The identification of vesicular glutamate transporter 3 suggests novel modes of signaling by glutamate - PubMed (original) (raw)

. 2002 Oct 29;99(22):14488-93.

doi: 10.1073/pnas.222546799. Epub 2002 Oct 18.

Jonathon Burman, Tayyaba Qureshi, Cindy H Tran, John Proctor, Juliette Johnson, Hui Zhang, David Sulzer, David R Copenhagen, Jon Storm-Mathisen, Richard J Reimer, Farrukh A Chaudhry, Robert H Edwards

Affiliations

The identification of vesicular glutamate transporter 3 suggests novel modes of signaling by glutamate

Robert T Fremeau Jr et al. Proc Natl Acad Sci U S A. 2002.

Abstract

Quantal release of the principal excitatory neurotransmitter glutamate requires a mechanism for its transport into secretory vesicles. Within the brain, the complementary expression of vesicular glutamate transporters (VGLUTs) 1 and 2 accounts for the release of glutamate by all known excitatory neurons. We now report the identification of VGLUT3 and its expression by many cells generally considered to release a classical transmitter with properties very different from glutamate. Remarkably, subpopulations of inhibitory neurons as well as cholinergic interneurons, monoamine neurons, and glia express VGLUT3. The dendritic expression of VGLUT3 by particular neurons also indicates the potential for retrograde synaptic signaling. The distribution and subcellular location of VGLUT3 thus suggest novel modes of signaling by glutamate.

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Figures

Fig 1.

Fig 1.

Discrete cell populations express VGLUT3 mRNA. (A) In situ hybridization with 35S-labeled VGLUT3 antisense RNA shows strong labeling of a superficial layer in the cortex, including the piriform cortex (Pir), and structures in the caudate-putamen (CPu). (B) In the hippocampus, pyramidal (CA1, CA3) and dentate gyrus (DG) granule cell layers contain VGLUT3 mRNA. The cortex, amygdala (Amy), and hypothalamus (Hypo) also label for VGLUT3. (C) A section adjacent to B shows no hybridization to 35S-labeled sense VGLUT3 RNA. (D_–_F) In the neocortex, dark-field microscopy of emulsion autoradiographs shows expression of VGLUT3 (D) by multiple layers, but most strongly by layer II. In contrast, VGLUT1 antisense RNA (E) labels all layers except layer I at high levels, and VGLUT2 (F) preferentially labels layer IV. cc, corpus callosum. (G and H) Fields CA3 (G), CA1 and dentate gyrus (H) of the hippocampus contain scattered cells (arrowheads) in stratum radiatum hybridizing strongly to VGLUT3 antisense RNA. Cells in the pyramidal and granule cell layers hybridize at lower levels. (I) In the midbrain, cells in the substantia nigra pars compacta (SNC) and ventral tegmental area (VTA) express VGLUT3 mRNA. (J) Cells in the dorsal raphe (DR), pontine raphe nuclei (RPn), and olivary nuclei hybridize strongly for VGLUT3. (K) In the cerebellum, the granule cell layer (g) labels most strongly for VGLUT3, but the molecular layer (m) also contains scattered hybridizing cells. The white matter (w) is unlabeled. p, Purkinje cell layer. [Bars = 1.25 mm (A_–_C), 200 μm (D_–_H and K), and 750 μm (I and J).]

Fig 2.

Fig 2.

Immunocytochemical localization of VGLUT3 to nerve terminals, inteneuron cell bodies, and glia. (A) In the hippocampus, the VGLUT3 antibody labels puncta in the pyramidal and granule cell layers, and at the border between stratum radiatum and lacunosum-moleculare. P, stratum pyramidale; R, stratum radiatum; LM, stratum lacunosum-moleculare; M, moleculare; Gr, granule cell layer of the dentate gyrus. (B) Preincubation with the GST-VGLUT3 fusion protein abolishes the staining for VGLUT3. (C) At high magnification, in CA3, labeled puncta surround unstained pyramidal cell bodies (p) (large arrowheads), and proximal parts of dendrites (small arrow). Similar puncta surround pyramidal cells in CA1 and granule cells of the dentate gyrus (not shown). (D) VGLUT3-immunoreactive terminals outlining a pair of unstained interneuron cell bodies (n) (large arrowheads) and their dendrites (small arrows) in stratum radiatum. (E) VGLUT3 in the perikaryon (but not the nucleus) of an interneuron in the hilus of the dentate gyrus. Stained interneuron-like cells, often decorated with stained nerve endings, occur scattered throughout the hippocampus (not shown). (F) In the striatum, a subpopulation of neurons (n) labels for VGLUT3 among many unstained cells. The labeling includes the dendritic compartment (arrows). (G) The neocortex contains scattered VGLUT3-positive interneuronal cells (n). Similar to the hippocampus, labeled perikarya and dendrites are often contacted by immunoreactive nerve endings (arrowheads). (H) Heavily stained terminals decorate the cell body (arrowhead) and dendrites (arrows) of a subpopulation of neurons (n) in layer II of the neocortex. (I and J) VGLUT3-positive glial cells (a) are seen most clearly in fiber tracts, in this case the corpus callosum. They occasionally contact capillaries (c) in association with perivascular staining (small arrowheads). C_–_J were examined under oil with a ×63 objective by differential interference contrast. [Bar in B indicates 50 μm (A and B), in C 25 μm (C_–_E), and in F 25 μm (F_–_J).]

Fig 3.

Fig 3.

GABAergic, cholinergic, and aminergic neurons express VGLUT3. (A_–_C) Scattered interneurons in stratum radiatum (Rad) of the hippocampus double stain for both VGLUT3 (A) and GAD (B and C). A subset of GAD-immunoreactive processes within the pyramidal cell layer (Py) also express VGLUT3. However, not all GAD-positive cells express VGLUT3 (arrow). (D_–_F) In the retina, a subset of cells in the inner nuclear layer (INL) and a discrete layer of synapses in the inner plexiform layer (IPL) label for VGLUT3 (D). Double staining for GAD (E) shows that the VGLUT3-positive cell bodies do not express GAD (F). (G_–_I) In the striatum, VGLUT3-immunoreactive perikarya and dendrites (G) also express choline acetyltransferase (H and I). (J_–_L) Processes of a raphe neuron grown in autaptic microculture label for VGLUT3 (J) as well as serotonin (K and L). Although most processes show colocalization, a subset contain serotonin but not VGLUT3 (arrow) whereas others contain VGLUT3 but not serotonin (arrowheads) (L). [Bars = 50 μm (A_–_C and G_–_I), 12.5 μm (D_–_F), and 10 μm (J_–_L).]

Fig 4.

Fig 4.

Ultrastructural localization of VGLUT3 to inhibitory nerve endings, dendrites, and astrocytic processes. (A) A VGLUT3-immunoreactive terminal (T) makes symmetric synapses (demarcated by arrowheads) onto two unlabeled pyramidal cells (p) in area CA1 of the hippocampus. (B) A labeled terminal (T) filled with synaptic vesicles makes a symmetric synapse (demarcated by arrowheads) with the proximal part of an apical dendrite in stratum radiatum of the hippocampus. An adjacent unlabeled terminal forms an asymmetric synapse onto a dendritic spine (S). (C) A subpopulation of dendrites (D) in the hippocampus exhibit staining of vesicle-like structures (*). Note the adjacent unlabeled terminal and dendrite. m, mitochondria. (D) In the striatum, multivesicular bodies (mvb) stain strongly for VGLUT3. (E and F) Dendrites in the striatum label strongly for VGLUT3, particularly in the vicinity of multiple synapses made by large terminals (demarcated by arrowheads) (E) and synapses made by smaller terminals (F). (G) The staining of dendrites extends to distal parts, including the thinnest branches. Note the labeling of vesicle-like structures (*) in proximity to an asymmetric synapse. (H) A subset of glial processes label for VGLUT3. A stained glial process (G) here surrounds the asymmetric synapse made onto a synaptic spine. (I) Glial end-feet (G) containing VGLUT3-immunoreactive membranes (*) also surround blood vessels (V). Glial filaments (f) and mitochondria (m) as well as endothelial cells (E) and myelinated axons (A) are unlabeled. (Bars = 250 nm.)

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