Vidar Gundersen

Astrocytes contain a vesicular compartment that is competent for regulated exocytosis of glutamate

Astrocytes establish rapid cell-to-cell communication through the release of chemical transmitters. The underlying mechanisms and functional significance of this release are, however, not well understood. Here we identify an astrocytic vesicular compartment that is competent for glutamate exocytosis. Using postembedding immunogold labeling of the rat hippocampus, we show that vesicular glutamate transporters (VGLUT1/2) and the vesicular SNARE protein, cellubrevin, are both expressed in small vesicular organelles that resemble synaptic vesicles of glutamatergic terminals. Astrocytic vesicles, which are not as densely packed as their neuronal counterparts, can be observed in small groups at sites adjacent to neuronal structures bearing glutamate receptors. Fluorescently tagged VGLUT-containing vesicles were studied dynamically in living astrocytes by total internal reflection fluorescence (TIRF) microscopy. After activation of metabotropic glutamate receptors, astrocytic vesicles underwent rapid (milliseconds) Ca2+- and SNARE-dependent exocytic fusion that was accompanied by glutamate release. These data document the existence of a Ca2+-dependent quantal glutamate release activity in glia that was previously considered to be specific to synapses.

Glutamate exocytosis from astrocytes controls synaptic strength

The release of transmitters from glia influences synaptic functions. The modalities and physiological functions of glial release are poorly understood. Here we show that glutamate exocytosis from astrocytes of the rat hippocampal dentate molecular layer enhances synaptic strength at excitatory synapses between perforant path afferents and granule cells. The effect is mediated by ifenprodil-sensitive NMDA ionotropic glutamate receptors and involves an increase of transmitter release at the synapse. Correspondingly, we identify NMDA receptor 2B subunits on the extrasynaptic portion of excitatory nerve terminals. The receptor distribution is spatially related to glutamate-containing synaptic-like microvesicles in the apposed astrocytic processes. This glial regulatory pathway is endogenously activated by neuronal activity–dependent stimulation of purinergic P2Y1 receptors on the astrocytes. Thus, we provide the first combined functional and ultrastructural evidence for a physiological control of synaptic activity via exocytosis of glutamate from astrocytes.

Expression of the vesicular glutamate transporters during development indicates the widespread corelease of multiple neurotransmitters

Three closely related proteins transport glutamate into synaptic vesicles for release by exocytosis. Complementary patterns of expression in glutamatergic terminals have been reported for VGLUT1 and VGLUT2. VGLUT3 shows expression by many cells not considered to be glutamatergic. Here we describe the changes in VGLUT expression that occur during development. VGLUT1 expression increases gradually after birth and eventually predominates over the other isoforms in telencephalic regions. Expressed at high levels shortly after birth, VGLUT2 declines with age in multiple regions, in the cerebellum by 14‐fold. In contrast, Coexpression of the two isoforms occurs transiently during development as well as permanently in a restricted subset of glutamatergic terminals in the adult. VGLUT3 is transiently expressed at high levels by select neuronal populations, including terminals in the cerebellar nuclei, scattered neurons in the cortex, and progenitor‐like cells, implicating exocytotic glutamate release in morphogenesis and development. VGLUT3 also colocalizes extensively during development with the neuronal vesicular monoamine transporter VMAT2, with the vesicular acetylcholine transporter VAChT, and with the vesicular γ‐aminobutyric acid transporter VGAT. Such coexpression occurs particularly at some specific developmental stages and is restricted to certain sets of cells. In skeletal muscle, VGLUT3 localizes to granular organelles in the axon terminal as well as in the muscle sarcoplasm. The results suggest novel mechanisms and roles for regulated transmitter release. J. Comp. Neurol. 480:264–280, 2004.

A QUANTITATIVE ASSESSMENT OF GLUTAMATE UPTAKE INTO HIPPOCAMPAL SYNAPTIC TERMINALS AND ASTROCYTES : NEW INSIGHTS INTO A NEURONAL ROLE FOR EXCITATORY AMINO ACID TRANSPORTER 2 (EAAT2)

The relative distribution of the excitatory amino acid transporter 2 (EAAT2) between synaptic terminals and astroglia, and the importance of EAAT2 for the uptake into terminals is still unresolved. Here we have used antibodies to glutaraldehyde-fixed d-aspartate to identify electron microscopically the sites of d-aspartate accumulation in hippocampal slices. About 3/4 of all terminals in the stratum radiatum CA1 accumulated d-aspartate-immunoreactivity by an active dihydrokainate-sensitive mechanism which was absent in EAAT2 glutamate transporter knockout mice. These terminals were responsible for more than half of all d-aspartate uptake of external substrate in the slices. This is unexpected as EAAT2-immunoreactivity observed in intact brain tissue is mainly associated with astroglia. However, when examining synaptosomes and slice preparations where the extracellular space is larger than in perfusion fixed tissue, it was confirmed that most EAAT2 is in astroglia (about 80%). Neither d-aspartate uptake nor EAAT2 protein was detected in dendritic spines. About 6% of the EAAT2-immunoreactivity was detected in the plasma membrane of synaptic terminals (both within and outside of the synaptic cleft). Most of the remaining immunoreactivity (8%) was found in axons where it was distributed in a plasma membrane surface area several times larger than that of astroglia. This explains why the densities of neuronal EAAT2 are low despite high levels of mRNA in CA3 pyramidal cell bodies, but not why EAAT2 in terminals account for more than half of the uptake of exogenous substrate by hippocampal slice preparations. This and the relative amount of terminal versus glial uptake in the intact brain remain to be discovered.

Demonstration of glutamate/aspartate uptake activity in nerve endings by use of antibodies recognizing exogenous d-aspartate

Nerve terminals as well as glial cells are thought to possess high-affinity Na(+)-dependent transport sites for excitatory amino acids. However, recent immunocytochemical results with antibodies against such a transporter isolated from rat brain showed a selective labelling of glial cells [Danbolt et al. (1992) Neuroscience 51, 295-310]. Critical evaluation of the literature indicates that previous evidence for nerve terminal uptake of acidic amino acids might possibly be attributed to glia. To find out whether there is indeed a glutamate transporter in nerve endings, we incubated hippocampal slices with D-aspartate (10 and 50 microM), a metabolically inert substrate for the high-affinity glutamate transport system. After fixation by glutaraldehyde/formaldehyde the slices were processed immunocytochemically with specific polyclonal antibodies raised against D-aspartate coupled to albumin by glutaraldehyde/formaldehyde. The electron-microscopic postembedding immunogold technique demonstrated a large accumulation of gold particles in nerve terminals making asymmetrical synapses, compared to their postsynaptic dendritic spines, as well as in glial cell processes. The labelled terminals include those of the glutamatergic Schaffer collaterals. Axosomatic boutons appeared unlabelled. Comparison with a test conjugate with known concentration of fixed D-aspartate (94 mM) suggests that the concentration attained in the terminals after incubation with 50 microM D-aspartate was in the lower millimolar range. The uptake was totally dependent on Na+, blocked by L-threo-3-hydroxyaspartate, and had a high affinity for D-aspartate (apparent Km about 20 microM). There was no labelling in slices incubated without D-aspartate. Compared to glia, the nerve terminals had a higher D-aspartate density and accounted for a much higher proportion of the total tissue uptake, but this relationship may be different in vivo. At the light-microscopic level the D-aspartate-like immunoreactivity showed a distinct laminar distribution, identical to that shown autoradiographically for D-[3H]aspartate and L-[3H]glutamate uptake sites [Taxt and Storm-Mathisen (1984) Neuroscience 11, 79-100], and corresponding to the terminal fields of the major excitatory fibre systems in the hippocampal formation. The novel approach described here establishes that glutamatergic nerve terminals as well as glia do sustain sodium-dependent high-affinity transport of excitatory amino acids, implying that more than one glutamate transporter must be present in the brain. Immunogold detection of D-aspartate gives a much higher anatomical resolution than electron microscopic autoradiography of D-[3H]aspartate or L-[3H]glutamate uptake, the only method that has been available previously for ultrastructural demonstration of uptake activity.(ABSTRACT TRUNCATED AT 400 WORDS)

Immunogold quantification of amino acids and proteins in complex subcellular compartments

An increasing number of imaging techniques are in use to study the localization of molecules involved in cell-to-cell signaling. Here we describe the use of immunogold procedures to detect and quantify molecules on electron micrographs. To measure the areas of the subcellular compartments under investigation, the protocol uses an overlay screen with an array of regularly spaced points. On the basis of this, the densities of the gold-labeled molecules can be calculated. Despite the limited lateral resolution of the immunogold method as used by many investigators (∼30 nm), it is possible to measure the content of molecules associated with tiny tissue compartments, e.g., synaptic vesicles and different types of membrane, such as plasma membranes and vesicle membranes. The quantification protocol can be carried out without using computer programs. The entire protocol can be completed in ∼15 d.

Morphological evidence for vesicular glutamate release from astrocytes

There is now growing evidence that astrocytes, like neurons, can release transmitters. One transmitter that in a vast number of studies has been shown to be released from astrocytes is glutamate. Although asytrocytic glutamate may be released by several mechanisms, the evidence in favor of exocytosis is most compelling. Astrocytes may respond to neuronal activity by such exocytotic release of glutamate. The astrocyte derived glutamate can in turn activate neuronal glutamate receptors, in particular N-methyl-D-aspartate (NMDA) receptors. Here we review the morphological data supporting that astrocytes possess the machinery for exocytosis of glutamate. We describe the presence of small synaptic-like microvesicles, SNARE proteins and vesicular glutamate transporters in astrocytes, as well as NMDA receptors situated in vicinity of the astrocytic vesicles.

GABA and GABAA receptors at hippocampal mossy fibre synapses

Anatomical and electrophysiological evidence has raised the possibility that corelease of GABA and glutamate occurs at hippocampal mossy fibre synapses which, however, lack the vesicular GABA transporter VGAT. Here, we apply immunogold cytochemistry to show that GABA, like glutamate, has a close spatial relation to synaptic vesicles in rat mossy fibre terminals, implying that a mechanism exists to package GABA in synaptic vesicles. We also show that GABAA and AMPA receptors are colocalized at mossy fibre synapses. The expression of GABA and GABAA receptors is, however, weaker than in inhibitory synapses. Electrical stimuli that recruit mossy fibres evoke monosynaptic GABAA receptor‐mediated signals in post‐synaptic targets that show marked frequency‐dependent facilitation and sensitivity to group II metabotropic receptors, two features that are characteristic of mossy fibre transmission. These results provide further evidence for GABA and glutamate cotransmission at mossy fibre synapses, although paired pre‐ and post‐synaptic recordings will be required to determine the role of GABA at this unusual synapse.

A quantitative electron microscopic immunocytochemical study of the distribution and synaptic handling of glutamate in rat hippocampus.

One of the major problems in glutamate immunocytochemistry has been the difficulty involved in separating immunocytochemical labelling due to metabolic glutamate from the labelling caused by transmitter glutamate. Another problem appears to be the accessibility of antigenic sites in conventional light microscopic preparations. In the present report, we have applied the primary glutamate antiserum onto ultrathin tissue sections, followed by the use of a colloidal gold detection system. The use of this postembedding immunogold procedure allows equal access of antibodies to all cellular compartments exposed at the section surface, allows quantitative assessment of the immunoreactivity, and affords a high resolution compatible with studies at the organelle level. When applied to slice preparations the immunogold procedure can be used to identify releasable pools of glutamate. These methodological advances have greatly increased the usefulness of glutamate immunocytochemistry as a tool to study putative glutamatergic terminals in the CNS.

GLYCINE, GABA AND THEIR TRANSPORTERS IN PANCREATIC ISLETS OF LANGERHANS: EVIDENCE FOR A PARACRINE TRANSMITTER INTERPLAY

To elucidate the possible roles of the CNS neurotransmitters glycine and GABA in neuroendocrine paracrine signalling, we investigated their localizations, and those of their transport proteins, by confocal immunofluorescence and quantitative post-embedding immuno-electron microscopy in the pancreatic islets of Langerhans. We show that A-cells contain glycine in synaptic-like microvesicles as well as in secretory granules. A-cells express the macromolecules necessary to: (1) concentrate glycine within both organelle types before release (the vesicular GABA/glycine transporter VGAT=VIAAT); and to (2) take up the transmitter from the extracellular space (the plasma membrane glycine transporter GLYT2). Also B-cells have glycine in their microvesicles and granules, but the microvesicle/cytosol ratio is lower than in A-cells, consistent with the presence of GABA (which competes with glycine for vesicular uptake) in the cytosol at a much higher concentration in B-cells than in A-cells. Both A- and B-cells contain GABA in their microvesicles and secretory granules, and the membranes of the two organelle types contain VGAT in both cell types. A-cells as well as B-cells express a plasma membrane transporter GAT3 that mediates uptake of GABA. The localization of VGAT in the cores of A-cell secretory granules, and in the secretory granule membranes in both cell types, indicates novel aspects of the mechanisms for release of glycine and GABA. The discovery that both A- and B-cells possess the molecular machinery for the evoked release of both glycine and GABA from synaptic-like microvesicles suggests that both of the principal inhibitory transmitters in the brain participate in paracrine signalling in the pancreas.