Vesicular glutamate and GABA transporters sort to distinct sets of vesicles in a population of presynaptic terminals

Abstract

Vesicular glutamate transporters (VGLUTs) 1 and 2 are expressed by neurons generally accepted to release glutamate as a neurotransmitter, whereas VGLUT3 appears in populations usually associated with a different classical transmitter. We now demonstrate VGLUT2 as well as the vesicular GABA transporter (VGAT) in a subset of presynaptic terminals in the dentate gyrus of the rat hippocampal formation. The terminals are distributed in a characteristic band overlapping with the outer part of the granule cell layer and the inner zone of the molecular layer. Within the terminals, which make asymmetric as well as symmetric synapses onto the somatodendritic compartment of the dentate granule cells, the 2 transporters localize to distinct populations of synaptic vesicles. Moreover, the axons forming these terminals originate in the supramammillary nucleus (SuM). Our data reconcile previous apparently conflicting reports on the physiology of the dentate afferents from SuM and demonstrate that both glutamate and GABA may be released from a single nerve terminal.

Figure 1.

VGLUT2 localizes to nerve terminals making asymmetrical as well as symmetrical synapses in the dentate gyrus. (A) Light microscopy of a hippocampal section stained for VGLUT2 by immunoperoxidase shows immunoreactivity in the outer part of the GCL and the immediately adjacent part of the inner zone, that is, the “VGLUT2-band” of the dentate molecular layer. (B, C) Electron microscopy reveals VGLUT2 staining in axon terminals contacting the proximal part of a dendrite (B), or the cell body of a dentate granule cell (C). The preservation of ultrastructure when performing immunoperoxidase staining does not allow for certain identification of the synaptic contacts, but the slender and parallel pre- and postsynaptic membranes are compatible with symmetrical synapses (cf. Fig. 2G). Despite dense staining, some vesicles are clearly outlined. (D, E) Electron microscopy of the VGLUT2-band shows VGLUT2 immunostained nerve terminals making asymmetrical synapses on a dendritic shaft (D) and on a spine (E). At, axon terminal; D, dendrite; Gr, granule cell; m, mitochondria; Mol, molecular layer; Nu, nucleus; S, dendritic spine. Scale bars: A, 10 μm; B, 200 nm; C, 200 nm; D, 200 nm; E, 125 nm.

Figure 2.

VGLUT2 colocalizes with VGAT in a subset of terminals in the hippocampal formation. (A) Immunofluorescence double staining for VGLUT2 (red) and VGAT (green) demonstrates punctate colocalization of VGLUT2 and VGAT (yellow) in the VGLUT2-band in the dentate gyrus. The inset depicts a three-dimensional reconstruction of a representative spot showing VGAT staining concentric with VGLUT2 staining. (B) Immunofluorescence staining for VGLUT2 (red) and VGAT (green) in CA2 shows colocalization in a subset of puncta (arrowheads) adjacent to cell bodies of pyramidal neurons. The inset shows some representative puncta at higher power demonstrating colocalization of the proteins. (C) Triple labeling for VGLUT2 (blue), VGAT (green) and GAD65 (red) was performed using secondary antibodies coupled to 3 different fluorochromes. Colocalization of the 3 proteins is detected in a subset of labeled spots (appears white) in the vicinity of cell bodies of granule cells (arrowheads). The inset in (C) shows the Z-stacks in the xy, xz, and yz planes demonstrating colocalization in a representative terminal-like punctum in all 3 planes. (D) VGLUT2 (red) and GAD65 (green) colocalize in terminal-like structures surrounding (arrowheads) dendrites stained for GABA_A receptor subunits α1–α2 (blue) (arrowheads). The inset shows a three-dimensional reconstruction of one of the punctum showing colocalization of VGLUT2 and GAD65 in contact with a dendritic process expressing GABA_A receptor. (E) VGLUT2 (red) and GAD65 (green) colocalize in the vicinity (arrowheads) of structures stained for NMDA receptor subunits 2A and B (blue). Note that the VGLUT2 and GAD65 colocalizing puncta impinge on the NMDA receptor labeled structures, as shown by three-dimensional reconstruction (inset). (F, G) Immunogold double labeling for VGLUT2 (large particles [10 nm], arrowheads) and VGAT (small particles [5 nm], arrows) was performed. Electron micrographs show colocalization of VGAT and VGLUT2 in terminals making an asymmetrical (F) and a symmetrical (G) synapse, respectively. At, axon terminal; D, dendrite; m, mitochondria; GCL, granule cell layer; Gr, granule cell; Mol, molecular layer; Py, pyramidal cell. Scale bars: (A and B) 120 μm; (C) 10 μm; (D) 5 μm; (E) 2 μm; (F) 200 nm; (G) 200 nm.

Figure 3.

Ontogeny of single and double stained terminals for VGLUT2 in the VGLUT2-band. The number of single stained terminals and double stained terminals at different developmental stages (P7, P14, and Ad [>P35]) is plotted as percentage of the total number of VGLUT2 terminals in adults, in the area sampled (see Fig. S1). At P7, the proportion of double labeled terminals is low. In the adult, the double labeled terminals constitute more than half of the VGLUT2-labeled terminals.

Figure 4.

VGLUT2 and VGAT define 2 distinct populations of synaptic vesicles. (A) Purified synaptic vesicles from a crude hippocampal LS1 fraction were prepared for EM and double stained for VGLUT2 (arrows; small gold particles) and VGAT (arrow heads; large gold particles). Of a total of 2617 vesicles observed, no double labeled vesicles were detected. Note that the halo resembling a vesicle around the big particles is a dehydration artifact (see Methods). (B) The left panel shows western blots of VGLUT2-containing vesicles immunoisolated from a crude hippocampal LS1 fraction (lane B). No VGAT or VGLUT1 signals were detected in this fraction, although it stained for VGLUT2 and the vesicular marker synaptophysin (p38). The supernatant left after immunoisolation of VGLUT2-containing vesicles (lane S) did not stain for VGLUT2, but it was stained for VGAT and VGLUT1 at the same levels as in the total LS1 fraction (lane T). The right panel shows western blots of VGAT-containing vesicles immunoisolated from an LS1 total fraction (lane B). This fraction stained for VGAT and the vesicular marker VAMP2, but not for VGLUT2 or VGLUT1. The supernatant shows only weak staining for VGAT (lane S). In contrast, the supernatant was as strongly stained for VGLUT2 and VGLUT1 as the total LS1 fraction. Scale bar: 100 nm.

Figure 5.

Terminals costained for VGLUT2 and VGAT are projections from the SuM. (A) A section double stained for VGLUT3 and VGAT shows that most of VGLUT3 stained terminals also stain for VGAT (arrowheads; inset). (B) Double labeling for VGLUT2 and VGLUT3 did not show colocalization. (C) A brain section triple labeled for VGLUT2, VGAT, and CCK. VGLUT2 colocalizes with VGAT in a subset of terminals (arrowheads; left inset), but is excluded from the CCK containing terminals (double arrowheads; right inset). (D) A tracer (Pha-I) was injected in the SuM. The distribution of tracer at the injection site is largely restricted to the SuM. (E, F) Double labeling with VGLUT2 or triple labeling with VGLUT2 and VGAT were followed by Z-stack confocal microscopy, deconvolution, and three-dimensional reconstruction. In (E), VGLUT2 costaining with the tracer occurs specifically in varicosities (nerve terminals) but not in the intervening axonal shaft. Inset shows a three-dimensional reconstruction of the area outlined by the white rectangle. In (F), VGLUT2 colocalizes with the tracer and VGAT in varicosities. Note that the axon stains only for the tracer. The lower inset shows a representative Z-stack where a triple stained nerve terminal is depicted in the xy, xz, and yz planes. Upper inset shows a three-dimensional reconstruction of the same terminal. Gr, granule cell; LM, lateral mammillary nucleus; MM, medial mammillary nucleus, median part; Mol, molecular layer of the dentate gyrus; MRe, mammillary recess of the third ventricle; 3V, third ventricle. Scale bars: (A) 5 μm; (B) 8 μm; (C) 5 μm; (D) 1000 μm; (E) 2.5 μm; (F) 1 μm.