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. 2010 Jun 2;30(22):7634-45.
doi: 10.1523/JNEUROSCI.0141-10.2010.

Synaptic and Vesicular Coexistence of VGLUT and VGAT in Selected Excitatory and Inhibitory Synapses

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Free PMC article

Synaptic and Vesicular Coexistence of VGLUT and VGAT in Selected Excitatory and Inhibitory Synapses

Johannes-Friedrich Zander et al. J Neurosci. .
Free PMC article

Abstract

The segregation between vesicular glutamate and GABA storage and release forms the molecular foundation between excitatory and inhibitory neurons and guarantees the precise function of neuronal networks. Using immunoisolation of synaptic vesicles, we now show that VGLUT2 and VGAT, and also VGLUT1 and VGLUT2, coexist in a sizeable pool of vesicles. VGAT immunoisolates transport glutamate in addition to GABA. Furthermore, VGLUT activity enhances uptake of GABA and monoamines. Postembedding immunogold double labeling revealed that VGLUT1, VGLUT2, and VGAT coexist in mossy fiber terminals of the hippocampal CA3 area. Similarly, cerebellar mossy fiber terminals harbor VGLUT1, VGLUT2, and VGAT, while parallel and climbing fiber terminals exclusively contain VGLUT1 or VGLUT2, respectively. VGLUT2 was also observed in cerebellar GABAergic basket cells terminals. We conclude that the synaptic coexistence of vesicular glutamate and GABA transporters allows for corelease of both glutamate and GABA from selected nerve terminals, which may prevent systemic overexcitability by downregulating synaptic activity. Furthermore, our data suggest that VGLUT enhances transmitter storage in nonglutamatergic neurons. Thus, synaptic and vesicular coexistence of VGLUT and VGAT is more widespread than previously anticipated, putatively influencing fine-tuning and control of synaptic plasticity.

Figures

Figure 1.
Figure 1.
Comparison of the amounts of abundant vesicular proteins and transmitter transporters on SVs immunoisolated from adult or juvenile rat brains by antibodies against VGLUT1, VGLUT2, and VGAT. A, Immunoisolated vesicles (adult brains) were obtained as given in Materials and Methods and detection was performed with the antibodies indicated or without antibody (w/o ab). Detection of the vesicular proteins indicated on the left side was performed using the respective polyclonal rabbit antibodies. A standard curve from the starting material (LS0) was performed in parallel to calculate the respective protein amounts in the individual immunoisolates. Relative amounts of Syp, Syg, and Syb as well as of VGLUT1, VGLUT2, and VGAT were quantified from three different immunoisolation experiments using standard curves performed from the initial LS0 fraction. Note that VGLUT2-immunoisolated vesicles harbor more VGAT than VGLUT1 isolates (*Student's t test, p < 0.03). B, Transporter-specific immunoisolated vesicles were obtained as given in methods using lysed synaptosomes (LS0) from rat brains at postnatal days 5 and 15. The amount of VGAT on VGLUT2-immunoisolated vesicles was the highest at P5. Relative amounts of Syp, VGLUT1, VGLUT2, and VGAT were quantified from three different immunoisolation experiments using P5 brains. Standard curves from the initial LS0 fraction of P5 brains were performed as in B (data not shown); the LS0 fraction presented in the graph corresponds to 4 μg of protein. Blots were developed for 30 s or 3 min (longer exposure).
Figure 2.
Figure 2.
Transmitter uptake into SVs and SV subpopulations. A, [3H]Serotonin or [3H]glutamate uptake was performed using SVs immunoisolated without (IgG) or with (Syp) the Syp antibody. B, [3H]Glutamate uptake was performed using SVs immunoisolated without (IgG) or with an antibody against either VGLUT1 or VGLUT2. C, [3H]GABA or [3H]glutamate uptake was performed using SVs immunoisolated without (IgG) or with (VGAT) the VGAT antibody. Note that VGAT immunoisolates take up GABA and glutamate. D, [3H]GABA uptake was performed into SVs (LP2 fraction) suspended in either gluconate containing KGC buffer or glutamate containing KG buffer in the absence (Con) or presence of 5 μm trypan blue (Trp). To avoid dilution of [3H]GABA by endogenously generated GABA, an inhibitor of GAD, 3-mercapto-propionic acid (10 μm), was present throughout. There was no Trp-mediated inhibition of uptake in KGC buffer (Gluconate, left columns). GABA uptake was significantly decreased (13%) in KG buffer (Glutamate) and further decreased (24%) when glutamate was partially replaced by 50 mm chloride (Glutamate + chloride), indicating a contribution of VGLUT activity. E, [3H]Serotonin uptake into SVs was performed in the absence (Con) or presence of Trp, either in KGC buffer (gluconate) or in KG buffer (glutamate). Trp significantly diminished serotonin uptake in KG buffer, indicating a contribution of VGLUT activity. Uptakes are given in dpm (decay/min) and were corrected for nonspecific uptake in the presence of 2 μm reserpine (serotonin uptake) or 2 μm bafilomycin (GABA or glutamate uptake). Values represent the mean of three determinations ± SD (*Student's t test, p < 0.05).
Figure 3.
Figure 3.
Identification of VGAT mRNA in hippocampal granular cells. A, Hippocampal granule cells of P15 and adult rats were identified electrophysiologically, based on their typical single action potential following depolarizing pulses, and single-cell PCR was performed using primers specific for VGLUT1 or VGAT. All granule cells at P15 (2 examples are shown) expressed both VGLUT1 and VGAT (arrowheads). Approximately 40% of the adult granule cells (15 cells in total; 2 examples are shown) showed a low expression of VGAT in addition to VGLUT1 (arrowheads). B, For comparison, interneurons isolated from the dentate gyrus and the hilar region were identified electrophysiologically. Interneurons typically fire repetitive action potentials throughout the depolarizing pulse without presenting accommodation. Single PCR was performed using both primers. In the two examples shown, a clear band for VGAT (arrowheads) was present, while VGLUT1 was completely absent.
Figure 4.
Figure 4.
Single and double immunogold labeling for VGLUT1, VGLUT2, and VGAT in terminals of the hippocampal CA3 region. A–C, Mossy fiber terminals (mt) are clearly labeled by rabbit antibodies directed against either VGLUT1 or VGLUT2 indicated by 10 (A, B) or 15 (C) nm gold particles. The asterisk indicates an axon bundle profile. Pd, Pyramidal cell dendrite. The insets in A and B represent details at a higher magnification. D, A mossy fiber terminal (mt) of the CA3 area of the hippocampus shows vesicle-associated immunoreactivity for the VGAT guinea pig antiserum indicated by 10 nm gold particles. The asterisk indicates an axon, pc pyramidal cell body. The inset represents a detail at a higher magnification. E–H, Mossy fiber terminals (mt) were double immunolabeled with the guinea pig antiserum against VGAT (10 nm gold particles) and rabbit antisera against either VGLUT1 or VGLUT2 (both 5 nm gold particles). G and H represent micrographs at a higher magnification, with arrowheads indicating VGAT and forked arrowheads indicating either VGLUT1 (G) or VGLUT2 (H). Note the SVs exhibiting putatively colocalized immunogold signals for VGAT and VGLUT2 shown in H. Thin arrows indicate asymmetric contacts. Sp, Spine; pd, pyramidal cell dendrite. Scale bars: A–D, 500 nm; E, F, 200 nm; G, H, insets in A, B, and D, 100 nm.
Figure 5.
Figure 5.
Double immunogold labeling for VGLUT1, VGLUT2, and VGAT in glutamatergic and GABAergic terminals of the cerebellar cortex. A–C, Double immunogold labeling of VGLUT1 (guinea pig antiserum, 5 nm gold particles, forked arrowheads in A′ and B′) and VGLUT2 (rabbit antiserum, 10 nm gold particles, arrowheads in A′, C′) in a mossy fiber terminal of the granular cell layer (cmt, A, A′), a parallel fiber terminal of the molecular layer (cpt, B, B′), and a climbing fiber terminal of the molecular layer (cct, C, C′). Details at higher magnification are given as insets (A′, B′, C′). Note that both VGLUT1 and VGLUT2 are present in the mossy fiber terminal (A, A′), while in the parallel fiber terminal only VGLUT1 (B, B′) and in the climbing fiber terminal only VGLUT2 (C, C′) could be detected. Asterisk denotes examples for axon profiles. The small thin arrow indicates an asymmetric synaptic contact. D, D′, Double immunogold labeling of VGLUT1 (rabbit antiserum, 5 nm gold particles, forked arrowheads) and VGAT (guinea pig antiserum, 10 nm gold particles, arrowheads) in a mossy fiber terminal (cmt) of the granular cell layer. A detail at higher magnification is given as an inset (D′). E, F, Cerebellar basket cell terminals (cbt) were subjected to a double immunogold labeling with VGAT (guinea pig antiserum, 10 nm gold particles, arrowheads) and rabbit antisera against either VGLUT1 or VGLUT2 (5 nm gold particles, forked arrowheads). Note that basket cell terminals have beside VGAT only VGLUT2. Asterisks denote examples for axon profiles; thick open arrows indicate symmetric contacts; pc, Purkinje cell body; pd, Purkinje cell dendrite; sp, dendritic spine. Scale bars: A–D (in A), E, F (in F), 200 nm; A′–D′, 100 nm.
Figure 6.
Figure 6.
Quantification of the number of gold particles staining for VGLUT1, VGLUT2, and VGAT in glutamatergic and GABAergic terminals of the hippocampal CA3 area and the cerebellum. A, The amounts of gold particles indicating either VGLUT1 (rabbit antiserum) or VGAT (guinea pig antiserum, 131004, Synaptic System) in HMFs were quantified in two to three sections of the CA3 area each from separately embedded tissue of two different animals. Single immunogold labeling was performed using the antibodies indicated and immunoreactivity was identified by secondary antibodies coupled to 10 nm gold particles. For VGAT, only HMFs yielding a labeling with at least 10 gold particles were included, which represent ∼40% of all HMFs analyzed per animal (70–80 in total). For each antibody, 30 terminals were analyzed. HMFs with an average area of 4.3 ± 0.3 μm2 were considered and values calculated as gold particles per 1 μm2. Nonspecific labeling (H background) obtained from dendritic cytoplasm and nuclei over an area of 20 μm2 for each section and antibody is given below and was subtracted. Note that background labeling was <10% under each condition. B, In a similar experimental design, the immunogold staining in the various terminals of cerebellum was quantified. For each animal, 40 [cerebellar mossy fibers (CMFs), cerebellar parallel fiber terminals (CPFs)] or 20 [cerebellar climbing fiber terminals (CCFs) or cerebellar basket cell terminals (CBCs)] were analyzed. The average areas for the terminals considered were the following: CMFs, 9.92 ± 0.68 μm2; CPFs, 0.49 ± 0.06 μm2; CCFs, 0.93 ± 0.12 μm2; CBFs, 1.27 ± 0.075 μm2. Values are given as gold particles per 1 μm2. There was no correction for background labeling. Note that CPFs were exclusively immunopositive for VGLUT1, while CCFs have only VGLUT2. CBFs have both VGAT and VGLUT2, while mossy fiber terminals contain all three transporters.

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