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A Possible Role of the Non-GAT1 GABA Transporters in Transfer of GABA From GABAergic to Glutamatergic Neurons in Mouse Cerebellar Neuronal Cultures

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Abstract

Cultures of dissociated cerebellum from 7-day-old mice were used to investigate the mechanism involved in synthesis and cellular redistribution of GABA in these cultures consisting primarily of glutamatergic granule neurons and a smaller population of GABAergic Golgi and stellate neurons. The distribution of GAD, GABA and the vesicular glutamate transporter VGlut-1 was assessed using specific antibodies combined with immunofluorescence microscopy. Additionally, tiagabine, SKF 89976-A, betaine, β-alanine, nipecotic acid and guvacine were used to inhibit the GAT1, betaine/GABA (BGT1), GAT2 and GAT3 transporters. Only a small population of cells were immuno-stained for GAD while many cells exhibited VGlut-1 like immuno-reactivity which, however, never co-localized with GAD positive neurons. This likely reflects the small number of GABAergic neurons compared to the glutamatergic granule neurons constituting the majority of the cells. GABA uptake exhibited the kinetics of high affinity transport and could be partly (20%) inhibited by betaine (IC50 142 μM), β-alanine (30%) and almost fully (90%) inhibited by SKF 89976-A (IC50 0.8 μM) or nipecotic acid and guvacine at 1 mM concentrations (95%). Essentially all neurons showed GABA like immunostaining albeit with differences in intensity. The results indicate that GABA which is synthesized in a small population of GAD-positive neurons is redistributed to essentially all neurons including the glutamatergic granule cells. GAT1 is not likely involved in this redistribution since addition of 15 μM tiagabine (GAT1 inhibitor) to the culture medium had no effect on the overall GABA content of the cells. Likewise the BGT1 transporter cannot alone account for the redistribution since inclusion of 3 mM betaine in the culture medium had no effect on the overall GABA content. The inhibitory action of β-alanine and high concentrations of nipecotic acid and guvacine on GABA transport strongly suggests that also GAT2 or GAT3 (HUGO nomenclature) could play a role.

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Acknowledgments

The secretarial (Ms. Hanne Danø) and the technical assistance (Lene Vigh, Heidi Nielsen and Sara Sánchez-Redondo) is cordially acknowledged. This work was supported by grants from FIS 061212 (Ministry of Health, Spain), 2009/SGR/214 (CIRIT, Generalitat de Catalunya, Spain) and The Danish Medical Research Council (22-03-250; 22-04-0314). Z. B. was recipient of a predoctoral fellowship from the Spanish Ministry of Education.

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Authors and Affiliations

  1. Department of Neurochemistry and Neuropharmacology, Institut d’Investigacions Biomèdiques de Barcelona, Consejo Superior de Investigaciones Científicas, CSIC-IDIBAPS, CIBERESP, Barcelona, Spain

    C. Suñol, Z. Babot & R. Cristòfol

  2. Department of Neuroscience, Faculty of Medicine, MTFS, NTNU, 7489, Trondheim, Norway

    U. Sonnewald

  3. Department of Pharmacology and Pharmacotherapy, Faculty of Pharmaceutical Sciences, University of Copenhagen, Universitetsparken 2, 2100, Copenhagen, Denmark

    H. S. Waagepetersen & A. Schousboe

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  1. C. Suñol
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  2. Z. Babot
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  3. R. Cristòfol
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  4. U. Sonnewald
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  5. H. S. Waagepetersen
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  6. A. Schousboe
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Correspondence to A. Schousboe.

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Suñol, C., Babot, Z., Cristòfol, R. et al. A Possible Role of the Non-GAT1 GABA Transporters in Transfer of GABA From GABAergic to Glutamatergic Neurons in Mouse Cerebellar Neuronal Cultures. Neurochem Res 35, 1384–1390 (2010). https://doi.org/10.1007/s11064-010-0196-1

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