Glutamate release from astrocytes as a non-synaptic mechanism for neuronal synchronization in the hippocampus.

Synchronization of activity of anatomically distributed groups of neurons represents a fundamental event in the processing of information in the brain. While this phenomenon is believed to result from dynamic interactions within the neuronal circuitry, how exactly populations of neurons become synchronized remains largely to be clarified. We propose that astrocytes are directly involved in the generation of neuronal synchrony in the hippocampus. By using a combination of experimental approaches in hippocampal slice preparations, including patch-clamp recordings and confocal microscopy calcium imaging, we studied the effect on CA1 pyramidal neurons of glutamate released from astrocytes upon various stimuli that trigger Ca2+ elevations in these glial cells, including Schaffer collateral stimulation. We found that astrocytic glutamate evokes synchronous, slow inward currents (SICs) and Ca2+ elevations in CA1 pyramidal neurons by acting preferentially, if not exclusively, on extrasynaptic NMDA receptors. Due to desensitization, AMPA receptors were not activated by astrocytic glutamate unless cyclothiazide was present. In the virtual absence of extracellular Mg2+, glutamate released from astrocytes was found to evoke, in paired recordings, highly synchronous SICs from two CA1 pyramidal neurons and, in Ca2+ imaging experiments, Ca2+ elevations that occurred synchronously in domains composed of 2-12 CA1 neurons. In the presence of extracellular Mg2+ (1 mM), synchronous SICs in two neurons as well as synchronous Ca2+ elevations in neuronal domains were still observed, although with a reduced frequency. Our results reveal a functional link between astrocytic glutamate and extrasynaptic NMDA receptors that contributes to the overall dynamics of neuronal synchrony. Our observations also raise a series of questions on possible roles of this astrocyte-to-neuron signaling in pathological changes in the hippocampus such as excitotoxic neuronal damage or the generation of epileptiform activity.     

Purinergic receptors mediate two distinct glutamate release pathways in hippocampal astrocytes

The purinergic P2X(7) receptor (P2X(7)R) can mediate glutamate release from cultured astrocytes. Using patch clamp recordings, we investigated whether P2X(7)Rs have the same action in hippocampal astrocytes in situ. We found that 2- and 3-O-(4-benzoylbenzoyl)ATP (BzATP), a potent, although unselective P2X(7)R agonist, triggers two different glutamate-mediated responses in CA1 pyramidal neurons; they are transient inward currents, which have the kinetic and pharmacological properties of previously described slow inward currents (SICs) due to Ca(2+)-dependent glutamate release from astrocytes, and a sustained tonic current. Although SICs were unaffected by P2X(7)Rs antagonists, the tonic current was inhibited, was amplified in low extracellular Ca(2+), and was insensitive to glutamate transporter and hemichannel inhibitors. BzATP triggered in astrocytes a large depolarization that was inhibited by P2X(7)R antagonists and amplified in low Ca(2+). In low Ca(2+) BzATP also induced lucifer yellow uptake into a subpopulation of astrocytes and CA3 neurons. Our results demonstrate that purinergic receptors other than the P2X(7)R mediate glutamate release that evokes SICs, whereas activation of a receptor that has features similar to the P2X(7)R, mediates a sustained glutamate efflux that generates a tonic current in CA1 neurons. This sustained glutamate efflux, which is potentiated under non-physiological conditions, may have important pathological actions in the brain.     

Glutamate, a neurotransmitter--and so much more. A synopsis of Wierzba III

It appears almost incredible that the first indications that glutamate excites brain tissue were obtained during the second half of the 20th century, that vesicles containing glutamate were demonstrated in glutamatergic neurons less than 25 years ago, and that glutamate was not accepted as the major excitatory transmitter until about the same time. During this span of time it has also become realized that glutamate is so much more than a conventional neurotransmitter: (1) astrocytes express vesicles accumulating glutamate by vesicular transporters akin to the vesicular glutamate transporters in glutamatergic neurons, and they release glutamate by exocytosis; (2) a series of metabolic processes in astrocytes (glutamate uptake, glutamine synthetase activity, glutamine release) are involved in neuronal reutilization of transmitter glutamate; (3) glutamine may also be utilized for synthesis of GABA, the major inhibitory transmitter; (4) de novo synthesis of glutamate accounts for 20% of cerebral glucose metabolism, all of which initially occurs in astrocytes, and at steady state a corresponding amount of glutamate is oxidatively degraded, mainly or exclusively in astrocytes; (5) tissue contents of glutamate/glutamine increase during enhanced glutamatergic activity, i.e., astrocytic de novo synthesis exceeds astrocytic metabolic degradation of glutamate.     

Two Distinct Modes of Exocytotic Fusion Pore Expansion in Large Astrocytic Vesicles

Formation of the fusion pore is a central question for regulated exocytosis by which secretory cells release neurotransmitters or hormones. Here, by dynamically monitoring exocytosis of large vesicles (2–7 μm) in astrocytes with two-photon microscopy imaging, we found that the exocytotic fusion pore was generated from the SNARE-dependent fusion at a ring shape of the docked plasma-vesicular membrane and the movement of a fusion-produced membrane fragment. We observed two modes of fragment movements, 1) a shift fragment that shifted to expand the fusion pore and 2) a fall-in fragment that fell into the collapsed vesicle to expand the fusion pore. Shift and fall-in modes are associated with full and partial collapses of large vesicles, respectively. The astrocytic marker, sulforhodamine 101, stained the fusion-produced membrane fragment more brightly than FM 1-43. Sulforhodamine 101 imaging showed that double fusion pores could simultaneously occur in a single vesicle (16% of large vesicles) to accelerate discharge of vesicular contents. Electron microscopy of large astrocytic vesicles showed shift and fall-in membrane fragments. Two modes of fusion pore formation demonstrate a novel mechanism underlying fusion pore expansion and provide a new explanation for full and partial collapses of large secretory vesicles.     

Neuronal synchrony mediated by astrocytic glutamate through activation of extrasynaptic NMDA receptors

Fast excitatory neurotransmission is mediated by activation of synaptic ionotropic glutamate receptors. In hippocampal slices, we report that stimulation of Schaffer collaterals evokes in CA1 neurons delayed inward currents with slow kinetics, in addition to fast excitatory postsynaptic currents. Similar slow events also occur spontaneously, can still be observed when neuronal activity and synaptic glutamate release are blocked, and are found to be mediated by glutamate released from astrocytes acting preferentially on extrasynaptic NMDA receptors. The slow currents can be triggered by stimuli that evoke Ca2+ oscillations in astrocytes, including photolysis of caged Ca2+ in single astrocytes. As revealed by paired recording and Ca2+ imaging, a striking feature of this NMDA receptor response is that it occurs synchronously in multiple CA1 neurons. Our results reveal a distinct mechanism for neuronal excitation and synchrony and highlight a functional link between astrocytic glutamate and extrasynaptic NMDA receptors.     

Computational modeling of paroxysmal depolarization shifts in neurons induced by the glutamate release from astrocytes

Recent experimental studies have shown that astrocytes respond to external stimuli with a transient increase of the intracellular calcium concentration or can exhibit self-sustained spontaneous activity. Both evoked and spontaneous astrocytic calcium oscillations are accompanied by exocytosis of glutamate caged in astrocytes leading to paroxysmal depolarization shifts (PDS) in neighboring neurons. Here, we present a simple mathematical model of the interaction between astrocytes and neurons that is able to numerically reproduce the experimental results concerning the initiation of the PDS. The timing of glutamate release from the astrocyte is studied by means of a combined modeling of a vesicle cycle and the dynamics of SNARE-proteins. The neuronal slow inward currents (SICs), induced by the astrocytic glutamate and leading to PDS, are modeled via the activation of presynaptic glutamate receptors. The dependence of the bidirectional communication between neurons and astrocytes on the concentration of glutamate transporters is analyzed, as well. Our numerical results are in line with experimental findings showing that astrocyte can induce synchronous PDSs in neighboring neurons, resulting in a transient synchronous spiking activity.     

Glia re-sealed particles freshly prepared from adult rat brain are competent for exocytotic release of glutamate

Glial subcellular re-sealed particles (referred to as gliosomes here) were purified from rat cerebral cortex and investigated for their ability to release glutamate. Confocal microscopy showed that the glia-specific proteins glial fibrillary acidic protein (GFAP) and S-100, but not the neuronal proteins 95-kDa postsynaptic density protein (PSD-95), microtubule-associated protein 2 (MAP-2) and beta-tubulin III, were enriched in purified gliosomes. Furthermore, gliosomes exhibited labelling neither for integrin-alphaM nor for myelin basic protein, which are specific for microglia and oligodendrocytes respectively. The Ca2+ ionophore ionomycin (0.1-5 microm) efficiently stimulated the release of tritium from gliosomes pre-labelled with [3H]d-aspartate and of endogenous glutamate in a Ca(2+)-dependent and bafilomycin A1-sensitive manner, suggesting the involvement of an exocytotic process. Accordingly, ionomycin was found to induce a Ca(2+)-dependent increase in the vesicular fusion rate, when exocytosis was monitored with acridine orange. ATP stimulated [3H]d-aspartate release in a concentration- (0.1-3 mm) and Ca(2+)-dependent manner. The gliosomal fraction contained proteins of the exocytotic machinery [syntaxin-1, vesicular-associated membrane protein type 2 (VAMP-2), 23-kDa synaptosome-associated protein (SNAP-23) and 25-kDa synaptosome-associated protein (SNAP-25)] co-existing with GFAP immunoreactivity. Moreover, GFAP or VAMP-2 co-expressed with the vesicular glutamate transporter type 1. Consistent with ultrastructural analysis, several approximately 30-nm non-clustered vesicles were present in the gliosome cytoplasm. It is concluded that gliosomes purified from adult brain contain glutamate-accumulating vesicles and can release the amino acid by a process resembling neuronal exocytosis.     

Mechanisms of glutamate release from astrocytes: gap junction "hemichannels", purinergic receptors and exocytotic release

Neuronal exocytotic release of glutamate at synapses involves a highly specialized vesicular apparatus, consisting of a variety of proteins connected to the vesicles or required for vesicular fusion to the presynaptic membrane. Astrocytes also release glutamate, and recent evidence indicates that this release can modify neuronal function. Several mechanisms have been proposed for astrocytic release of glutamate under pathological conditions, such as reversal of glutamate transporters and opening of volume sensitive ion channels. In this review we limit our discussion to findings supporting the exocytotic release of glutamate, as well as two new pathways implicated in this release, the ionotropic (P2X) purinergic receptors and gap junction hemichannels.     

Exocytotic release of ATP from cultured astrocytes

Astrocytes appear to communicate with each other as well as with neurons via ATP. However, the mechanisms of ATP release are controversial. To explore whether stimuli that increase [Ca(2+)](i) also trigger vesicular ATP release from astrocytes, we labeled ATP-containing vesicles with the fluorescent dye quinacrine, which exhibited a significant co-localization with atrial natriuretic peptide. The confocal microscopy study revealed that quinacrine-loaded vesicles displayed mainly non-directional spontaneous mobility with relatively short track lengths and small maximal displacements, whereas 4% of vesicles exhibited directional mobility. After ionomycin stimulation only non-directional vesicle mobility could be observed, indicating that an increase in [Ca(2+)](i) attenuated vesicle mobility. Total internal reflection fluorescence (TIRF) imaging in combination with epifluorescence showed that a high percentage of fluorescently labeled vesicles underwent fusion with the plasma membrane after stimulation with glutamate or ionomycin and that this event was Ca(2+)-dependent. This was confirmed by patch-clamp studies on HEK-293T cells transfected with P2X(3) receptor, used as sniffers for ATP release from astrocytes. Glutamate stimulation of astrocytes was followed by an increase in the incidence of small transient inward currents in sniffers, reminiscent of postsynaptic quantal events observed at synapses. Their incidence was highly dependent on extracellular Ca(2+). Collectively, these findings indicate that glutamate-stimulated ATP release from astrocytes was most likely exocytotic and that after stimulation the fraction of quinacrine-loaded vesicles, spontaneously exhibiting directional mobility, disappeared.     

Immunophilin deficiency augments Ca2+-dependent glutamate release from mouse cortical astrocytes

Immunophilins are receptors for immunosuppressive drugs such as the macrolides cyclosporin A (CsA) and FK506; correspondingly these immunophilins are referred to as cyclophilins and FK506-binding proteins (FKBPs). In particular, CsA targets cyclophilin D (CypD), which can modulate mitochondrial Ca(2+) dynamics. Since mitochondria have been implicated in the regulation of astrocytic cytosolic Ca(2+) (Ca(cyt)(2+)) dynamics and consequential Ca(2+)-dependent exocytotic release of glutamate, we investigated the role of CypD in this process. Cortical astrocytes isolated from CypD deficient mice Ppif(-/-) displayed reduced mechanically induced Ca(cyt)(2+) increases, even though these cells showed augmented exocytotic release of glutamate, when compared to responses obtained from astrocytes isolated from wild-type mice. Furthermore, acute treatment with CsA to inhibit CypD modulation of mitochondrial Ca(2+) buffering, or with FK506 to inhibit FKBP12 interaction with inositol-trisphosphate receptor of the endoplasmic reticulum, led to similar reductive effects on astrocytic Ca(cyt)(2+) dynamics, but also to an enhanced Ca(2+)-dependent exocytotic release of glutamate in wild-type astrocytes. These findings point to a possible role of immunophilin signal transduction pathways in astrocytic modulation of neuronal activity at the tripartite synapse.     

Adult astroglia is competent for Na+/Ca2+ exchanger-operated exocytotic glutamate release triggered by mild depolarization

Glutamate release induced by mild depolarization was studied in astroglial preparations from the adult rat cerebral cortex, that is acutely isolated glial sub-cellular particles (gliosomes), cultured adult or neonatal astrocytes, and neuron-conditioned astrocytes. K+ (15, 35 mmol/L), 4-aminopyridine (0.1, 1 mmol/L) or veratrine (1, 10 micromol/L) increased endogenous glutamate or [3H]D-aspartate release from gliosomes. Neurotransmitter release was partly dependent on external Ca2+, suggesting the involvement of exocytotic-like processes, and partly because of the reversal of glutamate transporters. K+ increased gliosomal membrane potential, cytosolic Ca2+ concentration [Ca2+]i, and vesicle fusion rate. Ca2+ entry into gliosomes and glutamate release were independent from voltage-sensitive Ca2+ channel opening; they were instead abolished by 2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiurea (KB-R7943), suggesting a role for the Na+/Ca2+ exchanger working in reverse mode. K+ (15, 35 mmol/L) elicited increase of [Ca2+]i and Ca2+-dependent endogenous glutamate release in adult, not in neonatal, astrocytes in culture. Glutamate release was even more marked in in vitro neuron-conditioned adult astrocytes. As seen for gliosomes, K+-induced Ca2+ influx and glutamate release were abolished by KB-R7943 also in cultured adult astrocytes. To conclude, depolarization triggers in vitro glutamate exocytosis from in situ matured adult astrocytes; an aptitude grounding on Ca2+ influx driven by the Na+/Ca2+ exchanger working in the reverse mode.     

Endothelial microsomal prostaglandin E synthase-1 facilitates neurotoxicity by elevating astrocytic Ca2+ levels

Recurrent seizures may cause neuronal damage in the hippocampus. As neurons form intimate interactions with astrocytes via glutamate, this neuron-glia circuit may play a pivotal role in neuronal excitotoxicity following such seizures. On the other hand, astrocytes contact vascular endothelia with their endfeet. Recently, we found kainic acid (KA) administration induced microsomal prostaglandin E synthase-1 (mPGES-1) and prostaglandin E(2) (PGE(2)) receptor EP3 in venous endothelia and on astrocytes, respectively. In addition, mice deficient in mPGES-1 exhibited an improvement in KA-induced neuronal loss, suggesting that endothelial PGE(2) might modulate neuronal damage via astrocytes. In this study, we therefore investigated whether the functional associations between endothelia and astrocytes via endothelial mPGES-1 lead to neuronal injury using primary cultures of hippocampal slices. We first confirmed the delayed induction of endothelial mPGES-1 in the wild-type (WT) slices after KA-treatment. Next, we examined the effects of endothelial mPGES-1 on Ca(2+) levels in astrocytes, subsequent glutamate release and neuronal injury using cultured slices prepared from WT and mPGES-1 knockout mice. Moreover, we investigated which EP receptor on astrocytes was activated by PGE(2). We found that endothelial mPGES-1 produced PGE(2) that enhanced astrocytic Ca(2+) levels via EP3 receptors and increased Ca(2+)-dependent glutamate release, aggravating neuronal injury. This novel endothelium-astrocyte-neuron signaling pathway may be crucial for neuronal damage after repetitive seizures, and hence could be a new target for drug development.     

Neuronal activity triggers calcium waves in hippocampal astrocyte networks.

The recent discovery that the neurotransmitter glutamate can trigger actively propagating Ca2+ waves in the cytoplasm of cultured astrocytes suggests the possibility that synaptically released glutamate may trigger similar Ca2+ waves in brain astrocytes in situ. To explore this possibility, we used confocal microscopy and the Ca2+ indicator fluo-3 to study organotypically cultured slices of rat hippocampus, where astrocytic and neuronal networks are intermingled in their normal tissue relationships. We find that astrocytic Ca2+ waves are present under these circumstances and that these waves can be triggered by the firing of glutamatergic neuronal afferents with latencies as short as 2 s. The Ca2+ waves closely resemble those previously observed in cultured astrocytes: they propagate both within and between astrocytes at velocities of 7-27 microns/s at 21 degrees C. The ability of tissue astrocyte networks to respond to neuronal network activity suggests that astrocytes may have a much more dynamic and active role in brain function than has been generally recognized.     

Prostaglandin E(2) stimulates glutamate receptor-dependent astrocyte neuromodulation in cultured hippocampal cells.

Recent Ca(2+) imaging studies in cell culture and in situ have shown that Ca(2+) elevations in astrocytes stimulate glutamate release and increase neuronal Ca(2+) levels, and that this astrocyte-neuron signaling can be stimulated by prostaglandin E(2) (PGE(2)). We investigated the electrophysiological consequences of the PGE(2)-mediated astrocyte-neuron signaling using whole-cell recordings on cultured rat hippocampal cells. Focal application of PGE(2) to astrocytes evoked a Ca(2+) elevation in the stimulated cell by mobilizing internal Ca(2+) stores, which further propagated as a Ca(2+) wave to neighboring astrocytes. Whole-cell recordings from neurons revealed that PGE(2) evoked a slow inward current in neurons adjacent to astrocytes. This neuronal response required the presence of an astrocyte Ca(2+) wave and was mediated through both N-methyl-D-aspartate (NMDA) and non-NMDA glutamate receptors. Taken together with previous studies, these data demonstrate that PGE(2)-evoked Ca(2+) elevations in astrocyte cause the release of glutamate which activates neuronal ionotropic receptors.     

Glutamate-stimulated ATP release from spinal cord astrocytes is potentiated by substance P

ATP has recently emerged as a key molecule mediating pathological pain. The aim of this study was to examine whether spinal cord astrocytes could be a source of ATP in response to the nociceptive neurotransmitters glutamate and substance P. Glutamate stimulated ATP release from these astrocytes and this release was greatly potentiated by substance P, even though substance P alone did not elicit ATP release. Substance P also potentiated glutamate-induced inward currents, but did not cause such currents alone. When glutamate was applied alone it acted exclusively through alpha-amino-3-hydroxy-5-methylisoxazole-4-proprionate receptors to stimulate Ca(2+) influx-dependent ATP release. However, when substance P was co-applied with glutamate, ATP release could be elicited by activation of NMDA and metabotropic glutamate receptors. Activation of neurokinin receptor subtypes, protein kinase C and phospholipases A(2), C and D were needed for substance P to bring about its effects. These results suggest that astrocytes may be a major source of ATP in the spinal cord on activation of nerve fibres that release substance P and glutamate.     

Regional difference of glutamate-induced swelling in cultured rat brain astrocytes

L-glutamate (glutamate) is an important neurotoxin as well as the major excitatory neurotransmitter. Extracellular glutamate levels are elevated following ischemia, hypoglycemia, and trauma. One consequence of elevated glutamate levels is cell swelling. Such swelling occurs primarily in astroglial cells. We characterized the regional difference in glutamate-induced swelling response of cultured astrocytes from rat cerebral cortex, hippocampus and cerebellum. Glutamate produced dose-dependent astrocytic swelling in both cerebral cortex and hippocampus, showing a maximal effect in 0.5 mM concentration, as measured by 3-O-methyl-D-[1-3H]glucose uptake. However, in cerebellum, glutamate did not produce astrocytic swelling. It has been suggested that Na+ -dependent glutamate uptake is a possible mechanism of glutamate-induced swelling. The Vmax for glutamate uptake into cerebellum astrocytes was significantly lower (6.7 nmol/mg protein/min) than those for cerebral cortex and hippocampus astrocytes (13.0 and 12.0 nmol/mg protein/min, respectively). In three regions, more than 90% of the cultured cells showed glial fibrillary acidic protein (GFAP) immunoreactivity. Immunoreactivity of GLT, one of the markers of glutamate transporters, which is expressed at low levels in cultured astrocytes, did not show any differences in three regions. However, immunoreactivities of GLAST, the other astroglial glutamate transporter, and aquaporin4 (APQ4), a water transporter, were significantly higher in cerebral cortex and hippocampus than in cerebellum. These results may explain the regional difference of glutamate-induced astrocytic swelling.     

Selective stimulation of astrocyte calcium in situ does not affect neuronal excitatory synaptic activity

Astrocytes are considered the third component of the synapse, responding to neurotransmitter release from synaptic terminals and releasing gliotransmitters--including glutamate--in a Ca(2+)-dependent manner to affect neuronal synaptic activity. Many studies reporting astrocyte-driven neuronal activity have evoked astrocyte Ca(2+) increases by application of endogenous ligands that directly activate neuronal receptors, making astrocyte contribution to neuronal effect(s) difficult to determine. We have made transgenic mice that express a Gq-coupled receptor only in astrocytes to evoke astrocyte Ca(2+) increases using an agonist that does not bind endogenous receptors in brain. By recording from CA1 pyramidal cells in acute hippocampal slices from these mice, we demonstrate that widespread Ca(2+) elevations in 80%-90% of stratum radiatum astrocytes do not increase neuronal Ca(2+), produce neuronal slow inward currents, or affect excitatory synaptic activity. Our findings call into question the developing consensus that Ca(2+)-dependent glutamate release by astrocytes directly affects neuronal synaptic activity in situ.     

Normal biogenesis and cycling of empty synaptic vesicles in dopamine neurons of vesicular monoamine transporter 2 knockout mice

The neuronal isoform of vesicular monoamine transporter, VMAT2, is responsible for packaging dopamine and other monoamines into synaptic vesicles and thereby plays an essential role in dopamine neurotransmission. Dopamine neurons in mice lacking VMAT2 are unable to store or release dopamine from their synaptic vesicles. To determine how VMAT2-mediated filling influences synaptic vesicle morphology and function, we examined dopamine terminals from VMAT2 knockout mice. In contrast to the abnormalities reported in glutamatergic terminals of mice lacking VGLUT1, the corresponding vesicular transporter for glutamate, we found that the ultrastructure of dopamine terminals and synaptic vesicles in VMAT2 knockout mice were indistinguishable from wild type. Using the activity-dependent dyes FM1-43 and FM2-10, we also found that synaptic vesicles in dopamine neurons lacking VMAT2 undergo endocytosis and exocytosis with kinetics identical to those seen in wild-type neurons. Together, these results demonstrate that dopamine synaptic vesicle biogenesis and cycling are independent of vesicle filling with transmitter. By demonstrating that such empty synaptic vesicles can cycle at the nerve terminal, our study suggests that physiological changes in VMAT2 levels or trafficking at the synapse may regulate dopamine release by altering the ratio of fillable-to-empty synaptic vesicles, as both continue to cycle in response to neural activity.     

Ca(2+) sources for the exocytotic release of glutamate from astrocytes

Astrocytes can exocytotically release the gliotransmitter glutamate from vesicular compartments. Increased cytosolic Ca(2+) concentration is necessary and sufficient for this process. The predominant source of Ca(2+) for exocytosis in astrocytes resides within the endoplasmic reticulum (ER). Inositol 1,4,5-trisphosphate and ryanodine receptors of the ER provide a conduit for the release of Ca(2+) to the cytosol. The ER store is (re)filled by the store-specific Ca(2+)-ATPase. Ultimately, the depleted ER is replenished by Ca(2+) which enters from the extracellular space to the cytosol via store-operated Ca(2+) entry; the TRPC1 protein has been implicated in this part of the astrocytic exocytotic process. Voltage-gated Ca(2+) channels and plasma membrane Na(+)/Ca(2+) exchangers are additional means for cytosolic Ca(2+) entry. Cytosolic Ca(2+) levels can be modulated by mitochondria, which can take up cytosolic Ca(2+) via the Ca(2+) uniporter and release Ca(2+) into cytosol via the mitochondrial Na(+)/Ca(2+) exchanger, as well as by the formation of the mitochondrial permeability transition pore. The interplay between various Ca(2+) sources generates cytosolic Ca(2+) dynamics that can drive Ca(2+)-dependent exocytotic release of glutamate from astrocytes. An understanding of this process in vivo will reveal some of the astrocytic functions in health and disease of the brain. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.     

Kinetic diversity in the fusion of exocytotic vesicles.

The speed at which secretory vesicles fuse with the plasma membrane is a key parameter for neuronal and endocrine functions. We determined the precise time courses for fusion of small clear and large dense-core vesicles in PC12 and chromaffin cells by simultaneously measuring both plasma membrane areas and release of vesicular contents. We found that instantaneous increases in cytosolic Ca2+ concentration evoked vesicle fusion, but with time constants that varied over four orders of magnitude among different types of vesicles and cells. This indicates that the molecular machinery for the final Ca2+-dependent fusion steps of exocytosis is highly variable and is as critical as Ca2+ signalling processes in determining the speed and amount of secretion of neurotransmitters and hormones. Our results suggest a new possibility that the molecules responsible for the final fusion reaction that leads to vesicle fusion are key determinants for neuronal plasticity and hormonal disorders.