Molecular, Structural, Functional, and Pharmacological Sites for Vesicular Glutamate Transporter Regulation.
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ABSTRACT:
SUBMITTER: Pietrancosta N
PROVIDER: S-EPMC7261050 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:A fundamental question in synaptic physiology is whether the unitary strength of a synapse can be regulated by presynaptic characteristics and, if so, what those characteristics might be. Here, we characterize a newly proposed mechanism for altering the strength of glutamatergic synapses based on the recently identified vesicular glutamate transporter VGLUT1. We provide direct evidence that filling in isolated synaptic vesicles is subject to a dynamic equilibrium that is determined by both the concentration of available glutamate and the number of vesicular transporters participating in loading. We observe that changing the number of vesicular transporters expressed at hippocampal excitatory synapses results in enhanced evoked and miniature responses and verify biophysically that these changes correspond to an increase in the amount of glutamate released per vesicle into the synaptic cleft. In addition, we find that this modulation of synaptic strength by vesicular transporter expression is endogenously regulated, both across development to coincide with a maturational increase in vesicle cycling and quantal amplitude and by excitatory and inhibitory receptor activation in mature neurons to provide an activity-dependent scaling of quantal size via a presynaptic mechanism. Together, these findings underscore that vesicular transporter expression is used endogenously to directly regulate the extent of glutamate release, providing a concise presynaptic mechanism for controlling the quantal efficacy of excitatory transmission during synaptic refinement and plasticity.
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Project description: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.
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Project description:Presynaptic terminal formation is a complex process that requires assembly of proteins responsible for synaptic transmission at sites of axo-dendritic contact. Accumulation of presynaptic proteins at developing terminals is facilitated by glutamate receptor activation. Glutamate is loaded into synaptic vesicles for release via the vesicular glutamate transporters VGLUT1 and VGLUT2. During postnatal development there is a switch from predominantly VGLUT2 expression to high VGLUT1 and low VGLUT2, raising the question of whether the developmental increase in VGLUT1 is important for presynaptic development. Here, we addressed this question using confocal microscopy and quantitative immunocytochemistry in primary cultures of rat neocortical neurons. First, in order to understand the extent to which the developmental switch from VGLUT2 to VGLUT1 occurs through an increase in VGLUT1 at individual presynaptic terminals or through addition of VGLUT1-positive presynaptic terminals, we examined the spatio-temporal dynamics of VGLUT1 and VGLUT2 expression. Between 5 and 12 days in culture, the percentage of presynaptic terminals that expressed VGLUT1 increased during synapse formation, as did expression of VGLUT1 at individual terminals. A subset of VGLUT1-positive terminals also expressed VGLUT2, which decreased at these terminals. At individual terminals, the increase in VGLUT1 correlated with greater accumulation of other synaptic vesicle proteins, such as synapsin and synaptophysin. When the developmental increase in VGLUT1 was prevented using VGLUT1-shRNA, the density of presynaptic terminals and accumulation of synapsin and synaptophysin at terminals were decreased. Since VGLUT1 knock-down was limited to a small number of neurons, the observed effects were cell-autonomous and independent of changes in overall network activity. These results demonstrate that up-regulation of VGLUT1 is important for development of presynaptic terminals in the cortex.
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Project description:The vesicular glutamate (GLU) transporter (VGLUT1) is a critical component of glutamatergic neurons that regulates GLU release. Despite the likely role of GLU release in drug abuse pathology, there is no information that links VGLUT1 with drugs of abuse. This study provides the first evidence that methamphetamine (METH) alters the dynamic regulation of striatal VGLUT1 function and expression through a polysynaptic pathway. METH increases cortical VGLUT1 mRNA, striatal VGLUT1 protein in subcellular fractions, and the Vmax of striatal vesicular GLU uptake. METH also increases glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein in the crude vesicle fraction. METH-induced increases in cortical VGLUT1 mRNA, as well as striatal VGLUT1 and GAPDH, are GABA(A) receptor-dependent because they are blocked by GABA(A) receptor antagonism in the substantia nigra. These results show that VGLUT1 can be dynamically regulated via a polysynaptic pathway to facilitate vesicular accumulation of GLU for subsequent release after METH.
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