Astrocyte volume regulation

This dissertation deals with the regulation of synapse formation between cultured hippocampal neurons, and the volume dynamics of astrocytes during hyposmotic-induced swelling;The developmental time course of synapse formation in cultured hippocampal neurons was examined. After 12 days in culture (DIC) inhibitory and excitatory synapses formed which were sensitive to the N-type calcium channel antagonist [omega]-conotoxin GVIA ([omega]-CgTx) while, at 4 DIC, immature connections were present in which spontaneous, but rarely evoked, synaptic currents were detected. A comparison of 4 and 12 DIC neurons revealed the presence of the synaptic proteins rab3a, synapsin I, and synaptotagmin, but the subcellular distribution changed from one in which immunoreactivity was initially distributed within the soma and neurites to a punctate varicose appearance. Correlated with these changes from immature to mature synaptic states was the development of [omega]-CgTx-sensitive calcium influx. These data suggest that the expression of functional [omega]-CgTx-sensitive calcium influx is temporally coincident with synapse formation, and that during the maturation of the synapse there is a redistribution of synaptic proteins;While astrocytes are known to actively regulate cell volume, the role of the F-actin cytoskeleton and membrane cycling in this process are ill-defined. A combination of scanning probe microscopy and a fluorescent dye dilution assay were utilized to dynamically examine hyposmotic-induced volume changes. Treatment of astrocytes with cytochalasin B to disrupt the F-actin cytoskeleton enhanced the hyposmotic-induced volume increase, but the rate of the compensatory regulatory volume decrease (RVD) was unaffected. Hyposmotic saline neither enhanced cell surface area nor caused altered membrane trafficking. These data are consistent with the hypothesis that astrocytes unfold plasma membrane upon treatment with hyposmotic saline, and reconvolute membrane during RVD. Furthermore, the F-actin based cytoskeleton may function to retard peak volume changes, presumably by restricting membrane unfolding, but does not function in controlling RVD. In addition to hyposmotic saline, glutamate release from astrocytes can also be stimulated by [alpha]-latrotoxin. Application of [alpha]-latrotoxin in the presence or absence of external calcium does not cause a significant increase in astrocyte volume, indicating that [alpha]-latrotoxin is not exerting its secretagogue effect through a swelling-activated pathway

Was the Scanner Calibration Slide used for its intended purpose?

In the article, Scanner calibration revisited, BMC Bioinformatics 2010, 11:361, Dr. Pozhitkov used the Scanner Calibration Slide, a key product of Full Moon BioSystems to generate data in his study of microarray scanner PMT response and proposed a mathematic model for PMT response [1]. In the end, the author concluded that "Full Moon BioSystems calibration slides are inadequate for performing calibration," and recommended "against using these slides." We found these conclusions are seriously flawed and misleading, and his recommendation against using the Scanner Calibration Slide was not properly supported

Receptor regulation of osmolyte homeostasis in neural cells

The capacity of cells to correct their volume in response to hyposmotic stress via the efflux of inorganic and organic osmolytes is well documented. However, the ability of cell-surface receptors, in particular G-protein-coupled receptors (GPCRs), to regulate this homeostatic mechanism has received much less attention. Mechanisms that underlie the regulation of cell volume are of particular importance to cells in the central nervous system because of the physical restrictions of the skull and the adverse impact that even small increases in cell volume can have on their function. Increases in brain volume are seen in hyponatraemia, which can arise from a variety of aetiologies and is the most frequently diagnosed electrolyte disorder in clinical practice. In this review we summarize recent evidence that the activation of GPCRs facilitates the volume-dependent efflux of osmolytes from neural cells and permits them to more efficiently respond to small, physiologically relevant, reductions in osmolarity. The characteristics of receptor-regulated osmolyte efflux, the signalling pathways involved and the physiological significance of receptor activation are discussed. In addition, we propose that GPCRs may also regulate the re-uptake of osmolytes into neural cells, but that the influx of organic and inorganic osmolytes is differentially regulated. The ability of neural cells to closely regulate osmolyte homeostasis through receptor-mediated alterations in both efflux and influx mechanisms may explain, in part at least, why the brain selectively retains its complement of inorganic osmolytes during chronic hyponatraemia, whereas its organic osmolytes are depleted.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/79149/1/jphysiol.2010.190777.pd

Rem2-Targeted shRNAs Reduce Frequency of Miniature Excitatory Postsynaptic Currents without Altering Voltage-Gated Ca2+ Currents

Ca2+ influx through voltage-gated Ca2+ channels (VGCCs) plays important roles in neuronal cell development and function. Rem2 is a member of the RGK (Rad, Rem, Rem2, Gem/Kir) subfamily of small GTPases that confers potent inhibition upon VGCCs. The physiologic roles of RGK proteins, particularly in the brain, are poorly understood. Rem2 was implicated in synaptogenesis through an RNAi screen and proposed to regulate Ca2+ homeostasis in neurons. To test this hypothesis and uncover physiological roles for Rem2 in the brain, we investigated the molecular mechanisms by which Rem2 knockdown affected synaptogenesis and Ca2+ homeostasis in cultured rat hippocampal neurons. Expression of a cocktail of shRNAs targeting rat Rem2 (rRem2) reduced the frequency of miniature excitatory postsynaptic currents (mEPSCs) measured 10 d after transfection (14 d in vitro), but did not affect mEPSC amplitude. VGCC current amplitude after rRem2-targeted knockdown was not different from that in control cells, however, at either 4 or 10 d post transfection. Co-expression of a human Rem2 that was insensitive to the shRNAs targeting rRem2 was unable to prevent the reduction in mEPSC frequency after rRem2-targeted knockdown. Over-expression of rRem2 resulted in 50% reduction in VGCC current, but neither the mEPSC frequency nor amplitude was affected. Taken together, the observed effects upon synaptogenesis after shRNA treatment are more likely due to mechanisms other than modulation of VGCCs and Ca2+ homeostasis, and may be independent of Rem2. In addition, our results reveal a surprising lack of contribution of VGCCs to synaptogenesis during early development in cultured hippocampal neurons

Dibucaine Mitigates Spreading Depolarization in Human Neocortical Slices and Prevents Acute Dendritic Injury in the Ischemic Rodent Neocortex

Spreading depolarizations that occur in patients with malignant stroke, subarachnoid/intracranial hemorrhage, and traumatic brain injury are known to facilitate neuronal damage in metabolically compromised brain tissue. The dramatic failure of brain ion homeostasis caused by propagating spreading depolarizations results in neuronal and astroglial swelling. In essence, swelling is the initial response and a sign of the acute neuronal injury that follows if energy deprivation is maintained. Choosing spreading depolarizations as a target for therapeutic intervention, we have used human brain slices and in vivo real-time two-photon laser scanning microscopy in the mouse neocortex to study potentially useful therapeutics against spreading depolarization-induced injury.We have shown that anoxic or terminal depolarization, a spreading depolarization wave ignited in the ischemic core where neurons cannot repolarize, can be evoked in human slices from pediatric brains during simulated ischemia induced by oxygen/glucose deprivation or by exposure to ouabain. Changes in light transmittance (LT) tracked terminal depolarization in time and space. Though spreading depolarizations are notoriously difficult to block, terminal depolarization onset was delayed by dibucaine, a local amide anesthetic and sodium channel blocker. Remarkably, the occurrence of ouabain-induced terminal depolarization was delayed at a concentration of 1 µM that preserves synaptic function. Moreover, in vivo two-photon imaging in the penumbra revealed that, though spreading depolarizations did still occur, spreading depolarization-induced dendritic injury was inhibited by dibucaine administered intravenously at 2.5 mg/kg in a mouse stroke model.Dibucaine mitigated the effects of spreading depolarization at a concentration that could be well-tolerated therapeutically. Hence, dibucaine is a promising candidate to protect the brain from ischemic injury with an approach that does not rely on the complete abolishment of spreading depolarizations

Two Distinct Modes of Hypoosmotic Medium-Induced Release of Excitatory Amino Acids and Taurine in the Rat Brain In Vivo

A variety of physiological and pathological factors induce cellular swelling in the brain. Changes in cell volume activate several types of ion channels, which mediate the release of inorganic and organic osmolytes and allow for compensatory cell volume decrease. Volume-regulated anion channels (VRAC) are thought to be responsible for the release of some of organic osmolytes, including the excitatory neurotransmitters glutamate and aspartate. In the present study, we compared the in vivo properties of the swelling-activated release of glutamate, aspartate, and another major brain osmolyte taurine. Cell swelling was induced by perfusion of hypoosmotic (low [NaCl]) medium via a microdialysis probe placed in the rat cortex. The hypoosmotic medium produced several-fold increases in the extracellular levels of glutamate, aspartate and taurine. However, the release of the excitatory amino acids differed from the release of taurine in several respects including: (i) kinetic properties, (ii) sensitivity to isoosmotic changes in [NaCl], and (iii) sensitivity to hydrogen peroxide, which is known to modulate VRAC. Consistent with the involvement of VRAC, hypoosmotic medium-induced release of the excitatory amino acids was inhibited by the anion channel blocker DNDS, but not by the glutamate transporter inhibitor TBOA or Cd2+, which inhibits exocytosis. In order to elucidate the mechanisms contributing to taurine release, we studied its release properties in cultured astrocytes and cortical synaptosomes. Similarities between the results obtained in vivo and in synaptosomes suggest that the swelling-activated release of taurine in vivo may be of neuronal origin. Taken together, our findings indicate that different transport mechanisms and/or distinct cellular sources mediate hypoosmotic medium-induced release of the excitatory amino acids and taurine in vivo

ATP signalling in epilepsy

This paper focuses on a role for ATP neurotransmission and gliotransmission in the pathophysiology of epileptic seizures. ATP along with gap junctions propagates the glial calcium wave, which is an extraneuronal signalling pathway in the central nervous system. Recently astrocyte intercellular calcium waves have been shown to underlie seizures, and conventional antiepileptic drugs have been shown to attenuate these calcium waves. Blocking ATP-mediated gliotransmission, therefore, represents a potential target for antiepileptic drugs. Furthermore, while knowledge of an antiepileptic role for adenosine is not new, a recent study showed that adenosine accumulates from the hydrolysis of accumulated ATP released by astrocytes and is believed to inhibit distant synapses by acting on adenosine receptors. Such a mechanism is consistent with a surround-inhibitory mechanism whose failure would predispose to seizures. Other potential roles for ATP signalling in the initiation and spread of epileptiform discharges may involve synaptic plasticity and coordination of synaptic networks. We conclude by making speculations about future developments

Large-Scale Mass Spectrometry Imaging Investigation of Consequences of Cortical Spreading Depression in a Transgenic Mouse Model of Migraine

Cortical spreading depression (CSD) is the electrophysiological correlate of migraine aura. Transgenic mice carrying the R192Q missense mutation in the Cacna1a gene, which in patients causes familial hemiplegic migraine type 1 (FHM1), exhibit increased propensity to CSD. Herein, mass spectrometry imaging (MSI) was applied for the first time to an animal cohort of transgenic and wild type mice to study the biomolecular changes following CSD in the brain. Ninety-six coronal brain sections from 32 mice were analyzed by MALDI-MSI. All MSI datasets were registered to the Allen Brain Atlas reference atlas of the mouse brain so that the molecular signatures of distinct brain regions could be compared. A number of metabolites and peptides showed substantial changes in the brain associated with CSD. Among those, different mass spectral features showed significant (t-test, P < 0.05) changes in the cortex, 146 and 377 Da, and in the thalamus, 1820 and 1834 Da, of the CSD-affected hemisphere of FHM1 R192Q mice. Our findings reveal CSD- and genotype-specific molecular changes in the brain of FHM1 transgenic mice that may further our understanding about the role of CSD in migraine pathophysiology. The results also demonstrate the utility of aligning MSI datasets to a common reference atlas for large-scale MSI investigations. [Figure: see text] ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s13361-015-1136-8) contains supplementary material, which is available to authorized users

Astrocyte volume regulation

This dissertation deals with the regulation of synapse formation between cultured hippocampal neurons, and the volume dynamics of astrocytes during hyposmotic-induced swelling;The developmental time course of synapse formation in cultured hippocampal neurons was examined. After 12 days in culture (DIC) inhibitory and excitatory synapses formed which were sensitive to the N-type calcium channel antagonist [omega]-conotoxin GVIA ([omega]-CgTx) while, at 4 DIC, immature connections were present in which spontaneous, but rarely evoked, synaptic currents were detected. A comparison of 4 and 12 DIC neurons revealed the presence of the synaptic proteins rab3a, synapsin I, and synaptotagmin, but the subcellular distribution changed from one in which immunoreactivity was initially distributed within the soma and neurites to a punctate varicose appearance. Correlated with these changes from immature to mature synaptic states was the development of [omega]-CgTx-sensitive calcium influx. These data suggest that the expression of functional [omega]-CgTx-sensitive calcium influx is temporally coincident with synapse formation, and that during the maturation of the synapse there is a redistribution of synaptic proteins;While astrocytes are known to actively regulate cell volume, the role of the F-actin cytoskeleton and membrane cycling in this process are ill-defined. A combination of scanning probe microscopy and a fluorescent dye dilution assay were utilized to dynamically examine hyposmotic-induced volume changes. Treatment of astrocytes with cytochalasin B to disrupt the F-actin cytoskeleton enhanced the hyposmotic-induced volume increase, but the rate of the compensatory regulatory volume decrease (RVD) was unaffected. Hyposmotic saline neither enhanced cell surface area nor caused altered membrane trafficking. These data are consistent with the hypothesis that astrocytes unfold plasma membrane upon treatment with hyposmotic saline, and reconvolute membrane during RVD. Furthermore, the F-actin based cytoskeleton may function to retard peak volume changes, presumably by restricting membrane unfolding, but does not function in controlling RVD. In addition to hyposmotic saline, glutamate release from astrocytes can also be stimulated by [alpha]-latrotoxin. Application of [alpha]-latrotoxin in the presence or absence of external calcium does not cause a significant increase in astrocyte volume, indicating that [alpha]-latrotoxin is not exerting its secretagogue effect through a swelling-activated pathway.</p