Ksenija Jeftinija

ATP stimulates calcium-dependent glutamate release from cultured astrocytes.

ATP caused a dose‐dependent, receptor‐mediated increase in the release of glutamate and aspartate from cultured astrocytes. Using calcium imaging in combination HPLC we found that the increase in intracellular calcium coincided with an increase in glutamate and aspartate release. Competitive antagonists of P2 receptors blocked the response to ATP. The increase in intracellular calcium and release of glutamate evoked by ATP were not abolished in low Ca2+‐EGTA saline, suggesting the involvement of intracellular calcium stores. Pre‐treatment of glial cultures with an intracellular Ca2+ chelator abolished the stimulatory effects of ATP. Thapsigargin (1 µm), an inhibitor of Ca2+‐ATPase from the Ca2+ pump of internal stores, significantly reduced the calcium transients and the release of aspartate and glutamate evoked by ATP. U73122 (10 µm), a phospholipase C inhibitor, attenuated the ATP‐stimulatory effect on calcium transients and blocked ATP‐evoked glutamate release in astrocytes. Replacement of extracellular sodium with choline failed to influence ATP‐induced glutamate release. Furthermore, inhibition of the glutamate transporters p‐chloromercuri‐phenylsulfonic acid and ltrans‐pyrolidine‐2,4‐dicarboxylate failed to impair the ability of ATP to stimulate glutamate release from astrocytes. However, an anion transport inhibitor, furosemide, and a potent Cl− channel blocker, 5‐nitro‐2(3‐phenylpropylamino)‐benzoate, reduced ATP‐induced glutamate release. These results suggest that ATP stimulates excitatory amino acid release from astrocytes via a calcium‐dependent anion‐transport sensitive mechanism.

Cultured astrocytes express proteins involved in vesicular glutamate release

Bradykinin induces receptor-mediated calcium-dependent release of glutamate from cultured astrocytes through a mechanism that is neither due to cell-swelling mechanism nor due to the reversal of the glutamate transporter. Astrocytes may thus release glutamate using a mechanism resembling the neuronal vesicular release of neurotransmitters. Synaptobrevin is a vesicular protein that together with plasma membrane proteins syntaxin and SNAP-25 participate in formation of the anchoring core complex required for initiation of exocytosis. Here, we demonstrate that synaptobrevin II is present in cultured astrocytes. Furthermore, we demonstrate that botulinus toxin type B and tetanus toxin cause a decrease in synaptobrevin II immunoreactivity and abolish bradykinin-induced release of glutamate from cultured astrocytes. While we were not able to demonstrate the presence of SNAP-25 or syntaxin immunoreactivity in cultured astrocytes, pretreatment with BoTx-A (which cleaves SNAP-25) and BoTx-C (which cleaves syntaxins) result in a decrease in the baseline release of glutamate and diminish the bradykinin-evoked release of glutamate from cultured astrocytes. These findings strongly support the notion that astrocytes may release neurotransmitters using a mechanism similar to the neuronal secretory process.

Neuroligand-Evoked Calcium-Dependent Release of Excitatory Amino Acids from Cultured Astrocytes

Abstract: The release of excitatory amino acids (EAAs) from neuron‐free cultures of neocortical astrocytes was monitored using HPLC. The neuroligand bradykinin caused a dose‐dependent receptor‐mediated increase in release of the EAAs glutamate and aspartate from type 1 astrocyte cell cultures obtained from rat cerebral cortex. Removal of calcium from the extracellular fluid prevented the bradykinin‐induced release of EAAs from astrocytes. The addition of the calcium ionophore ionomycin caused a calcium‐dependent release of EAAs. Inhibitors of the glutamate transporters p‐chloromercuriphenylsulfonic acid, l‐trans‐pyrrolidine‐2,4‐dicarboxylate, and dihydrokainate failed to impair the ability of bradykinin to stimulate glutamate release from astrocytes. α‐Latrotoxin, an active compound of black widow spider venom, caused a significant increase of the release of glutamate in calcium‐containing saline. In calcium‐depleted saline, α‐latrotoxin produced an initial increase in the concentration of glutamate followed by a decline in the concentration of glutamate indicating stimulation of exocytosis coupled with low calcium‐induced inhibition of endocytosis. Taken together, these data suggest that astrocytes may release neurotransmitter through a mechanism that is similar to the neuronal secretory process. Given the important role of glutamate in the induction of long‐term potentiation, learning, memory, and excitotoxicity, it will be important to determine external signals that control both the uptake and release of glutamate by astrocytes.

Excitatory amino acids are released from rat primary afferent neurons in vitro

Multiple lines of evidence implicate the excitatory amino acids (EAAs) (L-aspartate (L-Asp) and L-glutamate (L-Glu) as excitatory transmitters in the spinal cord. The specific objective of this study was to determine whether the EAAs are released from primary afferents. Dorsal root ganglia (DRG) from 2 to 18-day-old rats dissected and cultured for 1-2 weeks were washed in modified Ringers recording solution for a period of 1 h to allow equilibration. The mean +/- S.E.M. baseline concentrations of EAAs recovered during a 5 min interval were 533.29 +/- 65.59 nmol for L-Glu and 106.67 +/- 14.05 nmol for L-Asp. Stimulation of DRG organotypic cultures with potassium resulted in a significant concentration-dependent increase in the release of both EAAs. The concentration of Asp increased to 166 +/- 17% and 203 +/- 13% in response to 5 min exposure of the culture to 25 and 50 mM potassium, respectively. The concentration of Glu increased to 155 +/- 12% and 226 +/- 18% of control in response to the same stimuli. In response to application of 50 mM potassium for 25 min, peak concentrations increased to 465 +/- 53% for Asp and 312 +/- 51% for Glu of the basal concentration. Exposure of the cultures to 1 or 10 microM capsaicin also caused release of both EAAs. The concentrations of Asp and Glu significantly increased to 204 +/- 11% and 165 +/- 15% of basal concentrations, respectively, in response to a 5 min exposure to 1 microM capsaicin. High [K+]e failed to increase the release of EAAs from cultures where DRG cell bodies were removed 72 h prior to release experiments. These results confirm results demonstrating release of EAA from mammalian spinal cord tissues and directly demonstrate for the first time that primary afferent fibers are specifically involved in this release.

α-Latrotoxin stimulates glutamate release from cortical astrocytes in cell culture

The mechanism responsible for the ability of bradykinin to cause calcium‐dependent release of glutamate from astrocytes in vitro was investigated. The glutamate transport inhibitor, dihydrokainate, did not block bradykinin‐induced glutamate release, and bradykinin did not cause cell swelling. These data exclude the involvement of glutamate transporters or swelling mechanisms as mediating glutamate release in response to bradykinin. α‐Latrotoxin (3 nM), a component of black widow spider venom, stimulated calcium‐independent glutamate release from astrocytes. Since α‐latrotoxin induces vesicle fusion and calcium‐independent neuronal neurotransmitter release, our data suggest that astrocytes may release neurotransmitter using a mechanism similar to the neuronal secretory process.

Stimulatory Effect of Ghrelin on Isolated Porcine Somatotropes

Research on the mechanism for growth hormone secretagogue (GHS) induction of growth hormone secretion led to the discovery of the GHS receptor (GHS-R) and later to ghrelin, an endogenous ligand for GHS-R. The ability of ghrelin to induce an increase in the intracellular Ca2+ concentration – [Ca2+]i – in somatotropes was examined in dispersed porcine pituitary cells using a calcium imaging system. Somatotropes were functionally identified by application of human growth hormone releasing hormone. Ghrelin increased the [Ca2+]i in a dose-dependent manner in 98% of the cells that responded to human growth hormone releasing hormone. In the presence of (D-Lys3)-GHRP-6, a specific receptor antagonist of GHS-R, the increase in [Ca2+]i evoked by ghrelin was decreased. Pretreatment of cultures with somatostatin or neuropeptide Y reduced the ghrelin-induced increase of [Ca2+]i. The stimulatory effect of ghrelin on somatotropes was greatly attentuated in low-calcium saline and blocked by nifedipine, an L-type calcium channel blocker, suggesting involvement of calcium channels. In a zero Na+ solution, the stimulatory effect of ghrelin on somatotropes was decreased, suggesting that besides calcium channels, sodium channels are also involved in ghrelin-induced calcium transients. Either SQ-22536, an adenylyl cyclase inhibitor, or U73122, a phospholipase C inhibitor, decreased the stimulatory effects of ghrelin on [Ca2+]i transiently, indicating the involvement of adenylyl cyclase-cyclic adenosine monophosphate and phospholipase C inositol 1,4,5-trisphosphate pathways. The nonpeptidyl GHS, L-692,585 (L-585), induced changes in [Ca2+]i similar to those observed with ghrelin. Application of L-585 after ghrelin did not have additive effects on [Ca2+]i. Preapplication of L-585 blocked the stimulatory effect of ghrelin on somatotropes. Simultaneous application of ghrelin and L-585 did not cause an additive increase in [Ca2+]i. Our results suggest that the actions of ghrelin and synthetic GHS closely parallel each other, in a manner that is consistent with an increase of hormone secretion.

Effect of capsaicin and resiniferatoxin on peptidergic neurons in cultured dorsal root ganglion

The neurotoxic effect of capsaicin has been shown to be selective on a subpopulation of small dorsal root ganglion neurons in newborn animals. The aim of this study was to provide evidence of the long lasting effect of capsaicin and its ultrapotent analog resiniferatoxin (RTX) on sensory peptidergic neurons maintained in organotypic cultures. The effects of the two irritants were examined on neurons that contained substance P (SP) and calcitonin gene-related peptide (CGRP). Exposure of the cultures to 10 microM capsaicin and 100 nM RTX for periods of 2 days or longer resulted in almost complete elimination of SP-immunoreactive (IR) neurites and reduction, but not elimination, of CGRP-IR neurites. In addition, both 10 microM capsaicin and 100 nM RTX significantly reduced the number of SP- and CGRP-IR cell bodies within DRG explants. Capsaicin in 100 microM concentration produced complete elimination of SP-IR fibers and a greater decrease in the number of CGRP-IR fibers, but failed to completely eliminate IR cell bodies. Exposure of the cultures to the irritants in the same concentrations for 90 min did not produce a measurable effect on SP- or CGRP-IR in neurites or cell bodies. It is important to establish that the effect of capsaicin and RTX on cultured neurons was of long duration (longer than 4 days) and is therefore different from depletion of peptides. These findings demonstrate that processes of cultured sensory neurons are much more sensitive to capsaicin and RTX than cell bodies. Furthermore, our results show that SP-IR neuronal elements are more sensitive to capsaicin than CGRP-IR elements. These data suggest that cultured sensory neurons express the functional properties of differentiated sensory neurons in vivo.

Rotational dynamics of cargos at pauses during axonal transport.

Direct visualization of axonal transport in live neurons is essential for our understanding of the neuronal functions and the working mechanisms of microtubule-based motor proteins. Here we use the high-speed single particle orientation and rotational tracking technique to directly visualize the rotational dynamics of cargos in both active directional transport and pausing stages of axonal transport, with a temporal resolution of 2 ms. Both long and short pauses are imaged, and the correlations between the pause duration, the rotational behaviour of the cargo at the pause, and the moving direction after the pause are established. Furthermore, the rotational dynamics leading to switching tracks are visualized in detail. These first-time observations of cargos rotational dynamics provide new insights on how kinesin and dynein motors take the cargo through the alternating stages of active directional transport and pause.

Porosome in astrocytes

Secretion is a universal cellular process occurring in bakers yeast, to the complex multicellular organisms, to humans beings. Neurotransmission, digestion, immune response or the release of hormones occur as a result of cell secretion. Secretory defects result in numerous diseases and hence a molecular understanding of the process is critical. Cell secretion involves the transport of vesicular products from within cells to the outside. Porosomes are permanent cup‐shaped supramolecular structures at the cell plasma membrane, where secretory vesicles transiently dock and transiently fuse to release intravesicular contents to the outside. In the past decade, porosomes have been determined to be the universal secretory machinery in cells, present in the exocrine pancreas, endocrine and neuroendocrine cells, and in neurons. In this study, we report for the first time the presence of porosomes in rat brain astrocytes. Using atomic force microscopy on live astrocytes, cup‐shaped porosomes measuring 10–15 nm are observed at the cell plasma membrane. Further studies using electron microscopy confirm the presence of porosomes in astrocytes. Analogous to neuronal porosomes, there is a central plug in the astrocyte porosome complex. Immunoisolation and reconstitution of the astrocyte porosome in lipid membrane, demonstrates a structure similar to what is observed in live cells. These studies demonstrate that in astrocytes, the secretory apparatus at the cell plasma membrane is similar to what is found in neurons.

Development of Functional Neurons from Postnatal Stem Cells In Vitro

In order for stem cells to fulfill their clinical promise, we must understand their developmental transitions and it must be possible to control the differentiation of stem cells into specific cell fates. To understand the mechanism of the sequential restriction and multipotency of stem cells, we have established culture conditions that allow the differentiation of multipotential neural stem cells from postnatal stem cells. We used immunocytochemistry, fluorescence microscopy, and calcium imaging to demonstrate that progeny of adult rat neural stem cells develop into functional neurons that release excitatory neurotransmitters. We also found that the nontoxic heavy chain fragment of tetanus toxin, a toxin that targets neurons with high specificity, retained the specificity toward neural stem cell–derived neurons. These studies show that neural stem cells derived from adult tissues retain the potential to differentiate into functional neurons with morphological and functional properties of mature central nervous system neurons.