Yingchun Ni

Vesicular glutamate transporter-dependent glutamate release from astrocytes

Astrocytes exhibit excitability based on variations of their intracellular Ca2+ concentrations, which leads to glutamate release, that in turn can signal to adjacent neurons. This glutamate-mediated astrocyte–neuron signaling occurs at physiological intracellular Ca2+ levels in astrocytes and includes modulation of synaptic transmission. The mechanism underlying Ca2+-dependent glutamate release from astrocytes is most likely exocytosis, because astrocytes express the protein components of the soluble N-ethyl maleimide-sensitive fusion protein attachment protein receptors complex, including synaptobrevin 2, syntaxin, and synaptosome-associated protein of 23 kDa. Although these proteins mediate Ca2+-dependent glutamate release from astrocytes, it is not well understood whether astrocytes express functional vesicular glutamate transporters (VGLUTs) that are critical for vesicle refilling. Here, we find in cultured and freshly isolated astrocytes the presence of brain-specific Na+-dependent inorganic phosphate cotransporter and differentiation-associated Na+-dependent inorganic phosphate cotransporter that have recently been identified as VGLUTs 1 and 2. Indirect immunocytochemistry showed a punctate pattern of VGLUT immunoreactivity throughout the entire cell body and processes, whereas pharmacological inhibition of VGLUTs abolished mechanically and agonist-evoked Ca2+-dependent glutamate release from astrocytes. Taken together, these data indicate that VGLUTs play a functional role in exocytotic glutamate release from astrocytes.

Ca2+ entry through TRPC1 channels contributes to intracellular Ca2+ dynamics and consequent glutamate release from rat astrocytes

Astrocytes can respond to a variety of stimuli by elevating their cytoplasmic Ca2+ concentration and can in turn release glutamate to signal adjacent neurons. The majority of this Ca2+ is derived from internal stores while a portion also comes from outside of the cell. Astrocytes use Ca2+ entry through store‐operated Ca2+ channels to refill their internal stores. Therefore, we investigated what role this store‐operated Ca2+ entry plays in astrocytic Ca2+ responses and subsequent glutamate release. Astrocytes express canonical transient receptor potential (TRPC) channels that have been implicated in mediating store‐operated Ca2+ entry. Here, we show that astrocytes in culture and freshly isolated astrocytes from visual cortex express TRPC1, TRPC4, and TRPC5. Indirect immunocytochemistry reveals that these proteins are present throughout the cell; the predominant expression of functionally tested TRPC1, however, is on the plasma membrane. Labeling in freshly isolated astrocytes reveals changes in TRPC expression throughout development. Using an antibody against TRPC1 we were able to block the function of TRPC1 channels and determine their involvement in mechanically and agonist‐evoked Ca2+ entry in cultured astrocytes. Blocking TRPC1 was also found to reduce mechanically induced Ca2+‐dependent glutamate release. These data indicate that Ca2+ entry through TRPC1 channels contributes to Ca2+ signaling in astrocytes and the consequent glutamate release from these cells.

Vesicular release of glutamate mediates bidirectional signaling between astrocytes and neurons.

The major excitatory neurotransmitter in the CNS, glutamate, can be released exocytotically by neurons and astrocytes. Glutamate released from neurons can affect adjacent astrocytes by changing their intracellular Ca2+ dynamics and, vice versa, glutamate released from astrocytes can cause a variety of responses in neurons such as: an elevation of [Ca2+]i, a slow inward current, an increase of excitability, modulation of synaptic transmission, synchronization of synaptic events, or some combination of these. This astrocyte‐neuron signaling pathway might be a widespread phenomenon throughout the brain with astrocytes possessing the means to be active participants in many functions of the CNS. Thus, it appears that the vesicular release of glutamate can serve as a common denominator for two of the major cellular components of the CNS, astrocytes and neurons, in brain function.

Dual regulation of Ca2+-dependent glutamate release from astrocytes: Vesicular glutamate transporters and cytosolic glutamate levels

Vesicular glutamate transporters (VGLUTs) are responsible for vesicular glutamate storage and exocytotic glutamate release in neurons and astrocytes. Here, we selectively and efficiently overexpressed individual VGLUT proteins (VGLUT1, 2, or 3) in solitary astrocytes and studied their effects on mechanical stimulation‐induced Ca2+‐dependent glutamate release. Neither VGLUT1 nor VGLUT2 overexpression changed the amount of glutamate release, whereas overexpression of VGLUT3 significantly enhanced Ca2+‐dependent glutamate release from astrocytes. None of the VGLUT overexpression affected mechanically induced intracellular Ca2+ increase. Inhibition of glutamine synthetase activity by L‐methionine sulfoximine in astrocytes, which leads to increased cytosolic glutamate concentration, greatly increased their mechanically induced Ca2+‐dependent glutamate release, without affecting intracellular Ca2+ dynamics. Taken together, these data indicate that both VGLUT3 and the cytosolic concentration of glutamate are key limiting factors in regulating the Ca2+‐dependent release of glutamate from astrocytes.

Separation of individual neurons using dielectrophoretic alternative current fields

Experimental investigations into the dynamics of neuronal networks are a fundamental step towards understanding how the nervous system works. Memory formation and development are associated with changes in the electrical activity of the neurons. To understand the changes in the electrical activity, it is essential to conduct in vitro studies on individual neurons. Hence, there is an enormous need to develop novel ways for isolating and localizing individual neurons. To this end, we designed and fabricated a 4x4 multiple microelectrode array system to spatially arrange neurons by generating dielectrophoretic traps using gradient alternating current (AC) fields. We characterized the electric field distribution inside our test platform by using three-dimensional finite element modeling (FEM) and estimated the location of neurons over the electrode array. As the first stage in forming a neuronal network, dielectrophoretic AC fields were employed to separate the neurons from the glial cells and to position individual neurons over single electrodes. The extracellular electrical activity from a single neuron was recorded. The frequency spectrum of the electrical activity was generated using fast Fourier transformation analysis (FFT) to determine the characteristic burst rates of individual neurons.

Vesicular Glutamate Transporter Expression in Supraoptic Neurones Suggests a Glutamatergic Phenotype

Magnocellular neuroendocrine cells of the supraoptic nucleus (SON) release the peptides oxytocin (OT) and vasopressin (VP) from their dendrites and terminals. In addition to peptide‐containing large dense‐core vesicles, axon terminals from these cells contain clear microvesicles that have been shown to contain glutamate. Using multilabelling confocal microscopy, we investigated the presence of vesicular glutamate transporters (VGLUTs) in astrocytes as well as VP and OT neurones of the SON. Simultaneous probing of the SON with antibodies against VGLUT isoforms 1–3, OT, VP and glial fibrillary acidic protein (GFAP) revealed the presence of VGLUT‐2 in somata and dendrites of SON neurones. Immunoreactivity (‐ir) for VGLUT‐3 was also detected in both OT and VP neurones as well as in GFAP‐ir astrocytes and other cells of the ventral glial lamina. Colocalisation of VGLUT‐2 and VGLUT‐3 in individual SON neurones was also examined and VGLUT‐ir with both antibodies could be detected in both types of SON neurones. Although VGLUT‐1‐ir was strong lateral to the SON, only sparse labelling was apparent within the nucleus, and no colocalisation with either SON neurones or astrocytes was observed. The SON or the SON plus its surrounding perinuclear zone was probed using the reverse transcriptase‐polymerase chain reaction and the presence of mRNA for all three VGLUT isoforms was detected. These results suggest that similar arrangements of transmitters exist in SON neuronal dendrites and their neurohypophysial terminals and that magnocellular neuroendocrine somata and dendrites may be capable of glutamatergic transmission.