Analysis of Synaptotagmin, SV2, and Rab3 Expression in Cortical Glutamatergic and GABAergic Axon Terminals

Abstract

We investigated whether cortical glutamatergic and GABAergic release machineries can be differentiated on the basis of the nature and amount of proteins they express, by performing a quantitative analysis of the degree of co-localization of synaptotagmin (SYT) 1 and 2, synaptic vesicle protein 2 (SV2) A and B, and Rab3a and c in VGLUT1+, VGLUT2+, and VGAT+ terminals and synaptic vesicles (SVs) in rat cerebral cortex. Co-localization studies showed that VGLUT1 puncta had high levels of SV2A and B and of Rab3c, intermediate levels of SYT1, and low levels of SYT2 and Rab3c; VGLUT2 puncta exhibited intermediate levels of all presynaptic proteins studied; whereas vesicular GABA transporter (VGAT) puncta had high levels of SV2A and SYT2, intermediate levels of SYT1, Rab3a, and Rab3c, and low levels of SV2B. Since SV2B is reportedly expressed by glutamatergic neurons and we observed SV2B expression in VGAT puncta, we performed electron microscopic studies and found SV2B positive axon terminals forming symmetric synapses. Immunoisolation studies showed that the expression levels of the protein isoforms varied in the three populations of SVs. Expression of SYT1 was highest in VGLUT1-SVs, while SYT2 expression was similar in the three SV groups. Expression of SV2A was similarly high in all three SV populations, except for SV2B levels that were very low in VGAT SVs. Finally, Rab3a levels were similar in the three SV groups, while Rab3c levels were highest in VGLUT1-SVs. These quantitative results extend our previous studies on the differential expression of presynaptic proteins involved in neurotransmitter release in GABAergic and glutamatergic terminals and indicate that heterogeneity of the respective release machineries can be generated by the differential complement of SV proteins involved in distinct stages of the release process.

Keywords: Rab3; SV2; VGAT; VGLUT1; VGLUT2; synaptotagmin.

Figure 1

VGLUT1, VGLUT2, VGAT (R, rabbit; M, mouse), SYT1, SYT2, SV2A, SV2B, Rab3a, and Rab3c antibodies recognized bands of ~55, 60, 57, 65, 65, 100, 95, 25, and 25 kDa in the order, in crude membrane fractions of rat cerebral cortex.

Figure 2

Analysis of confocal microscopy images of cortical sections. The figure shows examples from VGLUT1/SYT1 [row(A)], VGLUT2/Rab3a [row (B)], and VGAT/SV2A series [row (C)]. To minimize the fusion of puncta, the contrast of each image was manually adjusted within the maximum range of levels for each color channel, which was examined separately to identify and count manually immunopositive puncta (first and second columns). Following merging, puncta were considered double-labeled when the overlap was complete or it occupied most of the area of the puncta and they were morphologically similar (arrowheads in fourth columns). Puncta not meeting these criteria (e.g., those indicated by arrows) were not considered double-labeled. Bars: 2 μm.

Figure 3

Co-localization of SYT1, SYT2, SV2A, SV2B, Rab3a, and Rab3c in VGLUT1+, VGLUT2+, and VGAT+ axon terminals in cerebral cortex. Values (means ± SEM) refer to the percentages of total puncta positive for the respective protein isoform for each of the three terminal populations identified based on the specific vesicular transporter.

Figure 4

Pre-embedding electron microscopy studies show that SV2B immunoreactivity [arrows in (A,B)] is present in axon terminals forming both asymmetric [arrowheads in (A); layer V] and symmetric [arrowheads in (B); layer V] synaptic contacts. AxT, axon terminal; Den, dendrite; N, pyramidal neuron. Scale bar: 500 nm (A,B).

Figure 5

Expression of SYT1, SYT2, SV2A, SV2B, and Rab3a and c in VGLUT1+, VGLUT2+, and VGAT+ SVs. (A) Glutamatergic and GABAergic SVs were immunoisolated from the LS1 fraction of rat cerebral cortex using beads coupled with either rabbit VGLUT1, VGLUT2, or VGAT antibodies. After immunoisolation, corresponding amounts of pellet and supernatant (SUP) fractions were subjected to immunoblotting with anti-SYT1 and -SYT2 antibodies, anti-SV2A/SV2B antibodies, or anti-Rab3a/Rab3c antibodies. (B) Quantification of the recovered immunoreactivities was carried out by densitometric scanning and interpolation of the data into a standard curve of rat brain LS1 fraction, and expressed as percent of the total input of LS1 added to the samples. The percentage of SYT1/SYT2, SV2A/SV2B, and Rab3a/Rab3c immunoreactivities (IR) detected in SVs immunoisolated with anti-VGLUT1 (VGLUT1–SV; upper left/right panel), anti-VGLUT2 (VGLUT2–SV; lower left/right panel), or anti-VGAT (VGAT SV; upper right/right panel) beads are shown as means (±SEM) of five independent experiments.

Figure 6

Hypothetical distribution of SYT1/2 in VGLUT1+ and VGLUT2+ glutamatergic and VGAT+ GABAergic cortical terminals. Based on a correlative analysis of confocal and immunoisolation data, the scenario proposed is highly schematic (e.g., the total amount of a SV-related protein is equally distributed among all positive terminals), and does not take into account the number of proteins/vesicles, but it emphasizes the concept that the amount of a SV-related protein is variable in positive terminals. Shaded terminals indicate terminals expressing a given protein; black circles indicate levels of expression of the protein in immunoisolated SVs. Four terminals make up 100%; similarly, 10 vesicles makes up 100%.