Real-Time Impedance-based Cell Analyzer as a Tool to Delineate Molecular Pathways Involved in Neurotoxicity and Neuroprotection in a Neuronal Cell Line

Many brain-related disorders have neuronal cell death involved in their pathophysiology. Improved in vitro models to study neuroprotective or neurotoxic effects of drugs and downstream pathways involved would help gain insight into the molecular mechanisms of neuroprotection/neurotoxicity and could potentially facilitate drug development. However, many existing in vitro toxicity assays have major limitations – most assess neurotoxicity and neuroprotection at a single time point, not allowing to observe the time-course and kinetics of the effect. Furthermore, the opportunity to collect information about downstream signaling pathways involved in neuroprotection in real-time would be of great importance. In the current protocol we describe the use of a real-time impedance-based cell analyzer to determine neuroprotective effects of serotonin 2A (5-HT_2A) receptor agonists in a neuronal cell line under label-free and real-time conditions using impedance measurements. Furthermore, we demonstrate that inhibitors of second messenger pathways can be used to delineate downstream molecules involved in the neuroprotective effect. We also describe the utility of this technique to determine whether an effect on cell proliferation contributes to an observed neuroprotective effect. The system utilizes special microelectronic plates referred to as E-Plates which contain alternating gold microelectrode arrays on the bottom surface of the wells, serving as cell sensors. The impedance readout is modified by the number of adherent cells, cell viability, morphology, and adhesion. A dimensionless parameter called Cell Index is derived from the electrical impedance measurements and is used to represent the cell status. Overall, the real-time impedance-based cell analyzer allows for real-time, label-free assessment of neuroprotection and neurotoxicity, and the evaluation of second messenger pathways involvement, contributing to more detailed and high-throughput assessment of potential neuroprotective compounds in vitro, for selecting therapeutic candidates.     

Mitochondria and Neuronal Death/Survival Signaling Pathways in Cerebral Ischemia

Apoptotic cell death pathways have been implicated in acute brain injuries, including cerebral ischemia, brain trauma, and spinal cord injury, and in chronic neurodegenerative diseases. Experimental ischemia and reperfusion models, such as transient focal/global ischemia in rodents, have been thoroughly studied and suggest the involvement of mitochondria and the cell survival/death signaling pathways in cell death/survival cascades. Recent studies have implicated mitochondria-dependent apoptosis involving pro- and anti-apoptotic protein binding, the release of cytochrome c and second mitochondria-derived activator of caspase, the activation of downstream caspases-9 and –3, and DNA fragmentation. Reactive oxygen species are known to be significantly generated in the mitochondrial electron transport chain in the dysfunctional mitochondria during reperfusion after ischemia, and are also implicated in the survival signaling pathway that involves phosphatidylinositol-3-kinase (PI3-K), Akt, and downstream signaling molecules, like Bad, 14-3-3, and the proline-rich Akt substrate (PRAS), and their bindings. Further studies of these survival pathways may provide novel therapeutic strategies for clinical stroke.Special issue dedicated to Lawrence F. Eng.     

Organotypic Hippocampal Slice Cultures: A Model System to Study Basic Cellular and Molecular Mechanisms of Neuronal Cell Death,Neuroprotection, and Synaptic Plasticity

The hippocampus has become one of the most extensively studied areas of the mammalian brain, and its proper function is of utmost importance, particularly for learning and memory. The hippocampus is the most susceptible brain region for damage, and its impaired function has been documented in many human brain diseases, e.g. hypoxia, ischemia, and epilepsy regardless of the age of the affected patients. In addition to experimental in vivo models of these disorders, the investigation of basic anatomical, physiological, and molecular aspects requires an adequate experimental in vitro model, which should meet the requirements for well-preserved representation of various cell types, and functional information processing properties in the hippocampus. In this review, the characteristics of organotypic hippocampal slice cultures (OHCs) together with the main differences between the in vivo and in vitro preparations are first briefly outlined. Thereafter, the use of OHCs in studies focusing on neuron cell death and synaptic plasticity is discussed. Special issue dedicated to Dr. Simo S. Oja     

Multi-electrode Array Recordings of Neuronal Avalanches in Organotypic Cultures

The cortex is spontaneously active, even in the absence of any particular input or motor output. During development, this activity is important for the migration and differentiation of cortex cell types and the formation of neuronal connections¹. In the mature animal, ongoing activity reflects the past and the present state of an animal into which sensory stimuli are seamlessly integrated to compute future actions. Thus, a clear understanding of the organization of ongoing i.e. spontaneous activity is a prerequisite to understand cortex function. Numerous recording techniques revealed that ongoing activity in cortex is comprised of many neurons whose individual activities transiently sum to larger events that can be detected in the local field potential (LFP) with extracellular microelectrodes, or in the electroencephalogram (EEG), the magnetoencephalogram (MEG), and the BOLD signal from functional magnetic resonance imaging (fMRI). The LFP is currently the method of choice when studying neuronal population activity with high temporal and spatial resolution at the mesoscopic scale (several thousands of neurons). At the extracellular microelectrode, locally synchronized activities of spatially neighbored neurons result in rapid deflections in the LFP up to several hundreds of microvolts. When using an array of microelectrodes, the organizations of such deflections can be conveniently monitored in space and time. Neuronal avalanches describe the scale-invariant spatiotemporal organization of ongoing neuronal activity in the brain^2,3. They are specific to the superficial layers of cortex as established in vitro^4,5, in vivo in the anesthetized rat ⁶, and in the awake monkey⁷. Importantly, both theoretical and empirical studies^2,8-10 suggest that neuronal avalanches indicate an exquisitely balanced critical state dynamics of cortex that optimizes information transfer and information processing.In order to study the mechanisms of neuronal avalanche development, maintenance, and regulation, in vitro preparations are highly beneficial, as they allow for stable recordings of avalanche activity under precisely controlled conditions. The current protocol describes how to study neuronal avalanches in vitro by taking advantage of superficial layer development in organotypic cortex cultures, i.e. slice cultures, grown on planar, integrated microelectrode arrays (MEA; see also ^11-14).     

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.     

Interfacing 3D Engineered Neuronal Cultures to Micro-Electrode Arrays: An Innovative In Vitro Experimental Model

Currently, large-scale networks derived from dissociated neurons growing and developing in vitro on extracellular micro-transducer devices are the gold-standard experimental model to study basic neurophysiological mechanisms involved in the formation and maintenance of neuronal cell assemblies. However, in vitro studies have been limited to the recording of the electrophysiological activity generated by bi-dimensional (2D) neural networks. Nonetheless, given the intricate relationship between structure and dynamics, a significant improvement is necessary to investigate the formation and the developing dynamics of three-dimensional (3D) networks. In this work, a novel experimental platform in which 3D hippocampal or cortical networks are coupled to planar Micro-Electrode Arrays (MEAs) is presented. 3D networks are realized by seeding neurons in a scaffold constituted of glass microbeads (30-40 µm in diameter) on which neurons are able to grow and form complex interconnected 3D assemblies. In this way, it is possible to design engineered 3D networks made up of 5-8 layers with an expected final cell density. The increasing complexity in the morphological organization of the 3D assembly induces an enhancement of the electrophysiological patterns displayed by this type of networks. Compared with the standard 2D networks, where highly stereotyped bursting activity emerges, the 3D structure alters the bursting activity in terms of duration and frequency, as well as it allows observation of more random spiking activity. In this sense, the developed 3D model more closely resembles in vivo neural networks.     

Organotypic Slice Cultures to Study Oligodendrocyte Dynamics and Myelination

NG2 expressing cells (polydendrocytes, oligodendrocyte precursor cells) are the fourth major glial cell population in the central nervous system. During embryonic and postnatal development they actively proliferate and generate myelinating oligodendrocytes. These cells have commonly been studied in primary dissociated cultures, neuron cocultures, and in fixed tissue. Using newly available transgenic mouse lines slice culture systems can be used to investigate proliferation and differentiation of oligodendrocyte lineage cells in both gray and white matter regions of the forebrain and cerebellum. Slice cultures are prepared from early postnatal mice and are kept in culture for up to 1 month. These slices can be imaged multiple times over the culture period to investigate cellular behavior and interactions. This method allows visualization of NG2 cell division and the steps leading to oligodendrocyte differentiation while enabling detailed analysis of region-dependent NG2 cell and oligodendrocyte functional heterogeneity. This is a powerful technique that can be used to investigate the intrinsic and extrinsic signals influencing these cells over time in a cellular environment that closely resembles that found in vivo.     

Opposite Effect of Protein Synthesis Inhibitors on Potassium Deficiency-Induced Apoptotic Cell Death in Immature and Mature Neuronal Cultures

Abstract: Typically, primary cultures of rat cerebellar granule neurons are grown in the presence of 25 m M KCl and are considered to mature by ∼7 days in vitro. Potassium deficiency was created by growing the neurons from days 1 to 4 in the presence of 12.5 m M KCl (immature cultures) or by switching the mature neurons grown with 25 m M KCl to 12.5 m M KCl. In both conditions we observed neuronal death that bears the signs of apoptosis, i.e., DNA fragmentation determined qualitatively by agarose gel electrophoresis of DNA and quantitatively by in situ terminal deoxynucleotidyl transferase assay. The protein synthesis inhibitors cycloheximide and anisomycin provided neuroprotection in the mature cultures but potentiated the toxic effect of KCl deprivation in the immature neurons. The results suggest that a prudent use of protein synthesis inhibitors is critical in experiments with primary neuronal cultures.     

撤除外源生长素诱发棉花胚性悬浮细胞程序性死亡

棉花胚性悬浮细胞在仅含生长素的MS培养基上培养时,生长良好;但当转入到不含生长素的MS培养基上培养时,大规模死亡.通过细胞学观察发现,在转入无生长素的MS培养基培养3～4 d后可见明显的核质浓缩、胞质收缩,而高温处理引起的细胞坏死无此现象.用抽提悬浮细胞基因组DNA进行凝胶电泳发现:这种细胞死亡还伴随有典型的DNA梯度出现,而坏死的细胞和对照无DNA梯度.表明这种由生长素撤除引起的细胞死亡是一种程序性死亡.这种细胞死亡能被水解酶抑制剂和半胱氨酸蛋白酶抑制剂抑制,表明水解酶和类半胱氨酸蛋白水解酶(CLP)参与细胞程序性死亡.     

Molecular Forms of Acetylcholinesterase in Pineal Cell and Sympathetic Neuronal Cultures

Abstract The activities of the various molecular forms of acetylcholinesterase (AChE) were measured in monolayer cultures of neonatal rat pineal cells grown alone and in co-culture with sympathetic neurons. AChE forms characterized by sedimentation coefficients of 4S, 6.5S, and 10S were found in the neuronal and pineal cultures, as well as in the co-cultures. The 16S AChE form was found only in the neuronal cultures. Total AChE activity increased with culture age in the co-cultures, but it decreased in pineal cells cultured alone. The low level of activity present in the neuronal cultures did not change markedly over the 27-day culture period. These results, which show bidirectional neuron-pineal cell effects, suggest that AChE molecular forms may be important markers to study the mechanisms underlying neuron-target cell interaction in the developing sympathetic nervous system.     

Activity Regulates Cell Death within Cortical Interneurons through a Calcineurin-Dependent Mechanism

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Interferon Suppresses Sympathetic Neuronal Cell Death Caused by Nerve Growth Factor Deprivation

Cultured rat sympathetic neurons die within 48 h after being deprived of nerve growth factor. Addition of interferons (IFN-alpha/beta or IFN-gamma) prevented the cell death in a dose-dependent manner. Upon longer periods of nerve growth factor deprivation, IFNs failed to maintain survival. Thus, IFNs retarded neuronal death, but did not prevent it. Ligand binding, autoradiography, and cross-linking experiments demonstrated the presence of specific IFN-gamma receptors on sympathetic neurons similar to those seen on other cell types. The possible relationships of the death-suppressing actions of IFNs are compared to the mechanisms of the antiviral or antiproliferative actions of IFNs.     

Expression of MCP-1 in the Hippocampus of SHRSP with Ischemia-Related Delayed Neuronal Death

1. The expression of monocyte chemoattractant protein-1 (MCP-1) was examined in stroke-prone spontaneously hypertensive rats with transient global ischemia in order to study the involvement of the infiltration of blood monocytes in the mechanism of ischemia-related neuronal death.2. The brains of the animals with occlusion of the bilateral carotid arteries for 10 min were removed at 8 h, 1, 2, 4 and 7 days after reperfusion. Frozen sections were used for in situ hybridization and tissue specimens from the hippocampus and the cerebral cortex were used to measure the concentration of MCP-1 by ELISA.3. No MCP-1 mRNA was detected in the hippocampus of the sham group animals. One day after ischemia-reperfusion, MCP-1 mRNA was clearly expressed in the CA4 subfield and the molecular layer of the dentate gyrus, while it was slightly expressed in the lacnosum moleculare of the CA1 subfield. A dramatic expression was demonstrated in the entire CA1 subfield at 2 days after the operation. Most of the cells expressing MCP-1 were astrocytes. At 4 and 7 days after reperfusion, no MCP-1 mRNA was detected in the hippocampus. The concentration of MCP-1 protein dramatically increased in the hippocampus at 2 days after reperfusion.4. Taken together with the findings of our previous study showing an increased permeability of the blood-brain barrier in the hippocampus from 12 h after ischemia-reperfusion, the astrocytes expressing MCP-1 might therefore induce the migration of monocytes into the brain parenchyma. As a result, such astrocytes expressing MCP-1 may therefore be related to the pathological events of delayed neuronal death in the pyramidal neurons.     

Ceramide Prevents Neuronal Programmed Cell Death Induced by Nerve Growth Factor Deprivation

Abstract: We examined the ability of ceramide and sphingomyelinase (SMase) to prevent neuronal programmed cell death (PCD). We found that a cell-permeable ceramide analogue prevented neuronal PCD when applied to established sympathetic neuron primary cultures at the time of nerve growth factor (NGF) deprivation. Other amphiphilic lipids such as oleic acid failed to prevent cell death. Exogenous SMase also showed the same effect, probably by raising the intracellular ceramide level by sphingomyelin (SM) breakdown. Phosphocholine, another hydrolytic product of SM by SMase, did not prevent cell death. Other phospholipases, such as phospholipase C and phospholipase A₂, could not prevent cell death. Given the recent findings that the SM cycle is activated to increase the intracellular ceramide level on NGF binding to the low-affinity NGF receptor (LNGFR) and that NGF binding to LNGFR suppresses apoptosis in neural cell lines, our results suggest the possibility of the SM cycle as a signaling mechanism transducing the PCD-preventing activity of NGF.     

Protocol for Culturing Sympathetic Neurons from Rat Superior Cervical Ganglia (SCG)

The superior cervical ganglia (SCG) in rats are small, glossy, almond-shaped structures that contain sympathetic neurons. These neurons provide sympathetic innervations for the head and neck regions and they constitute a well-characterized and relatively homogeneous population (4). Sympathetic neurons are dependent on nerve growth factor (NGF) for survival, differentiation and axonal growth and the wide-spread availability of NGF facilitates their culture and experimental manipulation (2, 3, 6). For these reasons, cultured sympathetic neurons have been used in a wide variety of studies including neuronal development and differentiation, mechanisms of programmed and pathological cell death, and signal transduction (1, 2, 5, and 6). Dissecting out the SCG from newborn rats and culturing sympathetic neurons is not very complicated and can be mastered fairly quickly. In this article, we will describe in detail how to dissect out the SCG from newborn rat pups and to use them to establish cultures of sympathetic neurons. The article will also describe the preparatory steps and the various reagents and equipment that are needed to achieve this.     

Vibratome Sectioning Mouse Retina to Prepare Photoreceptor Cultures

The retina is a part of the central nervous system that has organized architecture, with neurons in layers from the photoreceptors, both rods and cones in contact with the retinal pigmented epithelium in the most distant part on the retina considering the direction of light, and the ganglion cells in the most proximal distance. This architecture allows the isolation of the photoreceptor layer by vibratome sectioning. The dissected neural retina of a mouse aged 8 days is flat-embedded in 4% gelatin on top of a slice of 20% gelatin photoreceptor layer facing down. Using a vibratome and a double edged razor blade, the 100 µm thick inner retina is sectioned. This section contains the ganglion cells and the inner layer with notably the bipolar cells. An intermediary section of 15 µm is discarded before 200 µm of the outer retina containing the photoreceptors is recovered. The gelatin is removed by heating at 37 °C. Pieces of outer layer are incubated in 500 µl of Ringer''s solution with 2 units of activated papain for 20 min at 37 °C. The reaction is stopped by adding 500 µl 10% fetal calf serum (FCS) in Dulbecco''s Modified Eagle Medium (DMEM), then 25 units of DNAse I is added before centrifugation at RT, washed several times to remove serum and the cells are resuspended in 500 µl of DMEM and seeded at 1 x 10⁵ cells/cm². The cells are grown to 5 days in vitro and their viability scored using live/dead assay. The purity of the culture is first determined by microscopic observation during the experiment. The purity is then validated by seeding and fixing cells on a histological slide and analyzing using a rabbit polyclonal anti-SAG, a photoreceptor marker and mouse monoclonal anti-RHO, a rod photoreceptor specific marker. Alternatively, the photoreceptor layer (97% rods) can be used for gene or protein expression analysis and for transplantation.     

Preparation of Neuronal Co-cultures with Single Cell Precision

Microfluidic embodiments of the Campenot chamber have attracted great interest from the neuroscience community. These interconnected co-culture platforms can be used to investigate a variety of questions, spanning developmental and functional neurobiology to infection and disease propagation. However, conventional systems require significant cellular inputs (many thousands per compartment), inadequate for studying low abundance cells, such as primary dopaminergic substantia nigra, spiral ganglia, and Drosophilia melanogaster neurons, and impractical for high throughput experimentation. The dense cultures are also highly locally entangled, with few outgrowths (<10%) interconnecting the two cultures. In this paper straightforward microfluidic and patterning protocols are described which address these challenges: (i) a microfluidic single neuron arraying method, and (ii) a water masking method for plasma patterning biomaterial coatings to register neurons and promote outgrowth between compartments. Minimalistic neuronal co-cultures were prepared with high-level (>85%) intercompartment connectivity and can be used for high throughput neurobiology experiments with single cell precision.     

植物细胞程序化死亡研究进展

细胞程序化死亡 (PCD)是一种由基因控制的、主动的细胞死亡过程 ,它在植物正常生长发育过程中起着重要作用。本文就植物PCD的近期研究进展和其分子信号调控机制作一综合阐述