong to the chemical microenvironment of neural cells, even at the earliest stages of brain development. It is now established that specific neurotransmitter receptors are present on progenitor cells of the developing central nervous system and could play, during neural development, a role that has remained unsuspected until recently. The present review focuses on the occurrence of neurotransmitters and their corresponding ligand-gated ion channel receptors in immature cells, including neural stem cells of specific embryonic and neonatal brain regions. We summarize in vitro and in vivo data arguing that neurotransmitters could regulate morphogenetic events such as proliferation, growth, migration, differentiation and survival of neural precursor cells. The understanding of neurotransmitter function during early neural maturation could lead to the development of pharmacological tools aimed at improving adult brain repair strategies.     

Glial uptake of monoamines in primary cultures of rat median raphe nucleus and cerebellum

Summary Glial uptake of serotonin and dopamine was studied in primary cultures of the median raphe nucleus and cerebellum by using consecutive demonstration of monoamine fluorescence and glial fibrillary acidic protein immunofluorescence. Most of the glial cells taking up monoamines were glial fibrillary acidic protein positive. Astrocytes with a strong immunoreactivity exhibited monoamine fluorescence only occasionally, although such cells did take up L-dopa readily. Glial fibrillary acidic protein negative cells — morphologically identified as astrocytes — were seen to exhibit monoamine fluorescence after exposure. Glial uptake of serotonin at a concentration of 10^–4 M was detected in cerebellar cultures but not in cultures from the median raphe nucleus. When the concentration was 10^–3 M uptake of serotonin took place in both the areas but was weaker in cultures from the median raphe nucleus. At concentrations greater than 10^–5 M glial uptake of dopamine was detected in cultures from both the regions studied. No region dependent differences in glial uptake of dopamine was demonstrated, however. Based on these observations astrocytes and astrocyte-like glial cells take up dopamine and serotonin. Also glial cells with a remarkably high content of the glial fibrillary acidic protein are more resistant to monoamine uptake than cells exhibiting less intense or no glial fibrillary acidic protein immunofluorescence. The existence of regional differences in uptake of serotonin between the median raphe nucleus and cerebellum suggests that glial uptake of monoamines is not an entirely passive mechanism but may be actively controlled by glial cells in a region dependent manner.     

The effects of an anti-mitotic drug,bleomycin, on glial repair in an insect central nervous system

Summary The DNA-binding drug, bleomycin, has a profound effect on neural repair following selective glial disruption by ethidium bromide. The contribution of the granule-containing cells (which normally appear in the early stages of repair) is greatly reduced, the restoration of the blood-brain barrier is delayed and the ultrastructural organization of the reorganising perineurium is dramatically changed. The aberrant perineurial structure and function observed in the presence of bleomycin are postulated to result from the effects of the drug on haemocytes which, together with endogenous reactive cells, contribute to the normal process of glial repair.     

Cell proliferation in ectodermal explants from Xenopus embryos

Summary Dissociated prospective ectoderm cells from Xenopus laevis embryos divide autonomously up to the 17th division cycle of the embryo. To examine the requirements for the further proliferation of these cells, the continuation of cell division in compact ectodermal explants beyond the 17th division cycle has been studied. Such explants develop into aggregates of epidermal cells, as can be shown immunohistochemically with an anti-serum against Xenopus epidermal cytokeratin. Cell division in these explants is comparable to the in vivo proliferation rate at least during the first 24 h of cultivation, that is, well beyond the 17th division cycle. Thus, epidermal cells are provided with all the factors necessary for continued proliferation, but these can be effective only when the cells form tight aggregates. The long-term changes in cell number are complex. Mitotic figures are present until the explants disintegrate after 3–4 days. However, the total cell number per explant does not increase during later development. The production of cells by mitotic divisions is likely to be countered by the loss of cells due to cell death, which is indicated by the presence of pyknotic nuclei.     

Organ culture of adult cockroach CNS: ultrastructural and physiological characteristics

The abdominal nerve cord of adult Periplaneta americana has been maintained in culture for up to 5 weeks. The ultrastructural appearance of in vitro cords resembles that of in vivo cords although some degeneration of glia may occur during the first 2 weeks in culture. The glial cells most affected are those which make up the perineurium. The blood-brain barrier, formed by the perineurium in vivo, breaks down in vitro. Despite this, normal action potentials were recorded from giant axons in 1 month old cultured connectives.     

Integrin receptors are required for cell survival and proliferation during development of the peripheral glial lineage

Proliferation and survival of Schwann cells are important for nerve development and for disease processes in peripheral nerves. We have analyzed embryos lacking alpha4- or alpha5-integrins and show here that these integrins contribute to the control of glial cell numbers. To overcome early embryonic lethality an explant and grafting system that allows the study of isolated glial progenitor cells both in vitro and in vivo was used. Schwann cells differentiate in the absence of alpha5 but their numbers and the proliferation rate of early progenitor cells are reduced, suggesting that alpha5 is essential for normal proliferation. Survival, rather than proliferation, is compromised in alpha4-deficient explants. Conditional immortalization allowed further characterization and revealed that alpha4 contributes to survival in a cell-density-dependent fashion. In addition, transplants into chicken embryos were used to analyze in vivo cell migration and showed that cell death occurs mainly in highly motile, individually migrating cells. The cell death patterns in vitro and in vivo argue that alpha4-integrins play a role in survival during cell migration. Neural crest migration has been suggested to require these integrins; however, no defects in migration were observed in the absence of alpha4 or alpha5. We conclude that integrins can complement growth factors in the control of glial cell numbers.     

Dimerization of the largest subunit of chromatin assembly factor 1: importance in vitro and during Xenopus early development

To date, the in vivo importance of chromatin assembly factors during development in vertebrates is unknown. Chromatin assembly factor 1 (CAF-1) represents the best biochemically characterized factor promoting chromatin assembly during DNA replication or repair in human cell-free systems. Here, we identify a Xenopus homologue of the largest subunit of CAF-1 (p150). Novel dimerization properties are found conserved in both Xenopus and human p150. A region of 36 amino acids required for p150 dimerization was identified. Deletion of this domain abolishes the ability of p150 to promote chromatin assembly in vitro. A dominant-negative interference based on these dimerization properties occurs both in vitro and in vivo. In the embryo, nuclear organization was severely affected and cell cycle progression was impaired during the rapid early cleaving stages of Xenopus development. We propose that the rapid proliferation at early developmental stages necessitates the unique properties of an assembly factor that can ensure a tight coupling between DNA replication or repair and chromatin assembly.     

The influence of early protein-calorie malnutrition on neuronal and glial protein synthesis

The effect of pre- and postnatal undernutrition, produced according to the method of Chow and Lee (3), on the rate of protein synthesis in the brains of rats 11, 21, 34 and 90 days of age was studied by measuring the incorporation ofl-[¹⁴C]valine in vivo andl-[³H]lysine in vitro. Both in vivo and in vitro experiments were performed with high concentration of the precursor to decrease the effects of pool variations and protein degradation. Particular interest was given to the effects of this form of early protein-calorie malnutrition (PCM) on neuronal and glial cells which were isolated from the brains by gradient centrifugation. Brain protein synthesis measured in vivo which showed a peak at 21 days in both animal series, was depressed by PCM at 11 days but stimulated at 34 days of animal age. Small effect was observed in the 90-day-old animals. A similar response as in whole brain was seen for neuronal cells, while glial cells showed a different reaction. Studies of in vitro protein synthesis did not reveal appreciable effects of undernutrition in whole brain. Both neuronal and glial cells showed a moderate but not statistically significant elevation of protein synthesis in animals subjected to early PCM.     

Development of a glial network in the olfactory nerve: role of calcium and neuronal activity

In adult olfactory nerves of mammals and moths, a network of glial cells ensheathes small bundles of olfactory receptor axons. In the developing antennal nerve (AN) of the moth Manduca sexta, the axons of olfactory receptor neurons (ORNs) migrate from the olfactory sensory epithelium toward the antennal lobe. Here we explore developmental interactions between ORN axons and AN glial cells. During early stages in AN glial-cell migration, glial cells are highly dye coupled, dividing glia are readily found in the nerve and AN glial cells label strongly for glutamine synthetase. By the end of this period, dye-coupling is rare, glial proliferation has ceased, glutamine synthetase labeling is absent, and glial processes have begun to extend to enwrap bundles of axons, a process that continues throughout the remainder of metamorphic development. Whole-cell and perforated-patch recordings in vivo from AN glia at different stages of network formation revealed two potassium currents and an R-like calcium current. Chronic in vivo exposure to the R-type channel blocker SNX-482 halted or greatly reduced AN glial migration. Chronically blocking spontaneous Na-dependent activity by injection of tetrodotoxin reduced the glial calcium current implicating an activity-dependent interaction between ORNs and glial cells in the development of glial calcium currents.     

Blood cells contribute to glial repair in an insect

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.     

The glial regenerative response to central nervous system injury is enabled by pros-notch and pros-NFκB feedback

Organisms are structurally robust, as cells accommodate changes preserving structural integrity and function. The molecular mechanisms underlying structural robustness and plasticity are poorly understood, but can be investigated by probing how cells respond to injury. Injury to the CNS induces proliferation of enwrapping glia, leading to axonal re-enwrapment and partial functional recovery. This glial regenerative response is found across species, and may reflect a common underlying genetic mechanism. Here, we show that injury to the Drosophila larval CNS induces glial proliferation, and we uncover a gene network controlling this response. It consists of the mutual maintenance between the cell cycle inhibitor Prospero (Pros) and the cell cycle activators Notch and NFκB. Together they maintain glia in the brink of dividing, they enable glial proliferation following injury, and subsequently they exert negative feedback on cell division restoring cell cycle arrest. Pros also promotes glial differentiation, resolving vacuolization, enabling debris clearance and axonal enwrapment. Disruption of this gene network prevents repair and induces tumourigenesis. Using wound area measurements across genotypes and time-lapse recordings we show that when glial proliferation and glial differentiation are abolished, both the size of the glial wound and neuropile vacuolization increase. When glial proliferation and differentiation are enabled, glial wound size decreases and injury-induced apoptosis and vacuolization are prevented. The uncovered gene network promotes regeneration of the glial lesion and neuropile repair. In the unharmed animal, it is most likely a homeostatic mechanism for structural robustness. This gene network may be of relevance to mammalian glia to promote repair upon CNS injury or disease.     

Regulated vnd expression is required for both neural and glial specification in Drosophila.

The Drosophila embryonic CNS arises from the neuroectoderm, which is divided along the dorsal-ventral axis into two halves by specialized mesectodermal cells at the ventral midline. The neuroectoderm is in turn divided into three longitudinal stripes--ventral, intermediate, and lateral. The ventral nervous system defective, or vnd, homeobox gene is expressed from cellularization throughout early neural development in ventral neuroectodermal cells, neuroblasts, and ganglion mother cells, and later in an unrelated pattern in neurons. Here, in the context of the dorsal-ventral location of precursor cells, we reassess the vnd loss- and gain-of-function CNS phenotypes using cell specific markers. We find that over expression of vnd causes significantly more profound effects on CNS cell specification than vnd loss. The CNS defects seen in vnd mutants are partly caused by loss of progeny of ventral neuroblasts-the commissures are fused and the longitudinal connectives are aberrantly positioned close to the ventral midline. The commissural vnd phenotype is associated with defects in cells that arise from the mesectoderm, where the VUM neurons have pathfinding defects, the MP1 neurons are mis-specified, and the midline glia are reduced in number. vnd over expression results in the mis-specification of progeny arising from all regions of the neuroectoderm, including the ventral neuroblasts that normally express the gene. The CNS of embryos that over express vnd is highly disrupted, with weak longitudinal connectives that are placed too far from the ventral midline and severely reduced commissural formation. The commissural defects seen in vnd gain-of-function mutants correlate with midline glial defects, whereas the mislocalization of interneurons coincides with longitudinal glial mis-specification. Thus, Drosophila neural and glial specification requires that vnd expression by tightly regulated.     

The ganglion-connective junction in the central nervous system of the leech,Hirudo medicinalis

Classical studies of the nervous system of the leech revealed that there were specific types of very large glial cells associated with various parts of the neuron. Recent microelectrode studies demonstrated that there was a low resistance to the flow charge from any one of these large glial cells to another. The present study describes a previously unreported type of glial cell, the glial cell of the fascicles. These cells, which resemble the glial cells of the connectives but are smaller, are found in the fascicles of axons that unite the connectives to the neuropil. Thus, these cells are located between the glial cells of the connectives on the one hand and the glial cells of the neuropil and packets on the other and must be taken into account in considerations of the low resistance to the transfer of charge from one glial cell to another.     

An analysis of dorsal root ganglia differentiation using three tissue culture systems

Summary The histogenesis of the dorsal root ganglia of chick embryos (ages 3 to 9 days) was followed in three different tissue culture systems. Organotypic explants included dorsal root ganglia connected to the lumbosacral segment of the spinal cord or isolated explants of the contralateral ganglia. Additionally, dissociated monolayer cultures of ganglia tissue were established. The gradual differentiation of progenitor neuroblasts into distinct populations of large ventrolateral and small dorsomedial neurons was observed in vivo and in vitro. Neurites developed after 3 days in the presence or absence of nerve growth factor in the medium. In contrast, autoradiographic analysis indicates that [³H]thymidine incorporation in neuronal cultures differed significantly from intact embryos. In vivo, the number of neuronal progenitor cells labeled with [³H]thymidine decreased in older embryos; in vitro, uptake of [³H]thymidine label was not observed in ganglionic progenitor cells regardless of the age of the donor embryo or the type of culture system. Lack of proliferation in ganglionic progenitor cells was not due to degeneration because vital staining and uptake of [³H]deoxyglucose indicated that neurons were metabolically active. Furthermore, the block in mitotic activity in vitro was limited to presumptive ganglionic neuronal cells. In the ependyma of the spinal cord segment connected to the dorsal root ganglia, neuronal progenitor cells were heavily labeled as were non-neuronal cells within both spinal cord and ganglia. Our results suggest that in vitro conditions can promote the differentiation of sensory neurons from early embryos (E3.5–4.5) without proliferation of progenitor cells.     

MAPK signaling during Müller glial cell development in retina explant cultures

The Müller cell is the only glial cell type generated from the retinal neuroepithelium. This cell type controls normal retina homeostasis and has been suggested to play a neuroprotective role. Recent evidence suggests that mammalian Müller cells can de-differentiate and return to a progenitor or stem cell stage following injury or disease. In vivo exploration of the molecular mechanisms of Müller cell differentiation and proliferation will add essential information to manipulate Müller cell functions. Signal transduction pathways that regulate Müller cell responses and activity are a critical part of their cellular machinery. In this study, we focus on mitogen-activated protein kinase (MAPK) signaling pathway during Müller glial cell differentiation and proliferation. We found that both MAPK and STAT3 signaling pathways are present during Müller glial cell development. Ciliary neurotrophic factor (CNTF)-stimulated Müller glial cell proliferation is associated with early developmental stages. Specific inhibition of MAPK phosphorylation significantly reduced the number of Müller glial cells with or without CNTF stimulation. These results suggested that the MAPK signal transduction pathway is important in the formation of Müller glial cells during retina development.