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 - a functional study in an in situ model of cerebral ischemia.

The hippocampus is the region of the brain that is most susceptible to ischemic lesion because it contains pyramidal neurons that are highly vulnerable to ischemic cell death. A restricted brain neurogenesis limits the possibility of reversing massive cell death after stroke and, hence, endorses cell-based therapies for neuronal replacement strategies following cerebral ischemia. Neurons differentiated from neural stem/progenitor cells (NSPCs) can mature and integrate into host circuitry, improving recovery after stroke. However, how the host environment regulates the NSPC behavior 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 the maturation of electrophysiological properties and integration of the NSPC-derived neurons into the host circuits, with these cells developing appropriate activity 3 weeks or less after engraftment. In the tissue subjected to ischemia, the NSPC-derived neurons exhibited functional deficits, and differentiation of embryonic NSPCs to glial types - oligodendrocytes and astrocytes - was boosted. Our findings of the delayed neuronal maturation in post-ischemic conditions, while the NSPC differentiation was promoted towards glial cell types, provide new insights that could be applicable to stem cell therapy replacement strategies used after cerebral ischemia.

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 show spontaneous electrical activity that is 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 neurons 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 that were not correlated with neuronal activity. These events could be divided into fast-rising and slow-rising depolarizations, which showed different characteristics in satellite and non-satellite microglia. Fast-rising and slow-rising potentials differed with regard to voltage dependence. The frequency of these events was not affected by the application of tetrodotoxin, but the fast-rising event frequency decreased after application of GABA. We conclude that microglia show spontaneous electrical activity that is uncorrelated with the activity of adjacent neurons.

Long-term fate of grafted hippocampal neural progenitor cells following ischemic injury.

The adult CNS has a very limited capacity to regenerate neurons after insult. To overcome this limitation, the transplantation of neural progenitor cells (NPCs) has developed into a key strategy for neuronal replacement. This study assesses the long-term survival, migration, differentiation, and functional outcome of NPCs transplanted into the ischemic murine brain. Hippocampal neural progenitors were isolated from FVB-Cg-Tg(GFPU)5Nagy/J transgenic mice expressing green fluorescent protein (GFP). Syngeneic GFP-positive NPCs were stereotactically transplanted into the hippocampus of FVB mice following a transient global cerebral ischemia model. Behavioral tests revealed that ischemia/reperfusion induced spatial learning disturbances in the experimental animals. The NPC transplantation promoted cognitive function recovery after ischemic injury. To study the long-term fate of grafted GFP-positive NPCs in a host brain, immunohistochemical approaches were applied. Confocal microscopy revealed that grafted cells survived in the recipient tissue for 90 days following transplantation and differentiated into mature neurons with extensive dendritic trees and apparent spines. Immunoelectron microscopy confirmed the formation of synapses between the transplanted GFP-positive cells and host neurons that may be one of the factors underlying cognitive function recovery. Repair and functional recovery following brain damage represent a major challenge for current clinical and basic research. Our results provide insight into the therapeutic potential of transplanted hippocampal progenitor cells following ischemic brain injury.

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

The salivary acinar cells have unique Ca(2+) signaling machinery that ensures an extensive secretion. The agonist-induced secretion is governed by Ca(2+) signals originated from the endoplasmic reticulum (ER) followed by a store-operated Ca(2+) entry (SOCE). During tasting and chewing food a frequency of parasympathetic stimulation increases up to ten fold, entailing cells to adapt its Ca(2+) machinery to promote ER refilling and ensure sustained SOCE by yet unknown mechanism. By employing a combination of fluorescent Ca(2+) imaging in the cytoplasm and inside cellular organelles (ER and mitochondria) we described the role of mitochondria in adjustment of Ca(2+) 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 Ca(2+) handling system directing more Ca(2+) into mitochondria via microdomains of high [Ca(2+)] 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 Ca(2+)-ATPase-mediated Ca(2+) uptake within subplasmalemmal space. In conclusion, mitochondria play a key role in the maintenance of sustained SOCE and adequate ER refilling by regulating Ca(2+) fluxes within the cell that may represent an intrinsic adaptation mechanism to ensure a long-lasting secretion.

Functional coupling between ryanodine receptors, mitochondria and Ca(2+) ATPases in rat submandibular acinar cells.

Agonist stimulation of exocrine cells leads to the generation of intracellular Ca(2+) signals driven by inositol 1,4,5-trisphosphate receptors (IP(3)Rs) that rapidly become global due to propagation throughout the cell. In many types of excitable cells the intracellular Ca(2+) signal is propagated by a mechanism of Ca(2+)-induced Ca(2+) release (CICR), mediated by ryanodine receptors (RyRs). Expression of RyRs in salivary gland cells has been demonstrated immunocytochemically although their functional role is not clear. We used microfluorimetry to measure Ca(2+) signals in the cytoplasm, in the endoplasmic reticulum (ER) and in mitochondria. In permeabilized acinar cells caffeine induced a dose-dependent, transient decrease of Ca(2+) concentration in the endoplasmic reticulum ([Ca(2+)](ER)). This decrease was inhibited by ryanodine but was insensitive to heparin. Application of caffeine, however, did not elevate cytosolic Ca(2+) concentration ([Ca(2+)](i)) suggesting fast local buffering of Ca(2+) released through RyRs. Indeed, activation of RyRs produced a robust mitochondrial Ca(2+) transient that was prevented by addition of Ca(2+) chelator BAPTA but not EGTA. When mitochondrial Ca(2+) uptake was blocked, activation of RyRs evoked only a non-transient increase in [Ca(2+)](i) and substantially smaller Ca(2+) release from the ER. Upon simultaneous inhibition of mitochondrial Ca(2+) uptake and either plasmalemmal or ER Ca(2+) ATPase, activation of RyRs caused a transient rise in [Ca(2+)](i). Collectively, our data suggest that Ca(2+) released through RyRs is mostly "tunnelled" to mitochondria, while Ca(2+) ATPases are responsible for the fast initial sequestration of Ca(2+). Ca(2+) uptake by mitochondria is critical for maintaining continuous CICR. A complex interplay between RyRs, mitochondria and Ca(2+) ATPases is accomplished through strategic positioning of mitochondria close to both Ca(2+) release sites in the ER and Ca(2+) pumping sites of the plasmalemma and the ER.

CXCR3-dependent microglial recruitment is essential for dendrite loss after brain lesion.

Microglia are the resident macrophage population of the CNS and are considered its major immunocompetent elements. They are activated by any type of brain pathology and can migrate to the lesion site. The chemokine CXCL10 is expressed in neurons in response to brain injury and is a signaling candidate for activating microglia and directing them to the lesion site. We recently identified CXCR3, the corresponding receptor for CXCL10, in microglia and demonstrated that this receptor system controls microglial migration. We have now tested the impact of CXCR3 signaling on cellular responses after entorhinal cortex lesion. In wild-type mice, microglia migrate within the first 3 d after lesion into the zone of axonal degeneration, where 8 d after lesion denervated dendrites of interneurons are subsequently lost. In contrast, the recruitment of microglia was impaired in CXCR3 knock-out mice, and, strikingly, denervated distal dendrites were maintained in zones of axonal degeneration. No differences between wild-type and knock-out mice were observed after facial nerve axotomy, as a lesion model for assessing microglial proliferation. This shows that CXCR3 signaling is crucial in microglia recruitment but not proliferation, and this recruitment is an essential element for neuronal reorganization.

Differential properties of GABAergic synaptic connections in rat hippocampal cell cultures.

Based on the effect of prolonged tetanic stimulation (30 Hz, 4 sec), we divided GABAergic synaptic connections in hippocampal cell cultures into two groups: connections facilitated ( approximately 45%) and connections depressed ( approximately 55%) by the tetanic stimulation. In order to reveal possible reasons for the differential effect of the tetanization, we compared several properties of the connections belonging to both groups. We found that, on average, evoked IPSCs in the connections facilitated by the tetanization have a smaller amplitude and larger coefficient of variation (CV) of IPSC amplitude compared to connections depressed by the tetanization. We also estimated quantal parameters for both groups of connections assuming that transmitter release is reasonably described by a binomial distribution. We found that a background release probability (P) is substantially lower in the connections facilitated by the tetanization (P approximately 0.5) than in the connections depressed by the tetanization (P approximately 0.9) and suggest that this difference may underlie the differential effect of the tetanization. We also found that the tetanization induces the opposite effect on connections made by distinct presynaptic neurons with the same postsynaptic cell (convergent connections) in a fraction of postsynaptic neurons studied (3 out of 9). These results support the idea that properties of the presynaptic neuron are of primary importance for the observed differential effect of the tetanization, but they do not exclude a role of the postsynaptic neuron in this effect.

Post-tetanic depression of GABAergic synaptic transmission in rat hippocampal cell cultures.

The effect of tetanic stimulation (30 Hz, 4 s) on evoked GABAergic inhibitory postsynaptic currents (IPSCs) was studied in cell cultures of dissociated hippocampal neurons with established synaptic connections. It was found that tetanic stimulation elicited post-tetanic depression (PTD) of the evoked IPSCs with a duration of more than 50 s in about 60% of the connections tested; post-tetanic potentiation was induced in 25% of the connections. We propose that the opposite effects of tetanization on IPSC amplitude are due to differences in the type of the interneuron that was tetanized. Since PTD in our experiments was usually accompanied by changes in the IPSC coefficient of variation and changes of a paired pulse depression, which are thought to reflect presynaptic mechanisms of modulation, we suggest that part of the PTD is due to a presynaptic mechanism(s).