Elsevier

Neuroscience

Volume 165, Issue 3, 3 February 2010, Pages 934-943
Neuroscience

Sensory System
Research Paper
Heterogeneity of glutamatergic and GABAergic release machinery in cerebral cortex: analysis of synaptogyrin, vesicle-associated membrane protein, and syntaxin

https://doi.org/10.1016/j.neuroscience.2009.11.009Get rights and content

Abstract

To define whether cortical glutamatergic and GABAergic release machineries can be differentiated on the basis of the nature and amount of proteins they express, we studied the degree of co-localization of synaptogyrin (SGYR) 1 and 3, vesicle-associated membrane protein (VAMP) 1 and 2, syntaxin (STX) 1A and 1B in vesicular glutamate transporter (VGLUT)1-, VGLUT2- and vesicular GABA transporter (VGAT)-positive (+) puncta and synaptic vesicles in the rat cerebral cortex. Co-localization studies showed that SGYR1 and 3 were expressed in about 90% of VGLUT1+, 70% of VGLUT2+ and 80% of VGAT+ puncta; VAMP1 was expressed in approximately 45% of VGLUT1+, 55% of VGLUT2+, and 80% of VGAT+ puncta; VAMP2 in about 95% of VGLUT1+, 75% of VGLUT2+, and 80% of VGAT+ puncta; STX1A in about 65% of VGLUT1+, 30% of VGLUT2+, and 3% of VGAT+ puncta, and STX1B in approximately 45% of VGLUT1+, 35% of VGLUT2+, and 70% of VGAT+ puncta. Immunoisolation studies showed that while STX1A was completely segregated and virtually absent from VGAT synaptic vesicles, STX1B, VAMP1/VAMP2, SGYR1/SGYR3 showed a similar pattern with the highest expression in VGLUT1 immunoisolated vesicles and the lowest in VGAT immunoisolated vesicles. Moreover, we studied the localization of STX1B at the electron microscope and found that a population of axon terminals forming symmetric synapses were STX1B-positive.These results extend our previous observations on the differential expression of presynaptic proteins involved in neurotransmitter release in GABAergic and glutamatergic terminals and indicate that heterogeneity of glutamatergic and GABAergic release machinery can be contributed by both the presence or absence of a given protein in a nerve terminal and the amount of protein expressed by synaptic vesicles.

Section snippets

Animals and tissue preparation

Adult male Sprague–Dawley rats (190–220 g; Charles River, Milan, Italy) were used. All experiments were carried out in accordance with the European Community Council Directive dated November 24, 1986 (86/609 EEC) and were approved by the local authority veterinary service. All efforts were made to minimize animal suffering and the number of animals used. Animals were kept under a dark-light cycle of 12 h and permitted food and water ad libitum.

For Western blotting, rats were anesthetized with

Preliminary studies

In cortical crude membrane fractions, all antibodies recognized bands of predicted molecular mass (Fig. 2;Belizaire et al., 2004, Baumert et al., 1989, Baumert et al., 1990, Archer et al., 1990, Ruiz-Montasell et al., 1996, Bennett et al., 1992, Bellocchio et al., 1998, Chaudhry et al., 1998, Varoqui et al., 2002). The distribution of VGLUT1, VGLUT2, VGAT, SGYR1, SGYR3, VAMP1, VAMP2, STX1A and STX1B immunoreactivities was as previously described (Belizaire et al., 2004, Ruiz-Montasell et al.,

Discussion

The present studies showed that diverse proteins participating in neurotransmitter release are differentially expressed in glutamatergic and GABAergic terminals. In addition, they also showed that in terminals expressing a given protein participating in transmitter release, the percentage of synaptic vesicles expressing or interacting with that protein is variable.

Previous localization studies have shown that all VGAT+ puncta (e.g., Chaudhry et al., 1998, Minelli et al., 2003) and the vast

Acknowledgments

This work was supported by grants from Ministero Istruzione Università e Ricerca (PRIN) to FC and FB, and from Compagnia di San Paolo (2005.1964) and Telethon-Italy to FB. We thank R. Janz (University of Texas Medical School at Houston) for synaptogyrin antibodies, and M. Melone and G. Fattorini for help and insightful comments.

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