Immunogold Detection of L-glutamate and D-serine in Small Synaptic-Like Microvesicles in Adult Hippocampal Astrocytes

Abstract

Glutamate and the N-methyl-D-aspartate receptor ligand D-serine are putative gliotransmitters. Here, we show by immunogold cytochemistry of the adult hippocampus that glutamate and D-serine accumulate in synaptic-like microvesicles (SLMVs) in the perisynaptic processes of astrocytes. The estimated concentration of fixed glutamate in the astrocytic SLMVs is comparable to that in synaptic vesicles of excitatory nerve terminals (≈ 45 and ≈ 55 mM, respectively), whereas the D-serine level is about 6 mM. The vesicles are organized in small spaced clusters located near the astrocytic plasma membrane. Endoplasmic reticulum is regularly found in close vicinity to SLMVs, suggesting that astrocytes contain functional nanodomains, where a local Ca(2+) increase can trigger release of glutamate and/or D-serine.

Astrocyte Calcium Signaling Transforms Cholinergic Modulation to Cortical PlasticityIn Vivo

“…On the other hand, immunohistochemical studies using D-Ser antisera show the dominance of D-Ser in astrocytes (Schell et al, 1995;Wako et al, 1995;Williams et al, 2006). A recent study using immunogold electron microscopy detected D-Ser in synaptic-like microvesicles near the endoplasmic reticulum (ER), a major source of Ca 2ϩ for astrocytic [Ca 2ϩ ] i surges, in the perisynaptic processes of astrocytes (Bergersen et al, 2011), hinting at an ER activity-triggered release of D-Ser. In the present study, the extracellular concentration of D-Ser was significantly higher in the first 10 min in WT mice than IP 3 R2-KO mice, while the basal level of extracellular D-Ser concentration was similar between these mice.…”

Excitable Astrocytes: Ca2+- and cAMP-Regulated Exocytosis

“…SLMVs were found in close proximity to the plasma membrane in perisynaptic processes of astrocytes and have estimated diameters of 30-100 nm. They are present in much smaller numbers (2-15 vesicles) and less ordered groups than SVs of similar size in nerve terminals, where large pools of SVs exist with 100-1000s SVs per synapse [21,68,98,99]. In hippocampal slices, D-serine was shown to be released from much larger vesicles (1-3 lm in diameter) that are likely generated by intracellular fusion of SLMVs and/or other organelles following sustained increases in [Ca 2? ]…”

“…Glutamate and D-serine were suggested to be costored inside the same SLMV [31], since the SNARE protein VAMP2 is colocalized with both vGLUTs [21] and Dserine [31,32] in cultured astrocytes. Although an in situ study using immunogold labelling has shown that glutamate and D-serine are stored in a distinct SLMV population within the same astrocyte [99], a recent investigation on immunopurified astrocytic vesicles demonstrated that these vesicles can indeed co-store both gliosignal transmitters [98]. Comparison of isolated SLMVs from culture astrocytes [71,98] and isolated neuronal SVs shows that astrocytic SLMVs contain D-serine and glutamate, whereas isolated SVs contain glutamate, glycine and GABA but are devoid of D-serine [98,101], indicating a distinct physiological role of SLMVs and SVs in the CNS.…”

What do we know about gliotransmitter release from astrocytes?

“…However, different methods used to load organelles with dyes in culture have produced ambiguous results [65]. Based on electron microscopy (EM) analysis of astrocytes in rodent brain tissue, SLMVs have been suggested as the main storage compartment for gliotransmitters, including glutamate and d -serine [66]. In tissue, SLMVs range from 30 to 80 nm in diameter and are often found in close proximity to the plasma membrane in the perisynaptic processes of astrocytes [23,30,66].…”

“…Based on electron microscopy (EM) analysis of astrocytes in rodent brain tissue, SLMVs have been suggested as the main storage compartment for gliotransmitters, including glutamate and d -serine [66]. In tissue, SLMVs range from 30 to 80 nm in diameter and are often found in close proximity to the plasma membrane in the perisynaptic processes of astrocytes [23,30,66]. Whether other organelles, such as secretory lysosomes and dense core granules are involved in gliotransmitter release in situ has not been thoroughly investigated yet.…”