Analysis of synaptotagmin, SV2, and Rab3 expression in cortical glutamatergic and GABAergic axon terminals

Luca Bragina, Giorgia Fattorini, Silvia Giovedi, Marcello Melone, Federica Bosco, Fabio Benfenati, Fiorenzo Conti

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Abstract

We investigated whether ccrtical 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 thatVGLUT1 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 VGLUTI-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 presynap-tic 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.

Original languageEnglish
Article number32
JournalFrontiers in Cellular Neuroscience
Issue numberJANUARY
DOIs
Publication statusPublished - 2012

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rab3 GTP-Binding Proteins
Synaptotagmins
Synaptic Vesicles
Presynaptic Terminals
Synaptotagmin II
Proteins
Synaptotagmin I
Tics
Cerebral Cortex
Synapses
Population

Keywords

  • Rab3
  • SV2
  • Synaptotagmin
  • VGAT
  • VGLUT1
  • VGLUT2

ASJC Scopus subject areas

  • Cellular and Molecular Neuroscience

Cite this

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title = "Analysis of synaptotagmin, SV2, and Rab3 expression in cortical glutamatergic and GABAergic axon terminals",
abstract = "We investigated whether ccrtical 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 thatVGLUT1 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 VGLUTI-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 presynap-tic 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.",
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author = "Luca Bragina and Giorgia Fattorini and Silvia Giovedi and Marcello Melone and Federica Bosco and Fabio Benfenati and Fiorenzo Conti",
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T1 - Analysis of synaptotagmin, SV2, and Rab3 expression in cortical glutamatergic and GABAergic axon terminals

AU - Bragina, Luca

AU - Fattorini, Giorgia

AU - Giovedi, Silvia

AU - Melone, Marcello

AU - Bosco, Federica

AU - Benfenati, Fabio

AU - Conti, Fiorenzo

PY - 2012

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N2 - We investigated whether ccrtical 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 thatVGLUT1 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 VGLUTI-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 presynap-tic 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.

AB - We investigated whether ccrtical 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 thatVGLUT1 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 VGLUTI-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 presynap-tic 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.

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