Isolation of Neural Stem Cells from the embryonic mouse hippocampus for in vitro growth or engraftment into a host tissue

For both stem cell research and treatment of the central nervous system disorders, neural stem/progenitor cells (NSPCs) represent an important breakthrough tool. In the expanded stem cell-based therapy use, NSPCs not only provide a powerful cell source for neural cell replacement but a useful model for developmental biology research. Despite numerous approaches were described for isolation of NSPCs from either fetal or adult brain, the main issue remains in extending cell survival following isolation. Here we provide a simple and affordable protocol for making viable NSPCs from the fetal mouse hippocampi, which are capable of maintaining the high viability in a 2D monolayer cell culture or generating 3D neuro-spheroids of cell aggregates. Further, we describe the detailed method for engraftment of embryonic NSPCs onto a host hippocampal tissue for promoting multilinear cell differentiation and maturation within endogenous environment. Our experimental data demonstrate that embryonic NSPCs isolated using this approach show the high viability (above 88%). Within a host tissue, these cells were capable of differentiating to the main neural subpopulations (principal neurons, oligodendrocytes, astroglia). Finally, NSPC-derived neurons demonstrated matured functional properties (electrophysiological activity), becoming functionally integrated into the host hippocampal circuits within a couple of weeks after engraftment

Optimized Model of Cerebral Ischemia In situ for the Long-Lasting Assessment of Hippocampal Cell Death

Among all the brain, the hippocampus is the most susceptible region to ischemic lesion, with the highest vulnerability of CA1 pyramidal neurons to ischemic damage. This damage may cause either prompt neuronal death (within hours) or with a delayed appearance (over days), providing a window for applying potential therapies to reduce or prevent ischemic impairments. However, the time course when ischemic damage turns to neuronal death strictly depends on experimental modeling of cerebral ischemia and, up to now, studies were predominantly focused on a short time-window—from hours to up to a few days post-lesion. Using different schemes of oxygen-glucose deprivation (OGD), the conditions taking place upon cerebral ischemia, we optimized a model of mimicking ischemic conditions in organotypical hippocampal slices for the long-lasting assessment of CA1 neuronal death (at least 3 weeks). By combining morphology and electrophysiology, we show that prolonged (30-min duration) OGD results in a massive neuronal death and overwhelmed astrogliosis within a week post-OGD whereas OGD of a shorter duration (10-min) triggered programmed CA1 neuronal death with a significant delay—within 2 weeks—accompanied with drastically impaired CA1 neuron functions. Our results provide a rationale toward optimized modeling of cerebral ischemia for reliable examination of potential treatments for brain neuroprotection, neuro-regeneration, or testing neuroprotective compounds in situ

Satellite microglia display spontaneous electrical activity uncorrelated with activity of the attached neuron

Microglia are innate immune cells of the brain. We have studied a subpopulation of microglia, called satellite microglia. This cell type is defined by a close morphological soma-to-soma association with a neuron, indicative of a direct functional interaction. Indeed, ultrastructural analysis revealed closely attached plasma membranes of satellite microglia and neurons. However, we found no apparent morphological specializations of the contact and biocytin injection into satellite microglia showed no dye-coupling with the apposed neuron or any other cell. Likewise, evoked local field potentials or action potentials and postsynaptic potentials of the associated neuron did not lead to any transmembrane currents or non-capacitive changes in the membrane potential of the satellite microglia in the cortex and hippocampus. Both satellite and non-satellite microglia, however, showed spontaneous transient membrane depolarizations which were not correlated with neuronal activity. These events could be divided into fast and slow rising depolarisations, which exhibited different characteristics in satellite and non-satellite microglia. Fast and slow rising potentials differed with regard to voltage dependence. The frequency of these events was not affected by application of TTX, but fast rising event frequency decreased after application of GABA. We conclude that microglial cells show spontaneous electrical activity uncorrelated with the activity of adjacent neurons

Oligodendrocytes in the mouse corpus callosum maintain axonal function by delivery of glucose

In the optic nerve, oligodendrocytes maintain axonal function by supplying lactate as an energy substrate. Here, we report that, in acute brain slices of the mouse corpus callosum, exogenous glucose deprivation (EGD) abolished compound action potentials (CAPs), which neither lactate nor pyruvate could prevent. Loading an oligodendrocyte with 20 mM glucose using a patch pipette prevented EGD-mediated CAP reduction in about 70% of experiments. Loading oligodendrocytes with lactate rescued CAPs less efficiently than glucose. In mice lacking connexin 47, oligodendrocyte filling with glucose did not prevent CAP loss, emphasizing the importance of glial networks for axonal energy supply. Compared with the optic nerve, the astrocyte network in the corpus callosum was less dense, and loading astrocytes with glucose did not prevent CAP loss during EGD. We suggest that callosal oligodendrocyte networks provide energy to sustain axonal function predominantly by glucose delivery, and mechanisms of metabolic support vary across different white matter regions

Maturation of neural stem cells and integration into hippocampal circuits: functional study in post-ischemia in situ

The hippocampus is the most susceptible region of the brain to ischemic lesion, with highly vulnerable pyramidal interneurons to ischemic cell death. A restricted brain neurogenesis limits a withdrawal of massive cell death after stroke that endorses cell-based therapies for neuronal replacement strategies following cerebral ischemia. Neurons differentiated from neural stem/progenitor cells (NSPCs) matured and integrated into host circuitry, improving recovery after stroke. However, how host environment regulates the NSPC behaviour in post-ischemic tissue remains unknown. Here we studied functional maturation of NSPCs in control and post-ischemic hippocampal tissue after modelling cerebral ischemia in situ. We traced maturation of electrophysiological properties and integration of the NSPC-derived neurons into the host circuits, developing appropriate activity that takes 3 weeks or less after engraftment. In the ischemic-injured tissue, the NSPC-derived neurons exhibited functional deficits and differentiation of embryonic NSPCs was boosted to glial type – oligodendrocytes and astrocytes. Our findings of the delayed neuronal maturation whilst the promoted NSPC differentiation towards glial cell type in post-ischemic conditions provide new insights into stem-cell-therapy for replacement strategies in cerebral ischemia

Calcium Signals Driven by Single Channel Noise

Usually, the occurrence of random cell behavior is appointed to small copy numbers of molecules involved in the stochastic process. Recently, we demonstrated for a variety of cell types that intracellular Ca2+ oscillations are sequences of random spikes despite the involvement of many molecules in spike generation. This randomness arises from the stochastic state transitions of individual Ca2+ release channels and does not average out due to the existence of steep concentration gradients. The system is hierarchical due to the structural levels channel - channel cluster - cell and a corresponding strength of coupling. Concentration gradients introduce microdomains which couple channels of a cluster strongly. But they couple clusters only weakly; too weak to establish deterministic behavior on cell level. Here, we present a multi-scale modelling concept for stochastic hierarchical systems. It simulates active molecules individually as Markov chains and their coupling by deterministic diffusion. Thus, we are able to follow the consequences of random single molecule state changes up to the signal on cell level. To demonstrate the potential of the method, we simulate a variety of experiments. Comparisons of simulated and experimental data of spontaneous oscillations in astrocytes emphasize the role of spatial concentration gradients in Ca2+ signalling. Analysis of extensive simulations indicates that frequency encoding described by the relation between average and standard deviation of interspike intervals is surprisingly robust. This robustness is a property of the random spiking mechanism and not a result of control

G protein-coupled receptor-mediated calcium signaling in astrocytes

Astrocytes express a large variety of G~protein-coupled receptors (GPCRs) which mediate the transduction of extracellular signals into intracellular calcium responses. This transduction is provided by a complex network of biochemical reactions which mobilizes a wealth of possible calcium-mobilizing second messenger molecules. Inositol 1,4,5-trisphosphate is probably the best known of these molecules whose enzymes for its production and degradation are nonetheless calcium-dependent. We present a biophysical modeling approach based on the assumption of Michaelis-Menten enzyme kinetics, to effectively describe GPCR-mediated astrocytic calcium signals. Our model is then used to study different mechanisms at play in stimulus encoding by shape and frequency of calcium oscillations in astrocytes.Comment: 35 pages, 6 figures, 1 table, 3 appendices (book chapter

Astrocyte networks and intercellular calcium propagation

International audienceAstrocytes organize in complex networks through connections by gap junction channels that are regulated by extra-and intracellular signals. Calcium signals generated in individual cells, can propagate across these networks in the form of intercellular calcium waves, mediated by diffusion of second messengers molecules such as inositol 1,4,5-trisphosphate. The mechanisms underpinning the large variety of spatiotemporal patterns of propagation of astrocytic calcium waves however remain a matter of investigation. In the last decade, awareness has grown on the morphological diversity of astrocytes as well as their connections in networks, which seem dependent on the brain area, developmental stage, and the ultra-structure of the associated neuropile. It is speculated that this diversity underpins an equal functional variety but the current experimental techniques are limited in supporting this hypothesis because they do not allow to resolve the exact connectivity of astrocyte networks in the brain. With this aim we present a general framework to model intercellular calcium wave propagation in astrocyte networks and use it to specifically investigate how different network topologies could influence shape, frequency and propagation of these waves

Mitochondria adjust Ca(2+) signaling regime to a pattern of stimulation in salivary acinar cells

AbstractThe salivary acinar cells have unique Ca2+ signaling machinery that ensures an extensive secretion. The agonist-induced secretion is governed by Ca2+ signals originated from the endoplasmic reticulum (ER) followed by a store-operated Ca2+ entry (SOCE). During tasting and chewing food a frequency of parasympathetic stimulation increases up to ten fold, entailing cells to adapt its Ca2+ machinery to promote ER refilling and ensure sustained SOCE by yet unknown mechanism. By employing a combination of fluorescent Ca2+ imaging in the cytoplasm and inside cellular organelles (ER and mitochondria) we described the role of mitochondria in adjustment of Ca2+ signaling regime and ER refilling according to a pattern of agonist stimulation. Under the sustained stimulation, SOCE is increased proportionally to the degree of ER depletion. Cell adapts its Ca2+ handling system directing more Ca2+ into mitochondria via microdomains of high [Ca2+] providing positive feedback on SOCE while intra-mitochondrial tunneling provides adequate ER refilling. In the absence of an agonist, the bulk of ER refilling occurs through Ca2+-ATPase-mediated Ca2+ uptake within subplasmalemmal space. In conclusion, mitochondria play a key role in the maintenance of sustained SOCE and adequate ER refilling by regulating Ca2+ fluxes within the cell that may represent an intrinsic adaptation mechanism to ensure a long-lasting secretion