First direct demonstration of extensive GABA synthesis in mouse cerebellar neuronal cultures
Article first published online: 27 OCT 2004
Journal of Neurochemistry
Volume 91, Issue 4, pages 796–803, November 2004
How to Cite
Sonnewald, U., Olstad, E., Qu, H., Babot, Z., Cristòfol, R., Suñol, C., Schousboe, A. and Waagepetersen, H. (2004), First direct demonstration of extensive GABA synthesis in mouse cerebellar neuronal cultures. Journal of Neurochemistry, 91: 796–803. doi: 10.1111/j.1471-4159.2004.02794.x
- Issue published online: 27 OCT 2004
- Article first published online: 27 OCT 2004
- Received June 10, 2004; revised manuscript received July 7, 2004; accepted July 8, 2004.
- cerebellar granule neurones;
- γ-aminobutyric acid;
- kainic acid;
- nuclear magnetic resonance spectroscopy
Culturing mouse cerebellar neurones (predominantly glutamatergic) in the presence of [1−13C]glucose for 7 days resulted in a surprisingly extensive labelling of the inhibitory neurotransmitter GABA, the average content and labelling of which were 20 ± 4 nmol/mg protein and 20 ± 4%, respectively. Cultures of neocortical neurones (predominantly GABAergic) had under similar conditions a GABA content and labelling of 32 ± 2 nmol/mg protein and 21 ± 2%. The cerebellar cultures contained only 6% glutamate decarboxylase (GAD)-positive neurones when immunolabelled using a GAD67 antibody, while a dense network of neurones in the neocortical cultures stained positively for GAD67. Exposure of the cerebellar cultures to 50 µm kainic acid (KA) which is known to eliminate vesicular release of GABA, only marginally affected GABA labelling and cellular content. Likewise this treatment had no effect on the number of GAD67-positive neurones but a massive punctate immunostaining observed in control cultures was essentially eliminated. Increasing the KA concentration to 0.5 mm in the culture medium for 7 days led to a reduction of GABA labelling and content compared to cerebellar cultures not exposed to KA. Although it is likely that this large capacity for GABA synthesis resides in the relatively few GAD-positive neurones, it seems unlikely that they could account for the large average GABA content in the cultures. Therefore it must be concluded that the newly synthesized GABA is redistributed among the majority of the cells in these cultures, i.e. the glutamatergic neurones.