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.     

Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents

The marine gastropod mollusk Aplysia californica has a venerable history as a model of nervous system function, with particular significance in studies of learning and memory. The typical preparations for such studies are ones in which the sensory and motoneurons are left intact in a minimally dissected animal, or a technically elaborate neuronal co-culture of individual sensory and motoneurons. Less common is the isolated neuronal preparation in which small clusters of nominally homogeneous neurons are dissociated into single cells in short term culture. Such isolated cells are useful for the biophysical characterization of ion currents using patch clamp techniques, and targeted modulation of these conductances. A protocol for preparing such cultures is described. The protocol takes advantage of the easily identifiable glutamatergic sensory neurons of the pleural and buccal ganglia, and describes their dissociation and minimal maintenance in culture for several days without serum.     

Short-Term Functional and Morphological Changes in the Primary Cultures of Trigeminal Ganglion Cells

Several studies have proved that glial cells, as well as neurons, play a role in pain pathophysiology. Most of these studies have focused on the contribution of central glial cells (e.g., microglia and astrocytes) to neuropathic pain. Likewise, some works have suggested that peripheral glial cells, particularly satellite glial cells (SGCs), and the crosstalk between these cells and the sensory neurons located in the peripheral ganglia, play a role in the phenomenon that leads to pain. Nonetheless, the study of SGCs may be challenging, as the validity of studying those cells in vitro is still controversial. In this study, a research protocol was developed to examine the potential use of primary mixed neuronal–glia cell cultures obtained from the trigeminal ganglion cells (TGCs) of neonate mice (P10–P12). Primary cultures were established and analyzed at 4 h, 24 h, and 48 h. To this purpose, phase contrast microscopy, immunocytochemistry with antibodies against anti-βIII-tubulin and Sk3, scanning electron microscopy, and time-lapse photography were used. The results indicated the presence of morphological changes in the cultured SGCs obtained from the TGCs. The SGCs exhibited a close relationship with neurons. They presented a round shape in the first 4 h, and a more fusiform shape at 24 h and 48 h of culture. On the other hand, neurons changed from a round shape to a more ramified shape from 4 h to 48 h. Intriguingly, the expression of SK3, a marker of the SGCs, was high in all samples at 4 h, with some cells double-staining for SK3 and βIII-tubulin. The expression of SK3 decreased at 24 h and increased again at 48 h in vitro. These results confirm the high plasticity that the SGCs may acquire in vitro. In this scenario, the authors hypothesize that, at 4 h, a group of the analyzed cells remained undifferentiated and, therefore, were double-stained for SK3 and βIII-tubulin. After 24 h, these cells started to differentiate into SCGs, which was clearer at 48 h in the culture. Mixed neuronal–glial TGC cultures might be implemented as a platform to study the plasticity and crosstalk between primary sensory neurons and SGCs, as well as its implications in the development of chronic orofacial pain.     

Cryopreservation of Cortical Tissue Blocks for the Generation of Highly Enriched Neuronal Cultures

In this study, we outline a standardized protocol for the successful cryopreservation and thawing of cortical brain tissue blocks to generate highly enriched neuronal cultures. For this protocol the freezing medium used is 10% dimethyl sulfoxide (DMSO) diluted in Hank''s Buffered Salt Solution (HBSS). Blocks of cortical tissue are transferred to cryovials containing the freezing medium and slowly frozen at -1°C/min in a rate-controlled freezing container. Post-thaw processing and dissociation of frozen tissue blocks consistently produced neuronal-enriched cultures which exhibited rapid neuritic growth during the first 5 days in culture and significant expansion of the neuronal network within 10 days. Immunocytochemical staining with the astrocytic marker glial fibrillary acidic protein (GFAP) and the neuronal marker beta-tubulin class III, revealed high numbers of neurons and astrocytes in the cultures. Generation of neural precursor cell cultures after tissue block dissociation resulted in rapidly expanding neurospheres, which produced large numbers of neurons and astrocytes under differentiating conditions. This simple cryopreservation protocol allows for the rapid, efficient, and inexpensive preservation of cortical brain tissue blocks, which grants increased flexibility for later generation of neuronal, astrocyte, and neuronal precursor cell cultures.     

Effects of simulated microgravity on the development and maturation of dissociated cortical neurons

Although a wealth of evidence supports the hypothesis that some functions of the nervous system may be altered during exposure to microgravity, the possible changes in basic neuronal physiology are not easy to assess. Indeed, few studies have examined whether microgravity affects the development of neurons in culture. In the present study, a suspension of dissociated cortical cells from rat embryos were exposed to 24 h of simulated microgravity before plating in a normal adherent culture system. Both preexposed and control cells were used after a period of 7-10 d in vitro. The vitality and the level of reactive oxygen species of cultures previously exposed did not differ from those of normal cultures. Cellular characterization by immunostaining with a specific antibody displayed normal neuronal phenotype in control cells, whereas pretreatment in simulated microgravity revealed an increase of glial fibrillary acidic protein fluorescence in the elongated stellate glial cells. Electrophysiological recording indicated that the electrical properties of neurons preexposed were comparable with those of controls. Overall, our results indicate that a short time of simulated microgravity preexposure does not affect dramatically the ability of dissociated neural cells to develop and differentiate in an adherent culture system.     

Neuron culture from adult goldfish

Neurons and gla from the central nervous system of the adult teleost Carassius auratus have been grown as explant cultures of minced brain tissue and as trypsin dissociated cells. These cultures exhibit extensive neurite growth from two neuronal types, have organotypic ultrastructure, and contain electrically active cells. Autoradiographic data indicate that these neurons do not divide in culture, and histological evidence suggests that some mature neurons survive explantation and regenerate processes. However, explantation of brain fragments not containing undifferentiated cells, localized in the ventricular and subventricular zones in the brains of fish, resulted in mesenchymal and glial cell cultures only. Therefore, a contribution to the population of cells in culture by undifferentiated cells must be considered. The cultured neurons remained viable for at least 19 weeks and ultrastructural and electrophysiological data indicate synaptic interaction between cells in explant cultures.     

Characterisation of transverse slice culture preparations of postnatal rat spinal cord: preservation of defined neuronal populations

Spinal cord injury induces degenerative and regenerative processes and complex interactions of neurons with non-neuronal cells. In order to develop an in vitro tool for the investigation of such processes, we prepared and characterised spinal cord slice cultures (SCSC) from Wistar rats (p0–12). SCSC were sustained in vitro up to 12 days and characterised by immunohistochemistry. Calbindin⁺ neurons, distributed across the entire gray matter, were visible also after longer culture periods. NeuN⁺ neurons were best preserved in the dorsal horn whereas large NeuN⁺ and choline acetyltransferase⁺ motoneurons in the ventral horn vanished after 3 days in vitro. Nestin immunoreactivity was found in animals of all age groups, either in cells interspersed in the ependymal lining around the central canal or in cells resembling protoplasmic astrocytes. Glial fibrillary acidic protein⁺ astrocytes, initially restricted to the white matter, invaded the gray matter of SCSC early during the culture period. Microglial cells, stained by Griffonia simplicifolia isolectin B₄, were rapidly activated in the dorsal tract and in the gray matter but declined in number with time. SCSC derived from p0 or p3 animals showed a better preservation of the cytoarchitecture than cultures derived from older animals. In summary, SCSC undergo degenerative changes, but they contain defined neuronal populations, the cytoarchitecture is partially preserved and the glial reaction is limited.     

Nerve growth factor-inducible,large external (NILE) glycoprotein in developing rat cerebral cells in culture

Summary Fetal rat cerebral cells underwent neuronal differentiation in culture. This process was accompanied by distinct changes in the cellular glycoprotein pattern. The incorporation of [³H]-fucose into two proteins of apparent molecular weights of 30000 and 60000 daltons was significantly decreased and specific developmental changes were observed in a group of glycoproteins with high molecular weights (150000–250000 dalton). By means of indirect immunoprecipitation one of them was identified as NILE gp (nerve growth factor-inducible large external) glycoprotein (200000 dalton), a marker of central and peripheral neurons. Its developmental expression on neurons of dissociated rat cerebral cultures was studied using the indirect immunofluorescence technique and compared to the fluorescent-labeling pattern of other neuronal markers. Neurons expressing NILE gp were detected as early as after one day in culture. No preferential staining of neuntes versus cell bodies was observed. Two classes of NILE gp-positive cells were identified. One group consisted of a rounded cell-type, whereas the other group was represented by larger, more spindle-shaped neurons with a limited number of neuritic processes. In most cases one of these neuritic processes was preferentially labeled. Astroglia cells, as identified by immunolabeling with antisera against the glial acidic fibrillary protein, were observed to develop and mature after the first week in culture. NILE-positive neurons were found to be positioned in close association with glial cell processes.     

Neuron culture from adult goldfish.

Neurons and glia from the central nervous system of the adult teleost Carassius auratus have been grown as explant cultures of minced brain tissue and as trypsin dissociated cells. These cultures exhibit extensive neurite growth from two neuronal types, have organotypic ultrastructure, and contain electrically active cells. Autoradiographic data indicate that these neurons do not divide in culture, and histological evidence suggests that some mature neurons survive explantation and regenerate processes. However, explantation of brain fragments not containing undifferentiated cells, localized in the ventricular and subventricular zones in the brains of fish, resulted in mesenchymal and glial cell cultures only. Therefore, a contribution to the population of cells in culture by undifferentiated cells must be considered. The cultured neurons remained viable for at least 19 weeks and ultrastructural and electrophysiological data indicate synaptic interaction between cells in explant cultures.     

Effect of dissociative methods on cortisol binding and glutamyltransferase inducibility in chick embryo retina

The ability of the isolated embryonic chick retina (12 days) to bind a steroid (cortisol) decreases when the tissue is dissociated; the extent of this decrease depends upon the method of dissociation. Trypsin and mechanical dissociation decreased cortisol binding slightly; papain dissociation essentially eliminated it. Cortisol binding decreased with time in culture in both whole retina and monolayer cultures; this decrease may reflect, in part, a similar development decrease in ovo. Inducibility of glutamine synthetase in whole retinas and retinal monolayers prepared with either trypsin or papain also decreased with time in culture. For whole and trypsin-dissociated retinas, the drop in inducibility correlates with the drop in cortisol-binding capacity. This was not the case for monolayer cultures prepared by papain dissociation.     

Effect of dissociative methods on cortisol binding and glutamyltransferase inducibility in chick embryo retina

The ability of the isolated embryonic chick retina (12 day) to bind a steroid (cortisol) decreases when the tissue is dissociated; the extent of this decrease depends upon the method of dissociation. Trypsin and mechanical dissociation decreased cortisol binding slightly; papain dissociation essentially eliminated it. Cortisol binding decreased with time in culture in both whole retina and monolayer cultures; this decrease may reflect, in part, a similar developmental decreasein ovo. Inducibility of glutamine synthetase in whole retinas and retinal monolayers prepared with either trypsin or papain also decreased with time in culture. For whole and trypsin-dissociated retinas, the drop in inducibility correlates with a drop in cortisol-binding capacity. This was not the case for monolayer cultures prepared by papain dissociation.     

Primary cultures of human caudate nucleus

It is possible to grow functional primary dissociated cultures and explants from stereotactic biopsies of human parkinsonian caudate nuclei. Two major classes of cells were identified on morphological grounds. The culture cells appear to be stimulated by an unidentified soluble factor(s) obtained from human fetal neuronal cells in vitro. Culture of primary neuronal and glial cells from human adult cerebral nuclei seems to be a useful tool for several research purposes and in particular for studying both trophic factor action and target effects on afferent neurons for prospective human brain grafting.     

Characterization of cortical neuronal and glial alterations during culture of organotypic whole brain slices from neonatal and mature mice

Background

Organotypic brain slice culturing techniques are extensively used in a wide range of experimental procedures and are particularly useful in providing mechanistic insights into neurological disorders or injury. The cellular and morphological alterations associated with hippocampal brain slice cultures has been well established, however, the neuronal response of mouse cortical neurons to culture is not well documented.

Methods

In the current study, we compared the cell viability, as well as phenotypic and protein expression changes in cortical neurons, in whole brain slice cultures from mouse neonates (P4–6), adolescent animals (P25–28) and mature adults (P50+). Cultures were prepared using the membrane interface method.

Results

Propidium iodide labeling of nuclei (due to compromised cell membrane) and AlamarBlue™ (cell respiration) analysis demonstrated that neonatal tissue was significantly less vulnerable to long-term culture in comparison to the more mature brain tissues. Cultures from P6 animals showed a significant increase in the expression of synaptic markers and a decrease in growth-associated proteins over the entire culture period. However, morphological analysis of organotypic brain slices cultured from neonatal tissue demonstrated that there were substantial changes to neuronal and glial organization within the neocortex, with a distinct loss of cytoarchitectural stratification and increased GFAP expression (p<0.05). Additionally, cultures from neonatal tissue had no glial limitans and, after 14 DIV, displayed substantial cellular protrusions from slice edges, including cells that expressed both glial and neuronal markers.

Conclusion

In summary, we present a substantial evaluation of the viability and morphological changes that occur in the neocortex of whole brain tissue cultures, from different ages, over an extended period of culture.     

Towards Neuronal Organoids: A Method for Long-Term Culturing of High-Density Hippocampal Neurons

One of the goals in neuroscience is to obtain tractable laboratory cultures that closely recapitulate in vivo systems while still providing ease of use in the lab. Because neurons can exist in the body over a lifetime, long-term culture systems are necessary so as to closely mimic the physiological conditions under laboratory culture conditions. Ideally, such a neuronal organoid culture would contain multiple cell types, be highly differentiated, and have a high density of interconnected cells. However, before these types of cultures can be created, certain problems associated with long-term neuronal culturing must be addressed. We sought to develop a new protocol which may further prolong the duration and integrity of E18 rat hippocampal cultures. We have developed a protocol that allows for culturing of E18 hippocampal neurons at high densities for more than 120 days. These cultured hippocampal neurons are (i) well differentiated with high numbers of synapses, (ii) anchored securely to their substrate, (iii) have high levels of functional connectivity, and (iv) form dense multi-layered cellular networks. We propose that our culture methodology is likely to be effective for multiple neuronal subtypes–particularly those that can be grown in Neurobasal/B27 media. This methodology presents new avenues for long-term functional studies in neurons.     

GUANYL CYCLASE IN CHICK EMBRYO BRAIN CELL CULTURES: EVIDENCE OF NEURONAL LOCALIZATION

Abstract— Guanyl cyclase activity was studied in dissociated chick embryo brain cell cultures presenting different ratios of neuronal to glial elements. The cultures containing neurons in substantial numbers always had higher guanyl cyclase activities than those consisting mainly of glial cells. No guanyl cyclase activity could be found in cultures made up of pure glial or meningeal cells. These results provide further evidence for our conclusion based on subcellular fractionation studies (G oridis & M organ , 1973), that brain guanyl cyclase might be overwhelmingly concentrated in neurons. Guanyl cyclase activity of chick embryo cerebral hemispheres increased sixfold between day 12 and day 16 after fertilization; an increase, though of much smaller magnitude, was also seen in cultured cells of the same age.     

Toxoplasma gondii Migration within and Infection of Human Retina

Toxoplasmic retinochoroiditis is a common blinding retinal infection caused by the parasite, Toxoplasma gondii. Basic processes relating to establishment of infection in the human eye by T. gondii tachyzoites have not been investigated. To evaluate the ability of tachyzoites to navigate the human retina, we developed an ex vivo assay, in which a suspension containing 1.5×10⁷ parasites replaced vitreous in a posterior eyecup. After 8 hours, the retina was formalin-fixed and paraffin-embedded, and sections were immunostained to identify tachyzoites. To determine the preference of tachyzoites for human retinal neuronal versus glial populations, we infected dissociated retinal cultures, subsequently characterized by neuron-specific enolase or glial fibrillary acidic protein expression, and retinal cell lines, with YFP-expressing tachyzoites. In migration assays, retinas contained 110–250 live tachyzoites; 64.5–95.2% (mean  = 79.6%) were localized to the nerve fiber layer, but some were detected in the outer retina. Epifluorescence imaging of dissociated retinal cultures 24 hours after infection indicated preferential infection of glia. This observation was confirmed in growth assays, with significantly higher (p≤0.005) numbers of tachyzoites measured in glial verus neuronal cell lines. Our translational studies indicate that, after entering retina, tachyzoites may navigate multiple tissue layers. Tachyzoites preferentially infect glial cells, which exist throughout the retina. These properties may contribute to the success of T. gondii as a human pathogen.     

Immunocytochemical localization of Na+, K+-ATPase in primary cultures of rat retina

Conditions have been established which allow growth of embryonic rat retinal cells in dissociated cell culture for up to one month. Na⁺,K⁺-ATPase localization was stuied in both neuronal and mixed neuronal-glial (flat cell) cultures. High Na⁺,K⁺-ATPase-like-immunoreactivity was associated with plasma membranes of neuronal cell bodies and their processes. Markedly lower immunoreactivity was found in the underlying flat cells in mixed cultures. Staining was generally uniform over perikaryal plasma membranes and showed a bead-like appearance in neuronal processes, supporting previous studies in brain tissue which used histocytochemical procedures specific for the Na⁺,K⁺-ATPase. This system should be useful for examining distribution of the enzyme in developing nerve and glial cells and may help to resolve questions regarding Na⁺–K⁺ homeostasis by neurons and glia.Dedicated to Henry McIlwain.     

Fibronectin associated with the glial component of embryonic brain cell cultures

In a basic approach to investigations of neuronal–glial interactions during both normal brain development and its pathogenesis, embryonic brain cell populations were fractionated into purified neuronal and glial components. Using separation procedures based on differential adhesion and cytotoxicity, the isolated neuronal and glial phenotypes could be identified by distinct morphological and biochemical characteristics, including the visualization of glial fibrillary acid protein (GFA) within glial cells in immunohistochemical assays with monospecific anti-GFA serum. When unfractionated cerebrum cells dissociated from 10-day chick or 14-day mouse embryos were plated as monolayers and cultured for 1-14 days, monospecific antiserum against fibronectin (LETS glycoprotein) was found to react with many, but not all, of the cells as revealed by indirect immunofluorescence microscopy. The isolated neuronal and glial components of these populations were used to determine whether the appearance of membrane-associated fibronectin was characteristic of one cell type or the other, or both, and if neuronal–glial cell interaction was required for its expression. It was found that the surfaces of glial cells, completely isolated from neurons, showed an intense fluorescent reaction to the anti-fibronectin serum. In contrast, the purified neuronal cultures showed no fluorescence with either the anti-GFA or anti-fibronectin sera. These results demonstrate fibronectin as a cell surface protein associated primarily with glial cells and independent of neuronal–glial cell interaction for its expression. Furthermore, the results indicate that the fibronectin observed on glial cell surfaces in these cultures is produced endogenously and is not due to the preferential binding of fibronectin present in the culture medium. The role of fibronectin as an adhesive molecule in neuronal–glial interactions is discussed.     

Survival of hippocampal and cortical neurons in a mixture of MEM+ and B27-supplemented neurobasal medium

Serum-free B-27 supplemented neurobasal (NB) and a 10% fetal bovine serum-supplemented Eagle's minimum essential medium (MEM+) are used to culture rat embryonic hippocampal neurons for different purposes. Although NB medium leads to enhanced cell survival, it contains biological antioxidants and is not suitable for the study of free radical damage and oxidation in cultured neurons. MEM+ without additional antioxidants has been used widely in the study of free radical damage and oxidation, although it does not support optimum neuronal survival in culture. Serum in MEM+ leads to enhanced cell survival but also promotes glial cell proliferation. In this study, we used a new combination medium (NM-2) that consists of both NB and MEM+ for growing primary hippocampal and cortical neuronal cultures. NM-2 enhanced neuronal survival 78.9% for dissociated neurons at a density of 50 cells/mm(2) and 83.1% for 100 cells/mm(2), while decreasing glial cell proliferation to 2-3% and completely inhibiting oligodendrocytes. The NM-2 minimized the effectiveness of antioxidants in the medium to the neurotoxin 4-hydroxynonenal. It also decreased neuronal clumping and provided a more even distribution of neurons. Neurons survived for 4 weeks in NM-2 without changing the original medium. NM-2 provides a good environment for studies of free radical damage and oxidation of neurons. The combination incorporates the best of both NB and MEM+ that results in high neuron survival rate, low glial cell proliferation, reduced antioxidant level, and provides relatively pure cultures of hippocampal and cortical neurons.