嗅神经鞘细胞的培养纯化及体外生长特性

采用原代培养的方法,从2,5月成年大鼠的嗅球分离培养嗅神经鞘细胞（OECs),培养6天后,用阿糖胞苷（Ara-C)抑制,差速贴壁,Forskolin和BPE营养物质处理,根据P75蛋白免疫细胞化学染色和形态学特征分析了所得细胞的纯度,同时对不同培养时期的OECs 的形态进行观察和纯化后的活力测定。实验结果显示：（1）这种纯化方法简单,经济,快捷,所得的OECS纯度可达95％以上,并且随培养时间延长,细胞仍保持较高的纯度。（2）在培养早期2天到5天主要以巨噬细胞状,多极状,不规则状为主,培养中期7天到20天主要以扁平的双极,三极为主。晚期20天以后呈现双极,三极形态,其起上有许多细小的棘突。93）其中以培养早中期细胞的活力较好,培养20天以后,细胞活力较差,本研究为以OECs 作为移植材料对促进神经再生的研究获得丰富的细胞来源奠定了基础。     

Protein kinase C sensitizes olfactory adenylate cyclase

Effects of neurotransmitters on cAMP-mediated signal transduction in frog olfactory receptor cells (ORCs) were studied using in situ spike recordings and radioimmunoassays. Carbachol, applied to the mucosal side of olfactory epithelium, amplified the electrical response of ORCs to cAMP-generating odorants, but did not affect unstimulated cells. A similar augmentation of odorant response was observed in the presence of phorbol dibutyrate (PDBu), an activator of protein kinase C (PKC). The electrical response to forskolin, an activator of adenylate cyclase (AC), was also enhanced by PDBu, and it was attenuated by the PKC inhibitor Goe 6983. Forskolin-induced accumulation of cAMP in olfactory tissue was potentiated by carbachol, serotonin, and PDBu to a similar extent. Potentiation was completely suppressed by the PKC inhibitors Goe 6983, staurosporine, and polymyxin B, suggesting that the sensitivity of olfactory AC to stimulation by odorants and forskolin was increased by PKC. Experiments with deciliated olfactory tissue indicated that sensitization of AC was restricted to sensory cilia of ORCs. To study the effects of cell Ca2+ on these mechanisms, the intracellular Ca2+ concentration of olfactory tissue was either increased by ionomycin or decreased by BAPTA/AM. Increasing cell Ca2+ had two effects on cAMP production: (a) the basal cAMP production was enhanced by a mechanism sensitive to inhibitors of calmodulin; and (b) similar to phorbol ester, cell Ca2+ caused sensitization of AC to stimulation by forskolin, an effect sensitive to Goe 6983. Decreasing cell Ca2+ below basal levels rendered AC unresponsive to stimulation by forskolin. These data suggest that a crosstalk mechanism is functional in frog ORCs, linking the sensitivity of AC to the activity of PKC. At increased activity of PKC, olfactory AC becomes more responsive to stimulation by odorants, forskolin, and cell Ca2+. Neurotransmitters appear to use this crosstalk mechanism to regulate olfactory sensitivity.     

Degeneration and regeneration of olfactory cells induced by ZnSO4 and other chemicals

The necrotic effect of various salt solutions was tested on the catfish olfactory mucosa. Only zinc cations were able to induce an extensive degeneration of the olfactory cells. Two different modes of irrigation of the mucosa with zinc sulfate were investigated. (1) The olfactory cavity is flushed with the chemical for not more than a few seconds. At concentrations above 30 mM, the resulting damage is very reproducible, largely concentration independent and almost completely specific for the olfactory receptor cells. The non-sensory respiratory cells are unaffected, the sustentacular cells surrounding the receptor cells are affected mainly by a loss of microvilli. The olfactory receptor cells, on the contrary, start to degenerate within a few hours and by day 4 only 20% of the original receptor population remains. Division of the mucosal basal cells increases during days 3 and 4 on and day 6 olfactory receptor cells reach the bare surface of the lamella. After day 7, the receptor population reaches a level of more than 80% of its original value. Because of the absence of sustentacular processes covering the olfactory cell's knobs on day 6, it has been possible to confirm that each of the two types of olfactory receptor cells previously characterized are concentrated on each half of the mucosa. (2) The salt is maintained in contact with the tissue for several days. After this treatment most of the lamellae are irreversibly destroyed, some regeneration occurs in limited areas of the mucosa. In these small areas, indifferent respiratory cells reappear first between 20 and 35 days. It is only when the structure of the olfactory tissue is completely reorganized that the new receptor cells reappear between days 45 and 55. Regeneration is not completed before 60–65 days.     

Primary culture of antennal mechanoreceptor neurons of Manduca sexta

We have developed a primary cell culture system of antennal mechanoreceptor neurons from early-stage pupal sphinx moth Manduca sexta. Dissociated neurons from the moth antennae differentiated, grew and survived for several weeks in a conditioned culture medium. Bipolar neurons with soma diameters of 10-25 microns from the basal portion of the antennae could be positively identified as mechanoreceptor neurons, presumably derived from Johnston's organ, using a monoclonal antibody that recognizes neurofilaments in these neurons. The immunoreactivity was clear and specific from the first day after dissociation and became stronger during several days in culture. These neurons appeared healthy and showed normal whole-cell properties only a few days after plating. We found numerous mechanosensitive ion channels responding to both negative and positive pressures on the somata and neurites of differentiated neurons. This new culture system provides access to mechanoreceptor neurons that has never been possible before, allowing the use of both mechanical and electrical stimuli on neurons that are free from the accessory structures surrounding them in intact preparations.     

Cultured chick yolk sac epithelium: structure and IgG surface binding

The chick yolk sac endoderm transports maternal immunoglobulin G (IgG) from the yolk into the embryo during development, providing the newly hatched chick with passive immunity until it becomes immunocompetent. To study this transport process, chick yolk sac endodermal cells isolated from embryos of 6 to 18 days of incubation were grown in vitro on a collagen substrate. The cultured cells possessed a remarkable structural similarity to the in vivo tissue and reformed a polarized confluent epithelium with tight junctions and desmosomes joining the cells at their apical margins. In addition, the cells exhibited apical microvilli, numerous phagolysosomes in the cytoplasm and retained the expression of the yolk sac endoderm-specific enzyme marker, cysteine lyase. Importantly, the cultured cells retained the ability to specifically bind IgG as demonstrated by indirect immunofluorescence. Chicken IgG bound to the cultured cells at 4 degrees C in a diffuse pattern that clustered into a punctate pattern when a second antibody was used. Cultures from yolk sacs of day 6 through day 18 of development all demonstrated this immunofluorescent labeling for at least 14 days in culture. These results demonstrate that cultured yolk sac endoderm maintains its differentiated morphology and ability to bind IgG.     

Morphology and lactose synthesis in tissue culture of mammary alveoli isolated from lactating mice

Summary Mammary epithelial cells from lactating mice synthesize and secrete lactose in culture and retain many features of their in vivo morphology if mammary glands are only partially dissociated to alveoli, rather than completely dissociated to single cells. After 5 d in culture lactose synthesis by alveoli cultured on floating collagen gels is 10 to 20 times higher than in cultures of single cells on floating collagen gels. Moreover, mammary alveoli in culture retain sensitivity to lactogenic hormones; the synthesis of lactose by alveoli depends on the continued presence of insulin and either hydrocortisone or prolactin. In addition, within alveoli the original juxtaposition of constituent epithelial cells is retained, and cells are cuboidal and have many microvilli and fat droplets. In contrast, alveoli on attached gels flatten and lose their secretory morphology. These results indicate that the shape of the cells, presence of lactogenic hormones, and maintenance of epithelial:epithelial cell contacts are required for maintenance of mammary epithelial cell differentiation in culture. This research was supported by Grants CA-16392 and AG-02909 from the National Institutes of Health and Institutional Grant IN 119 from the American Cancer Society.     

Olfactory marker protein during ontogeny: Immunohistochemical localization

Specific immunohistochemical staining for the olfactory marker protein (OMP) is first demonstrated in rat olfactory receptor neurons at embryonic day 18, at which age no OMP can be seen in the olfactory bulb or vomeronasal epithelium. At 21 days OMP staining in the olfactory epithelium is more extensive and is evident in the fibrous and glomerular layers of the bulb as well. Staining intensity increases progressively until the full adult pattern is seen by 1 month postnatally. In the vomeronasal organ, staining is not observed until the fourth postnatal day and, even then, only with higher antiserum concentrations. In mice, very similar results are obtained, except for a much earlier appearance of OMP, on embryonic day 14. Olfactory epithelium from 12- and 13-day rat embryos maintained in organ culture for up to 2 weeks did not exhibit OMP staining, nor did several neural or nonneural tissues from adult animals. The temporal and causal interrelationships between OMP and other indicators of olfactory receptor cell maturation are considered.     

The retinal pigment epithelial cells of the adult newt and rat under conditions of in vitro organotypic culture

To understand why the retinal pigment epithelium (RPE) has different potentials for neural differentiation in lower and higher vertebrates, the RPEs of adult newts and rats were compared under similar in vitro cultivation conditions. The RPEs of both animal species were organotypically cultivated within the posterior eye wall under constant rotation in the serum medium free of growth factors. Comparison of the cell morphology, proliferation, and expression of pan-neural markers demonstrated that the RPE cells of adult newts and rats under similar in vitro conditions displayed both similarities and differemces. They were able to synthesize DNA but rarely divided mitotically. In addition, part of the RPE cells of both the newt and the rat were dislodged from the layer, migrated, and acquired a macrophage phenotype. However, the majority of the cells retained the initial morphology and remained within the layer. In several cases, these cells displayed the initial characteristics of neural differentiation, namely, expression of pan-neural proteins. The difference between the newt and rat RPE cells was in the ability of the former to generate in vitro an additional row of dedifferentiated NF-200-positive cells, characteristic of in vivo newt retinal regeneration. These data demonstrate that the RPE cells of the adult newt and rat retain the potential of manifesting neural cell traits; however, more advanced changes towards differentiation are characteristic of only the newt RPE.     

Love Is Blind: Indiscriminate Female Mating Responses to Male Courtship Pheromones in Newts (Salamandridae)

Internal fertilization without copulation or prolonged physical contact is a rare reproductive mode among vertebrates. In many newts (Salamandridae), the male deposits a spermatophore on the substrate in the water, which the female subsequently takes up with her cloaca. Because such an insemination requires intense coordination of both sexes, male newts have evolved a courtship display, essentially consisting of sending pheromones under water by tail-fanning towards their potential partner. Behavioral experiments until now mostly focused on an attractant function, i.e. showing that olfactory cues are able to bring both sexes together. However, since males start their display only after an initial contact phase, courtship pheromones are expected to have an alternative function. Here we developed a series of intraspecific and interspecific two-female experiments with alpine newt (Ichthyosaura alpestris) and palmate newt (Lissotriton helveticus) females, comparing behavior in male courtship water and control water. We show that male olfactory cues emitted during tail-fanning are pheromones that can induce all typical features of natural female mating behavior. Interestingly, females exposed to male pheromones of their own species show indiscriminate mating responses to conspecific and heterospecific females, indicating that visual cues are subordinate to olfactory cues during courtship.     

Neural cell differentiation from retinal pigment epithelial cells of the newt: an organ culture model for the urodele retinal regeneration.

Transdifferentiation from retinal pigment epithelium (RPE) to neural retina (NR) was studied under a new culture system as an experimental model for newt retinal regeneration. Adult newt RPEs were organ cultured with surrounding connective tissues, such as the choroid and sclera, on a filter membrane. Around day 7 in vitro, lightly pigmented "neuron-like cells" with neuritic processes were found migrating out from the explant onto the filter membrane. Their number gradually increased day by day. BrdU-labeling study showed that RPE cells initiated to proliferate under the culture condition on day 4 in vitro, temporally correlating to the time course of retinal regeneration in vivo. Histological observations of cultured explants showed that proliferating RPE cells did not form the stratified structure typically observed in the NR but they rather migrated out from the explants. Neuronal differentiation was examined by immunohistochemical detection of various neuron-specific proteins; HPC-1 (syntaxin), GABA, serotonin, rhodopsin, and acetylated tubulin. Immunoreactive cells for these proteins always possessed fine and long neurite-like processes. Numerous lightly pigmented cells with neuron-like morphology showed HPC-1 immunoreactivity. Fibroblast growth factor-2 (FGF-2), known as a potent factor for the transdifferentiation of ocular tissues in various vertebrates, substantially increased the numbers of both neuron-like cells and HPC-1-like immunoreactive cells in a dose-dependent manner. These results indicate that our culture method ensures neural differentiation of newt RPE cells in vitro and provides, for the first time, a suitable in vitro experimental model system for studying tissue-intrinsic factors responsible for newt retinal regeneration.     

Granule cell induction of 9-O-acetyl gangliosides on cerebellar glia in microcultures

In previous studies we have shown that the expression of acetylated gangliosides recognized by the JONES monoclonal antibody is correlated with regions of cell migration in the developing rat nervous system. In this study we have investigated the expression of these gangliosides in two different types of cultures prepared from dissociated postnatal rat cerebella. In the first type, cells are plated after dissociation under conditions where most of the glial cells develop a stellate morphology that anchors neurons but does not support their migration. In the second type of culture, cells are plated in a ratio of four neurons to one glial cell and under these conditions the predominant form of astroglia is an elongate form that supports the migration of granule neurons. Granule neurons express JONES antigens in dissociated cell suspensions and in cultures in which cells are plated either after dissociation or in a 4:1 neuron:glia ratio. On the other hand, glial cells grown in the absence of neurons are JONES negative. In addition, the expression of JONES gangliosides by glial cells is different in the two types of culture. In cultures where the astroglial cells display the stellate morphology only a small proportion show JONES staining. Cultures in which the glial cells assume the elongate morphology have a significantly higher number of JONES-positive astroglia.     

Ca2+-activated K+ currents regulate odor adaptation by modulating spike encoding of olfactory receptor cells

The olfactory system is thought to accomplish odor adaptation through the ciliary transduction machinery in olfactory receptor cells (ORCs). However, ORCs that have lost their cilia can exhibit spike frequency accommodation in which the action potential frequency decreases with time despite a steady depolarizing stimulus. This raises the possibility that somatic ionic channels in ORCs might serve for odor adaptation at the level of spike encoding, because spiking responses in ORCs encode the odor information. Here I investigate the adaptational mechanism at the somatic membrane using conventional and dynamic patch-clamp recording techniques, which enable the ciliary mechanism to be bypassed. A conditioning stimulus with an odorant-induced current markedly shifted the response range of action potentials induced by the same test stimulus to higher concentrations of the odorant, indicating odor adaptation. This effect was inhibited by charybdotoxin and iberiotoxin, Ca2+-activated K+ channel blockers, suggesting that somatic Ca2+-activated K+ currents regulate odor adaptation by modulating spike encoding. I conclude that not only the ciliary machinery but also the somatic membrane currents are crucial to odor adaptation.     

Developmental sequence of expression of voltage-dependent currents in embryonic Xenopus laevis myocytes.

Although the development of several of the voltage-dependent currents in embryonic amphibian myocytes has been described, the overall muscle electrical development, particularly the relative times of expression of different voltage-dependent currents, has not been addressed in a single study under one set of conditions. We have found that, in mesoderm isolated and cultured from neurula stage embryos, myocytes are identifiable before they express voltage-gated currents. These ionic currents are absent from all Xenopus mesodermal cells during the late gastrula/early neurula stages of embryonic development. At about the time of first somite segregation an inward rectifier K+ current is expressed in some myocytes, followed within 2 hr by a delayed rectifier K+ current. The density of both currents increases fourfold over the next 24 hr in culture. A Na+ current is not expressed in large numbers of myocytes until late in this culture period, at about the time that a slow Ca2+ current appears. Under our culture conditions the myocytes have a very low chloride conductance. A fast inactivating component to the outward K+ current is expressed in all myocytes by 24 hr in culture. In some experiments we dissociated embryos at later times and made recordings when all previously isolated myocytes expressed currents. In the late dissociations, most myocytes did not express currents, but developed them after a short period in culture. Because we have evidence that in vivo development is more closely approximated by the early dissociations, these results suggest that dissociation causes some degree of dedifferentiation.     

Dissociated Neurons and Glial Cells Derived from Rat Inferior Colliculi after Digestion with Papain

The formation of gliosis around implant electrodes for deep brain stimulation impairs electrode–tissue interaction. Unspecific growth of glial tissue around the electrodes can be hindered by altering physicochemical material properties. However, in vitro screening of neural tissue–material interaction requires an adequate cell culture system. No adequate model for cells dissociated from the inferior colliculus (IC) has been described and was thus the aim of this study. Therefore, IC were isolated from neonatal rats (P3_5) and a dissociated cell culture was established. In screening experiments using four dissociation methods (Neural Tissue Dissociation Kit [NTDK] T, NTDK P; NTDK PN, and a validated protocol for the dissociation of spiral ganglion neurons [SGN]), the optimal media, and seeding densities were identified. Thereafter, a dissociation protocol containing only the proteolytic enzymes of interest (trypsin or papain) was tested. For analysis, cells were fixed and immunolabeled using glial- and neuron-specific antibodies. Adhesion and survival of dissociated neurons and glial cells isolated from the IC were demonstrated in all experimental settings. Hence, preservation of type-specific cytoarchitecture with sufficient neuronal networks only occurred in cultures dissociated with NTDK P, NTDK PN, and fresh prepared papain solution. However, cultures obtained after dissociation with papain, seeded at a density of 2×10⁴ cells/well and cultivated with Neuro Medium for 6 days reliably revealed the highest neuronal yield with excellent cytoarchitecture of neurons and glial cells. The herein described dissociated culture can be utilized as in vitro model to screen interactions between cells of the IC and surface modifications of the electrode.     

Changes in the number of chick ciliary ganglion neuron processes with time in cell culture

The purpose of this study was to describe the shape of chick ciliary ganglion neurons dissociated from embryonic day 8 or 9 ganglia and maintained in vitro. Most of the neurons were multipolar during the first three days after plating, with an average of 6.0 processes extending directly from the cell body. The neurons became unipolar with time. The remaining primary process accounted for greater than 90% of the total neuritic arbor. This striking change in morphology was not due to the selective loss of multipolar cells, or to an obvious decline in the health of apparently intact cells. The retraction of processes was neither prevented nor promoted by the presence of embryonic muscle cells. Process pruning occurred to the same extent and over the same time course whether the cells were plated on a monolayer of embryonic myotubes or on a layer of lysed fibroblasts. Process retraction is not an inevitable consequence of our culture conditions. Motoneurons dissociated from embryonic spinal cords remained multipolar over the same period of time. We conclude that ciliary ganglion neurons breed true in dissociated cell culture in that the multipolar-unipolar transition reflects their normal, in vivo, developmental program.     

CNS glial cells support in vitro survival, division, and differentiation of dissociated olfactory neuronal progenitor cells.

Olfactory receptor neurons (ORNs) are replaced and differentiate in adult animals, but differentiation in dissociated cell culture has not been demonstrated. To test whether contact with the CNS regulates maturation, neonatal rat olfactory cells were grown on a culture substrate or on CNS astrocytes. Mature ORNs, immunopositive for olfactory marker protein (OMP), disappeared rapidly from both systems. Neurons positive for neuron-specific tubulin (immature and mature) disappeared from substrate-only cultures, but remained abundant in the cocultures. OMP-positive neurons reappeared after 10 days in vitro. Pulse labeling with [3H]thymidine showed extensive neurogenesis of both immature and mature olfactory neurons. This demonstrates, in vitro, both division and differentiation of olfactory progenitor cells.     

In vitro and in vivo characterization of blastemal cells from regenerating newt limbs.

To better characterize the cells involved in newt limb regeneration, blastemal cells from accumulation and differentiation phase blastemas were grown in dissociated cell culture, and their morphology and antigenic phenotype determined using a variety of antibodies directed against intermediate filaments, cell adhesion molecules, and extracellular matrix molecules. In addition to previously described blastemal cell morphologies, many of the cells in these cultures had a round cell body, with an eccentrically placed nucleus and a cytoplasm filled with autofluorescent granules. The majority of accumulation phase blastemal cells labeled with antibodies against GFAP, vimentin, 22/18 as well as with antibodies against NCAM, L-1, laminin, and fibronectin. The majority of differentiation phase blastemal cells had a similar phenotype but lacked expression of vimentin and fibronectin. Comparison of the blastemal phenotype in vitro and in vivo showed similar expression characteristics. However, in differentiation phase blastemas, laminin immunoreactivity was concentrated in specific locations. In addition, the proliferation of cultured blastemal cells is stimulated by the addition of a crude brain extract, consistent with previous studies in vivo and in vitro. Taken together, these observations suggest that dissociated cultures of newt limb blastemal cells provide a suitable model for the analysis of the cell and molecular mechanisms involved in limb regeneration.     

AN ANALYSIS OF DIFFERENTIATIVE CAPACITY OF PIGMENTED EPITHELIAL CELLS OF ADULT NEWT IRIS IN CLONAL CELL CULTURE

Singly dissociated cells from dorsal and ventral iris epithelia ( iris iridica ) of adult newts were cultured separately at clonal density to analyse their growth and differentiative capacity. Usually some attached cells began to proliferate on 12th day of culture, and grew with loss of melanosomes to form clonal cell colonies. Up to 30 days after inoculation, most of the clonal colonies formed typical epithelial monolayer sheets which consisted mostly of nonpigmented cells. Then, in some of those colonies, cells piled up together and form typical lens structures containing lens antigens. A month and a half after culturing, 30 to 40% of single iris cells, which had been previously marked, grew to form clonal colonies consisting of more than 100 cells. About 30% of these colonies expressed lens specificity and no significant differences in efficiency of colony formation and differentiation were detected between the dorsal cells and the ventral, suggesting that potent cells capable of transdifferentiating into lens cells are evenly distributed in all parts of the newt iris epithelium.     

Dissociated neurons of the pupal blowfly antenna in cell culture

Primary cell cultures are useful for studying the function of neurons in a simplified and controlled environment. We established a primary culture of antennal cells from pupal blowflies in order to investigate olfactory receptor neurons. In cultures, neuron-like cells were identified on the basis of morphology and immunocytochemical characterization with anti-HRP staining. Neuron-like cells showed variety in the extension pattern of neurites. Many neuron-like cells extended a single prominent long process, which reached about 200 mum after four days, and several short ones. However, some neuron-like cells differentiated in other ways; some exhibited bipolar or multipolar processes, distinct from intact olfactory receptor neurons. The size of cell bodies of neuron-like cells as divisible into two groups; approx. 7 mum diameter and 10-15 mum diameter. Neuron-like cells in culture will provide a good model for electrophysiological analysis and for developmental studies of olfactory receptor neurons.