Production of ceramides causes apoptosis during early neural differentiation in vitro

To investigate signal transduction pathways leading to apoptosis during the early phase of neurogenesis, we employed PCC7-Mz1 cells, which cease to proliferate and begin to differentiate into a stable pattern of neurons, astroglial cells, and fibroblasts upon incubation with retinoic acid (RA). As part of lineage determination, a sizable fraction of RA-treated cultures die by apoptosis. Applying natural long-chain C(16)-ceramides as well as membrane-permeable C(2)/C(6)-ceramide analogs caused apoptosis, whereas the biologically nonactive C(2)-dihydroceramide did not. Treating PCC7-Mz1 stem cells with a neutral sphingomyelinase or with the ceramidase inhibitor N-oleoylethanolamine elevated the endogenous ceramide levels and concomitantly induced apoptosis. Addition of RA caused an increase in ceramide levels within 3-5 h, which reached a maximum (up to 3.5-fold of control) between days 1 and 3 of differentiation. Differentiated PCC7-Mz1 cells did not respond with ceramide formation and apoptosis to RA treatment. The acidic sphingomyelinase contributed only weakly and the neutral Mg(2+)-dependent and Mg(2+)-independent sphingomyelinases not at all to the RA-mediated production of ceramides. However, ceramide increase was sensitive to the ceramide synthase inhibitor fumonisin B(1), suggesting a crucial role for the de novo synthesis pathway. Enzymatic assays revealed that ceramide synthase activity remained unaltered, whereas serine palmitoyltransferase (SPT), a key enzyme in ceramide synthesis, was activated approximately 2.5-fold by RA treatment. Activation of SPT seemed to be mediated via a post-translational mechanism because levels of the mRNAs coding for the two SPT subunits were unaffected. Expression of marker proteins shows that ceramide regulates apoptosis, rather than differentiation, during early neural differentiation.     

Bcl-2 inhibits cell death of serum-free mouse embryo cells caused by epidermal growth factor deprivation

SFME cells are brain-derived neural precursor cells that are acutely dependent on epidermal growth factor (EGF) for survival, undergoing apoptosis within 24 h after EGF withdrawal. Because the expression of the protooncogene bcl-2 inhibits apoptosis induced by the withdrawal of interleukins or nerve growth factor in some growth factor-dependent haematopoietic or neuronal cell cultures, we examined the effect of Bcl-2 expression on cell death of SFME cells in the absence of EGF. SFME cells expressing human Bcl-2 showed prolonged survival when deprived of EGF compared to control cells not expressing Bcl-2. A significant fraction of Bcl-2-expressing cells remained viable for 4 days in the absence of EGF and resumed proliferation upon readdition of EGF to the cultures. These results suggest that apoptosis induced by EGF withdrawal in SFME cells may share common mechanisms with other growth factor-related apoptotic systems.     

The effects of cell density, attachment substratum and dexamethasone on spontaneous apoptosis of rat hepatocytes in primary culture

Summary The rates of spontaneous cell detachment, cell viability, and apoptosis in primary cultures of rat hepatocytes plated at high and low density were compared. Apoptosis was frequent in detached cells, and the rates of cell detachment and apoptosis were greater in high-density than in low-density cultures. Among attached cells, more cells had condensed or fragmented nuclei in high-density than in low-density cultures. Further, ladder-like DNA fragmentation was not seen in low-cell-density cultures but was clearly evident in high-density cultures. Bax was more highly expressed in cells cultured at high density, and on collagen vs. matrigel, whereas changes of Bcl-2 and Fas expression observed in culture appeared unrelated to the rate of apoptosis. The rate of hepatocyte apoptosis appeared to be identical in low-density cultures on collagen 1 and matrigel, but when cells were cultured at high density, matrigel suppressed apoptosis by more than 50% at 36 h. In hepatocytes cultured on collagen 1, dexamethasone (0.1 μM) suppressed apoptosis in both low- and high-density cultures; higher doses had no further effects. In high density cultures, aurintricarboxylic acid (10 μM) suppressed apoptosis and this improved cell attachment at 48 h. It is concluded that cell viability in primary cultures of rat hepatocytes grown on collagen I is dependent on optimal culture density and that the cell population is regulated, at least in part, by apoptosis. Corticosteroids suppress spontaneous apoptosis of cultured hepatocytes in a non-dose-dependent manner, whereas matrigel abolishes apoptosis induced by increasing cell density. Bax may be an important protein in the cell density and cell matrix-dependent regulation of apoptosis in cultured hepatocytes.     

Human Bcl-2 protects against AMPA receptor-mediated apoptosis

Dysfunctions of the (S)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) subtype of ionotropic receptor for the brain's major excitatory neurotransmitter, L-glutamate, occur in various neurological conditions. We have previously demonstrated that AMPA receptor-mediated excitotoxicity occurs by apoptosis and here examined the influence of the expression of cell death repressor gene Bcl-2 on this excitotoxic insult. Using neuronal cortical cultures prepared from transgenic mice expressing the human Bcl-2 gene, the influence of Bcl-2 on AMPA receptor-mediated neuronal death was compared with that seen with staurosporine and H2O2. At day 6 cultures were exposed to AMPA (0.1-100 microM), and cellular injury was analyzed 48 h after insult using phase-contrast microscopy, a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide viability assay, and DNA staining with 4,6-diamidino-2-phenylindole and Sytox Green. AMPA produced a concentration-dependent increase in cell death that was significantly attenuated by human Bcl-2. AMPA (3 microM) increased the number of apoptotic nuclei to 60% of control in wild-type cultures, and human Bcl-2 significantly decreased the number of apoptotic nuclei to 30% of AMPA-treated cultures. Human Bcl-2 only provided significant neuroprotection against neuronal injury induced by low concentrations of staurosporine (1-10 nM) and H2O2 (0.1-30 microM) and where neuronal death was by apoptosis, but not against H2O2-induced necrosis. Our findings indicate that overexpression of Bcl-2 in primary cultured neurons protects in an insult-dependent manner against AMPA receptor-mediated apoptosis, whereas protection was not seen against more traumatic insults. This study provides new insights into the molecular therapeutics of neurodegenerative conditions.     

Anti-apoptotic defense of bcl-2 gene against hydroperoxide-induced cytotoxicity together with suppressed lipid peroxidation,enhanced ascorbate uptake,and upregulated Bcl-2 protein

Although it is well known that Bcl-2 can prevent apoptosis, the Bcl-2's anti-apoptotic mechanism is not fully understood. Here, we investigate the mechanism of oxidant-induced cell death and to investigate the role of Bcl-2 in the tert-butyl hydroperoxide (t-BuOOH)-induced oxidant injury in Rat-1 fibroblasts and their bcl-2 transfected counterparts, b5 cells. Treatment with t-BuOOH causes mitochondrial disfunction and induced morphological features consistent with apoptosis more markedly in Rat-1 cells than in b5 cells. The hydroperoxide t-BuOOH at concentrations less than 100 nM for as long as 48 h or with higher concentrations (up to 100 microM) for only 3 h induces death in Rat-1 cells, whereas their bcl-2 transfectants were significantly resistant to cytotoxicity by both time and all concentration other than 100 microM. The similar results were obtained also for DNA strand cleavages as detected by TUNEL stain. The bcl-2 transfectants significantly suppressed t-BuOOH-induced increases in both lipid peroxidation and caspase-3 activation 3 and 1 h after t-BuOOH exposure, respectively, but failed to suppress either caspase-1 activation or an enhanced production of the intracellular reactive oxygen species (ROS). Intracellular uptake of [1-(14)C] ascorbic acid (Asc) into the bcl-2 transfectants was superior to that into the non-transfectants always under examined conditions regardless of serum addition to culture medium and cell density. Upregulation of Bcl-2 proteins was rapidly induced after t-BuOOH exposure in the transfectants, but not in non-transfectants, and restored till 24 h to the normal Bcl-2 level. Thus suppressions of both lipid peroxidation and the subsequent cell death events such as caspase-3 activation and DNA cleavage were concerned with the inhibitory effects of Bcl-2 on the t-BuOOH-induced cytotoxicity. And some of these events may correlate with Bcl-2 expression-induced partial enhanced anti-oxidant cellular ability including enrichment of intracellular Asc and oxidative stress-induced upregulation of Bcl-2 protein. On the other hand, ROS production and caspase-1 activation were not related to cytoprotection by Bcl-2.     

Peroxisome proliferator-activated receptor gamma up-regulates the Bcl-2 anti-apoptotic protein in neurons and induces mitochondrial stabilization and protection against oxidative stress and apoptosis

Peroxisome proliferator-activated receptor gamma (PPARgamma) has been proposed as a therapeutic target for neurodegenerative diseases because of its anti-inflammatory action in glial cells. However, PPARgamma agonists preventbeta-amyloid (Abeta)-induced neurodegeneration in hippocampal neurons, and PPARgamma is activated by the nerve growth factor (NGF) survival pathway, suggesting a neuroprotective anti-inflammatory independent action. Here we show that the PPARgamma agonist rosiglitazone (RGZ) protects hippocampal and dorsal root ganglion neurons against Abeta-induced mitochondrial damage and NGF deprivation-induced apoptosis, respectively, and promotes PC12 cell survival. In neurons and in PC12 cells RGZ protective effects are associated with increased expression of the Bcl-2 anti-apoptotic protein. NGF-differentiated PC12 neuronal cells constitutively overexpressing PPARgamma are resistant to Abeta-induced apoptosis and morphological changes and show functionally intact mitochondria and no increase in reactive oxygen species when challenged with up to 50 microM H2O2. Conversely, cells expressing a dominant negative mutant of PPARgamma show increased Abeta-induced apoptosis and disruption of neuronal-like morphology and are highly sensitive to oxidative stress-induced impairment of mitochondrial function. Cells overexpressing PPARgamma present a 4- to 5-fold increase in Bcl-2 protein content, whereas in dominant negative PPARgamma-expressing cells, Bcl-2 is barely detected. Bcl-2 knockdown by small interfering RNA in cells overexpressing PPARgamma results in increased sensitivity to Abeta and oxidative stress, further suggesting that Bcl-2 up-regulation mediates PPARgamma protective effects. PPARgamma prosurvival action is independent of the signal-regulated MAPK or the Akt prosurvival pathways. Altogether, these data suggest that PPARgamma supports survival in neurons in part through a mechanism involving increased expression of Bcl-2.     

Antisense inhibition of BCL-2 expression induces retinoic acid-mediated cell death during differentiation of human NT2N neurons

Changes in expression of the proto-oncogene Bcl-2 are well known in the developing brain, with a high expression level in young post-mitotic neurons that are beginning the outgrowth of processes. The physiological significance of the Bcl-2 up-regulation in these neurons is not fully understood. We used a differentiation model for human CNS neurons to study the expression and function of Bcl-2. NT2/D1 human neuronal precursor cells differentiated into a neuronal phenotype in the presence of 10 microM retinoic acid for 3-5 weeks. This concentration of retinoic acid was not toxic to undifferentiated NT2/D1 cells but was sufficient to up-regulate the BCL-2 protein in 6 days. The BCL-2 levels increased further after 3 weeks, i.e. when the cells started to show neuronal morphology. Inhibition of the accumulation of endogenous BCL-2 with vectors expressing the antisense mRNA of Bcl-2 caused extensive apoptosis after 3 weeks of the retinoic acid treatment. The loss of neuron-like cells from differentiating cultures indicated that the dead cells were those committed to neuronal differentiation. Death was related to the presence of retinoic acid since withdrawal of retinoic acid after 16 days of treatment dramatically increased cell surviving. The ability of BCL-2 to prevent retinoic acid-induced cell death was also confirmed in undifferentiated NT2/D1 cells that were transfected with a vector containing Bcl-2 cDNA in sense orientation and exposed to toxic doses (40-80 microM) of retinoic acid. Furthermore, down-regulation of BCL-2 levels by an antisense oligonucleotide in neuronally differentiated NT2/D1 cells increased their susceptibility to retinoic acid-induced apoptosis. These results indicate that one function of the up-regulation of endogenous BCL-2 during neuronal differentiation is to regulate the sensitivity of young post-mitotic neurons to retinoic acid-mediated apoptosis.     

ATP induces the death of developing avian retinal neurons in culture via activation of P2X7 and glutamate receptors

Previous data suggest that nucleotides are important mitogens in the developing retina. Here, the effect of ATP on the death of cultured chick embryo retina cells was investigated. In cultures obtained from retinas of 7-day-old chick embryos (E7) that were cultivated for 2 days (E7C2), both ATP and BzATP induced a ～30 % decrease in cell viability that was time- and dose-dependent and that could be blocked by 0.2 mM oxidized ATP or 0.3 μM KN-62. An increase in cleaved caspase-3 levels and in the number of TUNEL-positive cells was observed when cultures were incubated with 3 mM ATP and immunolabeling for cleaved-caspase 3 was observed over neurons but not over glial cells. ATP-dependent cell death was developmentally regulated, the maximal levels being detected by E7C2-3. Nucleotides were able to increase neuronal ethidium bromide and sulforhodamine B uptake in mixed and purified neuronal cultures, an effect that was blocked by the antagonists Brilliant Blue G and oxidized ATP. In contrast, nucleotide-induced cell death was observed only in mixed cultures, but not in purified cultures of neurons or glia. ATP-induced neuronal death was blocked by the glutamatergic antagonists MK801 and DNQX and activation of P2X7 receptors by ATP decreased the uptake of [³H]-d-aspartate by cultured glial cells with a concomitant accumulation of it in the extracellular medium. These results suggest that ATP induces apoptosis of chick embryo retinal neurons in culture through activation of P2X7 and glutamate ionotropic receptors. Involvement of a P2X7 receptor-mediated inhibition of the glial uptake of glutamate is suggested.     

Expression of muscarinic acetylcholine receptors and choline acetyltransferase enzyme in cultured antennal sensory neurons and non‐neural cells of the developing moth Manduca sexta

Antennal sensory neurons of Manduca sexta emerge from epidermal cells that also give rise to sheath cells surrounding the peripheral parts of the neurons and to glial cells that enwrap the sensory axons in the antennal nerve. Reciprocal interactions between sensory neurons and glial cells are believed to aid in axon growth and guidance, but the exact nature of these interactions is not known. We investigated the possibility of cholinergic interactions in this process by locating muscarinic acetylcholine receptors (mAChRs) and choline acetyltransferase (ChAT) enzyme in cultured antennal sensory neurons and non‐neural cells. ChAT and mAChRs were present in the sensory neurons from the first day in culture. Therefore, the sensory neurons are probably cholinergic, as previously suggested, but they may also be controlled by ACh. In 7‐day‐old cultures a subgroup of small non‐neural cells with processes expressed ChAT activity, and in 14‐day‐old cultures non‐neural cells that formed lamellipodia and scaffoldlike structures on the culture substrate were labeled with ChAT antibody. mAChR activity was detected in similar non‐neural cells but only in areas surrounding the nuclei. In addition, mAChRs were found in flat lamellipodia and filopodia forming cells that were present in 1‐day‐old cultures and grew in size during the 2 week investigation period. These findings suggest muscarinic cholinergic interactions between the neural and non‐neural cells during the development of Manduca antenna. © 2004 Wiley Periodicals, Inc. J Neurobiol, 2005     

Cisplatin effects on F-actin and matrix proteins precede renal tubular cell detachment and apoptosis in vitro

In primary cultures of porcine proximal tubular kidney cells and LLC-PK1 cells cisplatin (5 - 50 microM) caused apoptosis and cell detachment; in both systems cell detachment occurred, preceded by a loss of cytoskeletal F-actin stress fibers within 4 - 6 h, and a reduction of mRNA encoding for fibronectin, collagen a2 type (IV) and laminin B2 within 17 - 41 h. Prevention of F-actin damage by phalloidin prevented nuclear fragmentation, suggesting a relation between F-actin damage and apoptosis. Overexpression of Bcl-2 also prevented apoptosis, but did not prevent damage to the F-actin skeleton or the reduction of mRNA expression of the matrix proteins. These results suggest that Bcl-2 overexpression interferes with apoptotic signals downstream of F-actin. The relevance of these results for cell detachment in kidney toxicity is discussed.     

Controlled adhesion and growth of long term glial and neuronal cultures on Parylene-C

This paper explores the long term development of networks of glia and neurons on patterns of Parylene-C on a SiO(2) substrate. We harvested glia and neurons from the Sprague-Dawley (P1-P7) rat hippocampus and utilized an established cell patterning technique in order to investigate cellular migration, over the course of 3 weeks. This work demonstrates that uncontrolled glial mitosis gradually disrupts cellular patterns that are established early during culture. This effect is not attributed to a loss of protein from the Parylene-C surface, as nitrogen levels on the substrate remain stable over 3 weeks. The inclusion of the anti-mitotic cytarabine (Ara-C) in the culture medium moderates glial division and thus, adequately preserves initial glial and neuronal conformity to underlying patterns. Neuronal apoptosis, often associated with the use of Ara-C, is mitigated by the addition of brain derived neurotrophic factor (BDNF). We believe that with the right combination of glial inhibitors and neuronal promoters, the Parylene-C based cell patterning method can generate structured, active neural networks that can be sustained and investigated over extended periods of time. To our knowledge this is the first report on the concurrent application of Ara-C and BDNF on patterned cell cultures.     

Combined effects of aphidicolin and retinoic acid on proliferation and differentiation of human leukaemic (HL-60) cells

The relationships between replicative DNA synthesis and retinoic acid (RA)-induced differentiation of human promyelocytic leukaemic (HL-60) cells are evaluated with the use of Aphidicolin, a specific and reversible inhibitor of DNA polymerase alpha (alpha). Addition of a sublethal concentration of Aphidicolin (0.4 microM) in culture for 3 days suppresses DNA synthesis to a similar level of the resting stage (day 8) in control cultures. DNA synthesis is reactivated to the level observed in the growing stage of control cultures once Aphidicolin is removed after 3 days in culture. The level of DNA synthesis at the early stage of RA-induction (day 3) is suppressed by only 17% when compared to control cultures. The inhibitory effect of Aphidicolin on DNA synthesis in both control cultures and RA-induced cell cultures is similar. However, no reactivation of DNA synthesis is observed after removal of Aphidicolin on day 3 from RA-induced cell cultures. Flow cytometric analysis of DNA content on day 3 reveals that cells accumulate in G1 and early S phases of the cell cycle after exposure to Aphidicolin with or without RA. Of interest is the fact that, while Aphidicolin alone did not induce cells to differentiate, neither did it interfere with RA-induced cell differentiation (the rate of RA-induced cell differentiation in the presence of Aphidicolin is similar to that of RA-treated cultures in the absence of Aphidicolin). These results suggest that the combined use of Aphidicolin and RA may inhibit leukaemic cell proliferation more effectively without causing severe cytotoxicity and without interfering with RA-induced cell differentiation.     

Protective effect of Bcl-2 in NSO myeloma cell culture is greater in more stressful environments

In the present study, the protective effects of Bcl-2 over-expression in a suspension culture (without any adaptation) and spent medium (low nutrient and high toxic metabolite conditions) were investigated. In the suspension culture without prior adaptation, the viability of the control cell line fall to 0% by day 7, whereas the Bcl-2 cell line had a viability of 65%. The difference in the viability and viable cell density between the Bcl-2 and control cell lines was more apparent in the suspension culture than the static culture, and became even more apparent on day 6. Fluorescence microscopic counting revealed that the major mechanism of cell death in the control cell line in both the static and suspension cultures was apoptosis. For the Bcl-2 cell lines, necrosis was the major mode of cell death in the static culture, but apoptosis became equally important in the suspension culture. When the NSO 6A1 cell line was cultured in spent medium taken from a 14 day batch culture, the control cell line almost completely lost its viability by day 5, whereas, the Bcl-2 still had a viability of 73%. The viable cell density and viability of the Bcl-2 cell line cultivated in fresh medium were 2.2 and 2.7 fold higher, respectively, than those of the control cultures. However, the viable cell density and viability of the Bcl-2 cultivated in the spent medium were 8.7 and 7.8 fold higher, respectively, than those of the control cultures. Most of the dead cells in the control cell line were apoptotic; whereas, the major cell death mechanisms in the Bcl-2 cell line were necrotic.     

Preconditioning-induced protection against cyanide-induced neurotoxicity is mediated by preserving mitochondrial function.

The central nervous system is one of the main target organs in cyanide toxicity. In this study, primary cultures of chick embryonic neurons were used to characterize sodium cyanide (NaCN)-induced cell death and to investigate the mechanism of NaCN-mediated preconditioning. After treatment of the cells with 1mM NaCN for 1h followed by a NaCN-free incubation period of 23 h, we observed features of apoptosis such as a reduction in nuclear size, chromatin condensation and nuclear fragmentation as evaluated by nuclear staining with Hoechst 33258 and electron microscopy. In addition, NaCN-induced neurotoxicity was reduced by the protein synthesis inhibitor cycloheximide (CHX) suggesting an active type of cell death. Most of the neurons with condensed chromatin and a shrunken nuclei also showed membrane damage at a late stage. Mitochondrial membrane potential as well as the protein levels of Bcl-2 and Bcl-x(L) decreased 15-60 min and 1-3 h after the exposure to NaCN (1mM, 1h), respectively. Preconditioning caused by incubating chick neurons with 100 microM NaCN for 30 min followed by a NaCN-free interval of 24h significantly protected the neurons against subsequent NaCN (1mM, 1h)-induced damage. Preconditioning prevented NaCN-induced decrease in the mitochondrial membrane potential as well as in the protein levels of Bcl-2 and Bcl-x(L) suggesting that preconditioning-induced neuroprotection is mediated by preserving mitochondrial function.     

The Fetal Hypothalamus Has the Potential to Generate Cells with a Gonadotropin Releasing Hormone (GnRH) Phenotype

Background

Neurospheres (NS) are colonies of neural stem and precursor cells capable of differentiating into the central nervous system (CNS) cell lineages upon appropriate culture conditions: neurons, and glial cells. NS were originally derived from the embryonic and adult mouse striatum subventricular zone. More recently, experimental evidence substantiated the isolation of NS from almost any region of the CNS, including the hypothalamus.

Methodology/Findings

Here we report a protocol that enables to generate large quantities of NS from both fetal and adult rat hypothalami. We found that either FGF-2 or EGF were capable of inducing NS formation from fetal hypothalamic cultures, but that only FGF-2 is effective in the adult cultures. The hypothalamic-derived NS are capable of differentiating into neurons and glial cells and most notably, as demonstrated by immunocytochemical detection with a specific anti-GnRH antibody, the fetal cultures contain cells that exhibit a GnRH phenotype upon differentiation.

Conclusions/Significance

This in vitro model should be useful to study the molecular mechanisms involved in GnRH neuronal differentiation.     

MUCOPOLYSACCHARIDES SYNTHESIZED BY CULTURED GLIAL CELLS DERIVED FROM A PATIENT WITH SANFILIPPO A SYNDROME

—Glial cells were cultured from brain tissue obtained at autopsy of a patient with Sanfilippo A syndrome. Mucopolysaccharides were labeled by culturing the cells in the presence of [³⁵S]sulfate. After proteolysis, intracellular and media-elaborated mucopolysaccharides were fractionated by Dowex 1 chromatography. One fraction, identified as heparan sulfate by chromatographic, electrophoretic, and enzyme susceptibility properties, accumulated in Sanfilippo glial cells in greater amounts than in controls. Heparan sulfate was also excreted into the culture media by both Sanfilippo and normal cultures, and it constituted a major fraction of the sulfated mucopolysaccharides synthesized by glial cells. Sanfilippo and normal fibroblasts were also included in these studies for comparative purposes. Sanfilippo fibroblasts accumulated significantly increased amounts of heparan sulfate as compared to normal fibroblasts. Heparan sulfate was excreted into the culture media by Sanfilippo and normal fibroblasts in equivalent amounts, but in contrast to glial cells, it was only a minor component of the sulfated mucopolysaccharides produced. Cultured glial cells should provide a useful system for investigating the role of heparan sulfate in glial cell function.     

Expression of muscarinic acetylcholine receptors and choline acetyltransferase enzyme in cultured antennal sensory neurons and non-neural cells of the developing moth Manduca sexta

Antennal sensory neurons of Manduca sexta emerge from epidermal cells that also give rise to sheath cells surrounding the peripheral parts of the neurons and to glial cells that enwrap the sensory axons in the antennal nerve. Reciprocal interactions between sensory neurons and glial cells are believed to aid in axon growth and guidance, but the exact nature of these interactions is not known. We investigated the possibility of cholinergic interactions in this process by locating muscarinic acetylcholine receptors (mAChRs) and choline acetyltransferase (ChAT) enzyme in cultured antennal sensory neurons and non-neural cells. ChAT and mAChRs were present in the sensory neurons from the first day in culture. Therefore, the sensory neurons are probably cholinergic, as previously suggested, but they may also be controlled by ACh. In 7-day-old cultures a subgroup of small non-neural cells with processes expressed ChAT activity, and in 14-day-old cultures non-neural cells that formed lamellipodia and scaffoldlike structures on the culture substrate were labeled with ChAT antibody. mAChR activity was detected in similar non-neural cells but only in areas surrounding the nuclei. In addition, mAChRs were found in flat lamellipodia and filopodia forming cells that were present in 1-day-old cultures and grew in size during the 2 week investigation period. These findings suggest muscarinic cholinergic interactions between the neural and non-neural cells during the development of Manduca antenna.     

Recent advances in neural cell culture

Cells isolated from the avian and mammalian central and peripheral nervous system and cultured in vitro provide an opportunity to study in situ properties of neurons and glial cells under relatively simple and carefully controlled conditions. Since Harrison's success in maintaining in vitro embryonic frog spinal cord 80 years ago, neural tissue culture has developed into an important and versatile discipline of neuroscience. The techniques developed in the past fall into four broad classes: Explant cultures, which are explanted from specific neuroanatomic loci to substrates as small tissue fragments. Dissociated cell cultures, which involve the seeding of enzymatically or mechanically dispersed cells on various attachment substrates. Reaggregate cultures, which require re-association of dissociated cells into small aggregates. Purified cell populations, which are prepared by the isolation of different cell types by gradient centrifugation or other separation techniques. These cultures have been utilized in studying various aspects of brain development and function. In this review several areas of significant and stimulating development in neural cell culture have been documented. They include formulation of serum-free medium, effects of growth factors, utilization of cell type-specific markers, and isolation and culture of purified neuronal/glial cells.