Changing interactions between astrocytes and neurons during CNS maturation

The environments of the developing brain and injured adult brain differ in their abilities to support axonal growth. To determine if astrocytes contribute to this difference, neurons were plated onto astrocytes cultured from the neonatal rat cortex and from the injured adult brain. Two patterns of neurite growth were observed in these two astrocyte culture systems. Neurons contacting the neonatal astrocytes had neurites that were twice as long as those contacting the injured adult astrocytes. Furthermore, in cultures with neonatal astrocytes, neurites faithfully followed the astrocytic processes, maximizing their contact, while in cultures of injured adult astrocytes, the neurites had a tendency to cross the processes orthogonally, minimizing their interaction with the astrocytes. When neurons were grown suspended over either neonatal or injured adult astrocytes, no difference in neurite length or the pattern of neurite growth was observed, indicating that neurite growth was not differentially affected by soluble factors released from the two populations of astrocytes. The addition of fetal calf serum, which is known to contain protease inhibitors, did not alter neurite growth when compared to serum-free medium, suggesting that a substantial difference in protease activity does not account for the variations in neurite length observed. Based on these results, it appears that the molecular components of the external surface of injured adult astrocytes do not support neurite growth to the same extent as those found on neonatal astrocytes. The differing abilities of these two populations of cultured astrocytes to support neurite growth in culture may reflect a change in the functional role of these cells that occurs during the development of the central nervous system.     

Cell cultures derived from early zebrafish embryos differentiate in vitro into neurons and astrocytes

The zebrafish is a polular nonmammalian model for studies of neural development. We have derived cell cultures, initiated from blastula-stage zebrafish embryos, that differentiate in vitro into neurons and astrocytes. Cultures were initiated in basal nutrient medium supplemented with bovine insulin, trout serum, trout embryo extract and fetal bovine serum. After two weeks in culture the cells exhibited extensive neurite outgrowth and possessed elevated levels of acetylcholinesterase enzyme activity. Ultrastructural analysis revealed that the neurites possessed microtubules, synaptic vessicles and areas exhibiting growth cone morphology. The cultures expressed proteins recognized by antibodies to the neuronal and astrocyte-specific markers, neurofilament and glial fibrillary acidic protein (GFAP). Poly-D-lysine substrate stimulated neurite outgrowth in the cultures and inhibited the growth of nonneuronal cells. Medium conditioned by the buffalo rat liver line, BRL, promoted the growth and survival of the cells in culture. Mitotically active cells were identified in cultures that had undergone extensive differentiation. The embryo cell cultures provide an in vitro system for investigations of biochemical parameters influencing zebrafish neuronal cell growth and differentiation.     

Temperature and pH dependence of nerve growth factor-mediated neurite outgrowth of PC12 pheochromocytoma cells

Nerve growth factor-mediated neurite outgrowth of PC12 pheochromocytoma cells was dependent on medium pH and temperature. Optimal pH was 6.8-7.1. No neurites were formed below 25 degrees C, and the number of cells having neurites increased upon elevating temperature. In contrast, the cells pretreated with nerve growth factor in suspension culture developed neurites even at 25 degrees C when they were transferred to monolayer culture. Temperature dependence of rates of the neurite formation indicates that apparent activation energy for this process is 44.6 kJ/mol.     

Mouse acetylcholinesterase enhances neurite outgrowth of rat R28 cells through interaction with laminin-1

The enzyme acetylcholinesterase (AChE) terminates synaptic transmission at cholinergic synapses by hydrolyzing the neurotransmitter acetylcholine, but can also exert 'non-classical', morpho-regulatory effects on developing neurons such as stimulation of neurite outgrowth. Here, we investigated the role of AChE binding to laminin-1 on the regulation of neurite outgrowth by using cell culture, immunocytochemistry, and molecular biological approaches. To explore the role of AChE, we examined fiber growth of cells overexpressing different forms of AChE, and/or during their growth on laminin-1. A significant increase of neuritic growth as compared with controls was observed for neurons over-expressing AChE. Accordingly, addition of globular AChE to the medium increased total length of neurites. Co-transfection with PRIMA, a membrane anchor of AChE, led to an increase in fiber length similar to AChE overexpressing cells. Transfection with an AChE mutant that leads to the retention of AChE within cells had no stimulatory effect on neurite length. Noticeably, the longest neurites were produced by neurons overexpressing AChE and growing on laminin-1, suggesting that the AChE/laminin interaction is involved in regulating neurite outgrowth. Our findings demonstrate that binding of AChE to laminin-1 alters AChE activity and leads to increased neurite growth in culture. A possible mechanism of the AChE effect on neurite outgrowth is proposed due to the interaction of AChE with laminin-1.     

Neuroserpin regulates neurite outgrowth in nerve growth factor-treated PC12 cells

Neuroserpin is a serine protease inhibitor widely expressed in the developing and adult nervous systems and implicated in the regulation of proteases involved in processes such as synaptic plasticity, neuronal migration and axogenesis. We have analysed the effect of neuroserpin on growth factor-induced neurite outgrowth in PC12 cells. We show that small changes in neuroserpin expression result in changes to the number of cells extending neurites and total neurite length following NGF treatment. Increased expression of neuroserpin resulted in a decrease in the number of cells extending neurites and a reduction in total free neurite length whereas reduced levels of neuroserpin led to a small increase in the number of neurite extending cells and a significant increase in total free neurite length compared to the parent cell line. Neuroserpin also altered the response of PC12 cells to bFGF and EGF treatment. Neuroserpin was localised to dense cored secretory vesicles in PC12 cells but was unable to complex with its likely enzyme target, tissue plasminogen activator at the acidic pH found in these vesicles. These data suggest that modulation of neuroserpin levels at the extending neurite growth cone may play an important role in regulating axonal growth.     

Automated screening of neurite outgrowth

Outgrowth of neurites in culture is used for assessing neurotrophic activity. Neurite measurements have been performed very slowly using manual methods or more efficiently with interactive image analysis systems. In contrast, medium-throughput and noninteractive image analysis of neurite screens has not been well described. The authors report the performance of an automated image acquisition and analysis system (IN Cell Analyzer 1000) in the neurite assay. Neuro-2a (N2a) cells were plated in 96-well plates and were exposed to 6 conditions of retinoic acid. Immunofluorescence labeling of the cytoskeleton was used to detect neurites and cell bodies. Acquisition of the images was automatic. The image set was then analyzed by both manual tracing and automated algorithms. On 5 relevant parameters (number of neurites, neurite length, total cell area, number of cells, neurite length per cell), the authors did not observe a difference between the automated analysis and the manual analysis done by tracing. These data suggest that the automated system addresses the same biology as human scorers and with the same measurement precision for treatment effects. However, throughput of the automated system is orders of magnitude higher than with manual methods.     

Geometry of isolated sensory neurons in culture. Effects of embryonic age and culture substratum

Sensory neurons were dissociated from lumbar dorsal root ganglia of embryonic chick and put into culture, either directly or after removing non-neuronal cells by density gradient centrifugation. The cells were grown on culture substrata of various kinds in medium containing nerve growth factor (NGF). After 24 h the cultures were fixed, mounted and analysed. Lengths of neurites were measured, and the numbers of primary processes formed at the cell body and of growth cones were counted. From these values, the rates of growth cone advance and frequency of growth cone branching were calculated. Neuronal outgrowths increased strikingly in length and complexity with embryonic age; there was a 3.5-fold increase in total neurite length and a 3-fold increase in the number of growth cones when neurons from 15-day embryos (E15) were compared with those from 8-day embryos (E8) grown on the same substratum (glass). Growth was markedly greater on surfaces prepared with laminin or conditioned medium compared with plain glass or air-dried collagen. When E15 neurons grown on glass were compared with those grown on laminin, for example, a 2.5-fold increase in total neurite length and a 3-fold increase in the number of growth cones was observed. Calculations showed that a major factor in these changes was an increase in the frequency of growth cone branching. The number of initial processes emanating from the cell body changed with age, but not with the different substrata tested. Non-neuronal cells when present in low numbers and in contact with neurons did not appear to influence neuronal geometry in a systematic way. Our results document the fact that both external factors (in this case, the nature of the culture substratum) and intrinsic factors (stage of development of the neuron) can influence the geometry of neurite outgrowth.     

Immunocytochemical Study of Microtubules in Chromaffin Cells in Culture and Evidence That Tubulin Is Not an Integral Protein of the Chromaffin Granule Membrane

Abstract Bovine adrenal chromaffin cells were maintained in culture in Dulbecco's modified Eagle's medium containing 20% foetal calf serum and 10 units per ml of Nerve Growth Factor. Under these conditions, chromaffin cells developed up to five neurites per cell. The neurites showed lateral branches and varicosities along their trunk which ended with thick growth cone-like structures. Cultures of chromaffin cells were stained by indirect immunofluorescence with antibodies against (a) chromogranin A to follow the distribution of chromaffin granules, the catecholamine-storing organelles, and (b) tubulin, to study the microtubular system during outgrowth of neurites. Chromogranin A antibodies showed a very intensely staining punctate pattern, not randomly distributed but localized in neurites. Chromaffin granules were found to migrate from the cell body to reach neurite endings where they were densely packed. Intense staining was also observed in varicosities; a linear arrangement of granules was evident along neurite trunks. Tubulin antibodies decorated a complex network, clearly visible at the cell periphery and also in the growth cone-like structures, in the palm region of the growth cone. Colchicine treatment effected retraction of neurites and disappearance of organized microtubule networks; chromaffin granules were found in the perinuclear region of the cell. Some tubulin (0.2% of total membrane proteins) was found in the purified chromaffin granule membrane preparation; however, this tubulin is probably associated with contaminating plasma membranes. By the criteria of morphology and staining with antitubulin antibodies, adult bovine chromaffin cells in culture display characteristics similar to those of sympathetic neurones. In addition, they showed an exaggerated transport of granules. Adult bovine chromaffin cells in culture offer an excellent model for studying the role of microtubules and the contractile apparatus in relation to cell morphological changes and neurosecretion.     

The possible role of "sibling neurite bias" in the coordination of neurite extension, branching, and survival

In this review we consider a novel mechanism, "sibling neurite bias," which may explain aspects of the coordination of elongation, branching, and resorption among different neurites growing from the same neuronal cell body. In this model, growing neurites which incorporate structural precursors at higher rates would deplete the cellular pool of precursors available to their "sibling" neurites; neurites would compete for survival, but in addition they would bias each other's behavior during active growth. Evidence is reviewed that "sibling neurite bias" may contribute to the establishment and stabilization of specific neural connections. Specific examples examined include the loss of polyinnervation at the developing neuromuscular junction, contextual mapping in the retino-tectal system, and selective neurite growth patterns and synaptic connections in nerve tissue culture model systems.     

A novel function of transcription factor alpha-Pal/NRF-1: increasing neurite outgrowth

Alpha-Pal/NRF-1 is a critical regulator of the promoter of human IAP/CD47 gene, a gene related to memory formation in rodents. However, its function in neurons was unknown. We found that stable or transient expression of full-length alpha-Pal/NRF-1 in human neuroblastoma IMR-32 cells significantly induced neurite outgrowth and increased the length of neurites both in medium containing 10% fetal bovine serum and in serum-free medium. In contrast, the dominant-negative mutant of alpha-Pal/NRF-1 inhibited the induction and extension of neurites. Ectopic expression of full-length alpha-Pal/NRF-1 also increased the induction of neurite outgrowth in primary mouse cortical neurons. The IAP antisense cDNA significantly inhibited the increase of neurite outgrowth by alpha-Pal/NRF-1. These findings indicate that a novel function of alpha-Pal/NRF-1 is to regulate neuronal differentiation, and that this function is mediated partly via its downstream IAP gene.     

The possible role of “sibling neurite bias” in the coordination of neurite extension,branching, and survival

In this review we consider a novel mechanism, “sibling neurite bias,” which may explain aspects of the coordination of elongation, branching, and resorption among different neurites growing from the same neuronal cell body. In this model, growing neurites which incorporate structural precursors at higher rates would deplete the cellular pool of precursors available to their “sibling” neurites; neurites would compete for survival, but in addition they would bias each other's behavior during active growth. Evidence is reviewed that “sibling neurite bias” may contribute to the establishment and stabilization of specific neural connections. Specific examples examined include the loss of polyinnervation at the developing neuromuscular junction, contextual mapping in the retino-tectal system, and selective neurite growth patterns and synaptic connections in nerve tissue culture model systems.     

Neurite development in vitro. I. The effects of adenosine 3′5′-cyclic monophosphate (cyclic AMP)

Elevated levels of 3′5′ adenosine monophosphate (cyclic AMP) stimulate a wide variety of cellular events including aggregation, differentiation, morphological expression, pigment migration, and secretion. The role of cyclic AMP in these events prompted our present study of embryonic chick dorsal root ganglia. Test substances were applied to cultures during the routine feeding procedure. Their development was quantitatively evaluated on the basis of explant size, length of glial-like outgrowth, distribution of growth, neurite number, length, diameter, and degree of arborization. These parameters were all shown to be independent of each other. The high variability of in vitro neurite development necessitated the use of over 100 cultures per treatment group. Cultures treated with 5′ AMP exhibited no significant differences from controls. Those treated with cyclic AMP, dibutyryl cyclic AMP, or Nerve Growth Factor (NGF) exhibited statistically significant increases in area of outgrowth, the number of neurites per culture, and in diameters, lengths, and degree of neurite arborization. The growth promoting activity of dibutyryl cyclic AMP and NGF were greater than those of cyclic AMP. Electron microscopic study shows neurites formed under the influence of cyclic AMP or its dibutyryl derivative to resemble those grown in NGF. These studies suggest the possibility that cyclic AMP stimulates neurite growth by mediating the process of microtubule (MT) assembly. They further prompt us to speculate that one way NGF enhances neurite development is by stimulating MT assembly via a “Second Messenger System”.     

Rapid growth cone translocation on laminin is supported by lamellipodial not filopodial structures

To determine the relationship between growth cone structure and motility, we compared the neurite extension rate, the form of individual growth cones, and the organization of f-actin in embryonic (E21) and postnatal (P30) sympathetic neurons in culture. Neurites extended faster on laminin than on collagen, but the P30 nerites were less than half as long as E21 neurites on both substrata. Growth cone shape was classified into one of five categories, ranging from fully lamellipodial to blunt endings. The leading margins of lamellipodia advanced smoothly across the substratum ahead of any filopodial activity and contained meshworks of actin filaments with no linear f-actin bundles, indicating that filopodia need not underlie lamellipodia. Rapid translocation (averaging 0.9-1.4 microns/min) was correlated with the presence of lamellipodia; translocation associated with filopodia averaged only 0.3-0.5 microns/min. This relationship extended to growth cones on a branched neurite where the translocation of each growth cone was dependent on its shape. Growth cones with both filopodial and lamellipodial components moved at intermediate rates. The prevalence of lamellipodial growth cones depended on age of the neurites; early in culture, 70% of E21 growth cones were primarily lamellipodial compared to 38% of P30 growth cones. A high percentage of E21 lamellipodial growth cones were associated with rapid neurite elongation (1.2 mm/day), whereas a week later, only 16% were lamellipodial, and neurites extended at 0.5 mm/day. Age-related differences in neurite extension thus reflected the proportion of lamellipodial growth cones present rather than disparities in basic structure or in the rates at which growth cones of a given type moved at different ages. Filopodia and lamellipodia are each sufficient to advance the neurite margin; however, rapid extension of superior cervical ganglion neurites was supported by lamellipodia independent of filopodial activity.     

The enhancement of growth and differentiation of rat adrenal nerve cells by the addition of conditioned medium from human fibroblast cultures

The growth of rat adrenal nerve cells was remarkably enhanced by supplementing the cultured medium from the human fibroblast cell line, Hs 68. Maximum specific growth rate and length of the neurites were observed as 0.076 (1/hr) and 0.026 mm, respectively in 20% supplement of five day old medium. In adding more than 20% of the cultured medium both cell and neurite growth was severely decreased. It was interesting that the cultured medium from Hs 68 cells could play a role in the extension of the neurites rather than in the growth of neurite cells. It was also found that molecules lower than 50,000 daltons in the conditioned medium could improve the growth of neurite bearing cells and the extension of the neurites than larger molecules. The efficacy of the proteins (<50,000 MW) was similar to that of human nerve growth factor and much better than that of basic fibroblast growth factor which was mainly secreted from human fibroblast cells. This revised version was published online in August 2006 with corrections to the Cover Date.     

Steps in the formation of a bipolar outgrowth pattern by cultured neurons, and their substrate dependence.

We studied the steps in the formation of the bipolar outgrowth pattern of cultured adult Anterior Pagoda (AP) neurons of the leech growing on a central nervous system (CNS) homogenate as substrate. This pattern, which consists of two primary neurites directed in opposite directions plus some bifurcations, resembles their embryonic pattern but is different from the patterns they develop in culture on leech laminin or Concanavalin A as substrates. In eight neurons that were studied, one primary neurite formed and branched several hours before the second one. Time-lapse video analysis showed that between 12 and 36 h of growth, the more proximal branch of the early neurite migrated retrogradely, rotated, and formed the second primary branch. Both neurites elongated until the total neurite length reached 130-160 microm, when the elongation of primary neurites became synchronous with the retraction of secondary processes, suggesting competition. The substrate dependence of these events was tested by plating AP neurons on leech laminin. On this substrate AP neurons produced multiple independent primary neurites with branches. Retraction of some large branches was followed by their regrowth, and did not correlate with the changes in other neurites. We propose that the dynamics in the formation of the bipolar outgrowth pattern of AP neurons arise from inhibitory extracellular matrix molecules, which reduce the synthesis of precursors for neurite formation.     

Ginkgolide B promotes axonal growth of retina ganglion cells by anti-apoptosis in vitro

One common feature of glaucoma, optic neuritis and some other optic nerve diseases is sustained and irreversible apoptosis of retinal ganglion cells (RGCs). Ginkgolide B is believed to protect neurons in brain and contribute to neurite outgrowth and synapse formation. The aim of the present study was to explore the effects of Ginkgo biloba extract (EGB761) and ginkgolide B on axonal growth of RCGs. Retina explants were cultured in three-dimensional tissue culture system, and the number and length of neurites were analyzed. Immunohistochemistry staining was performed to confirm that the neurite observed was axon of RGCs. TUNEL and activated caspase-3 staining were also applied to observe RGCs apoptosis. The result shows that neurites of RGCs treated with EGB761 or ginkgolide B were more and longer than those in control. The neurite is proved to be the axon of RGCs by immunostaining. Furthermore, compared with control group, RGCs treated with ginkgolide B showed decreased cellular apoptosis and inhibited caspase-3 activation. These results suggest ginkgolide B can promote RGCs axon growth by protecting RGCs against apoptosis.     

Neurotoxicity caused by didanosine on cultured dorsal root ganglion neurons

The purpose of the present study was to investigate whether didanosine (ddI) directly causes morphological and ultrastructural abnormalities of dorsal root ganglion (DRG) neurons in vitro. Dissociated DRG cells and organotypic DRG explants from embryonic 15-day-old Wistar rats were cultured for 3 days and then exposed to ddI (1 μg/ml, 5 μg/ml, 10 μg/ml, and 20 μg/ml) for another 3 days and 6 days, respectively. Neurons cultured continuously in medium served as normal controls. The diameter of the neuronal cell body and neurite length were measured in dissociated DRG cell cultures. Neuronal ultrastructural changes were observed in both culture models. ddI induced dose-dependent decreases in neurite number, length of the longest neurite in each neuron, and total neurite length per neuron in dissociated DRG cell cultures with 3 days treatment. There were no morphological changes seen in organotypic DRG cultures even with longer exposure time (6 days). But ddI induced ultrastructural changes in both culture models. Ultrastructural abnormalities included loss of cristae in mitochondria, clustering of microtubules and neurofilaments, accumulation of glycogen-like granules, and emergence of large dense particles between neurites or microtubules. Lysosome-like large particles emerged inconstantly in neurites. ddI induced a neurite retraction or neurite loss in a dose-dependent manner in dissociated DRG neurons, suggesting that ddI may partially contribute to developing peripheral neuropathy. Cytoskeletal rearrangement and ultrastructural abnormalities caused by ddI in both culture models may have a key role in neurite degeneration.     

Hydrogel design for supporting neurite outgrowth and promoting gene delivery to maximize neurite extension

Hydrogels capable of gene delivery provide a combinatorial approach for nerve regeneration, with the hydrogel supporting neurite outgrowth and gene delivery inducing the expression of inductive factors. This report investigates the design of hydrogels that balance the requirements for supporting neurite growth with those requirements for promoting gene delivery. Enzymatically-degradable PEG hydrogels encapsulating dorsal root ganglia explants, fibroblasts, and lipoplexes encoding nerve growth factor were gelled within channels that can physically guide neurite outgrowth. Transfection of fibroblasts increased with increasing concentration of Arg-Gly-Asp (RGD) cell adhesion sites and decreasing PEG content. The neurite length increased with increasing RGD concentration within 10% PEG hydrogels, yet was maximal within 7.5% PEG hydrogels at intermediate RGD levels. Delivering lipoplexes within the gel produced longer neurites than culture in NGF-supplemented media or co-culture with cells exposed to DNA prior to encapsulation. Hydrogels designed to support neurite outgrowth and deliver gene therapy vectors locally may ultimately be employed to address multiple barriers that limit regeneration.     

Neurite outgrowth from hippocampal neurons is promoted by choroid plexus ependymal cells <Emphasis Type="Italic">in vitro</Emphasis>

Choroid plexus ependymal cells (CPECs) were known to promote axonal growth when choroid plexus is grafted into the adult rat spinal cord. The present study was carried out to examine whether CPECs promote axonal outgrowth from neurons derived from the CNS in vitro. Hippocampal neurons were cocultured on CPEC monolayers. After 24 h, neurite extension was evaluated using various parameters in comparison with cultures grown on poly-L-lysine (PLL)-coated plates and cocultures grown on astrocyte monolayers. The primary neurite length and total neurite length were longest in the cocultures with CPECs. The number of primary neurites and the number of branches were larger in the cultures with CPECs than in the cultures on PLL-coated plates, but almost the same as in the cocultures with astrocytes. Next, we examined whether the neurite extension-promoting effect occurring within 24 h is due primarily to contact with the CPECs or to factors secreted by CPECs into the culture medium. The CPEC monolayers were killed by ethanol fixation, and neurons cultured on them. The neurons extended long neurites with elaborate branching, as in the case of cocultures grown on living CPECs. On the other hand, CPEC-conditioned medium exhibited less promoting effect on neurite outgrowth from hippocampal neurons. These results indicate that CPECs have a capacity to promote neurite outgrowth from CNS neurons in vitro, and that surface plasma membrane-bound components of CPECs strongly contribute to the enhancement of neurite outgrowth in the present coculture system.