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Keywords:

  • cerebellar granule neurones;
  • γ-aminobutyric acid;
  • glutamate;
  • kainic acid;
  • metabolism;
  • nuclear magnetic resonance spectroscopy

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Cerebral cortical and cerebellar granule neurones
  6. NMR spectroscopy and HPLC
  7. GAD immunostaining
  8. Data analysis
  9. Results
  10. Immunocytochemistry of GAD
  11. Discussion
  12. Acknowledgements
  13. References

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.

Abbreviations used
AMPA

α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid

DMEM

Dulbecco's minimum essential medium

GAD

glutamate decarboxylase

KA

kainic acid

LSD

least significant difference

PBS

phosphate-buffered saline

PDH

pyruvate dehydrogenase

TCA

tricarboxylic acid

Brain function is tightly coupled to a fine-tuned balance between excitatory glutamatergic and inhibitory GABAergic neurotransmission. Brain areas such as hippocampus and cerebellum are characterized by neuronal networks consisting of glutamatergic and GABAergic neurones. These neurones are classically thought to express enzymes and other entities associated specifically with either one of these neuronal phenotypes. In particular, glutamate decarboxylase (GAD) and GABA transporters in vesicles and plasma membranes are considered GABAergic characteristics (Saito et al. 1974; Borden 1996; Chaudhry et al. 1998).

In cerebellum, the glutamatergic granule neurones (Young et al. 1974) receive inhibitory GABAergic inputs from Golgi neurones as well as a tonic inhibition which may represent non-vesicular GABA release (Rossi et al. 2003). Cultures of dissociated cerebellum constitute a model system in which the association of GABAergic parameters with glutamatergic and GABAergic neurones, respectively, can be investigated. Such cultures consist primarily of glutamatergic granule cells, with a minor contribution of GABAergic stellate and basket neurones, i.e. GABAergic as well as glutamatergic characteristics are expressed in these cultures (Pearce et al. 1981; Hertz et al. 1985; Hertz and Schousboe 1987; Kovacs et al. 2003). The neuronal composition of such cultures can be influenced by exposure of the cells to kainic acid (KA; Seil et al. 1979; Drejer and Schousboe 1989; Damgaard et al. 1996).

On the basis of this, it was investigated if cerebellar granule cells in culture synthesize and contain GABA and if these parameters are affected by KA. The synthesis of GABA in cells can be determined by a variety of methods. Using 13C-labelled precursors such as [1−13C]glucose followed by NMR spectroscopy of cell extracts, it was demonstrated that GABAergic cerebral cortical neurones synthesize GABA, which is extensively labelled with 13C (Sonnewald et al. 1993). In analogy, to determine if GABA was synthesized in cerebellar granule neurones, these cells were cultured in a medium containing [1−13C]glucose and subjected to KA under various conditions. The rationale for exposure to KA (50 µm), a potent agonist at both the KA and α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors (for review see Lerma 1998), was based on the findings by Drejer and Schousboe (1989) and Simmons and Dutton (1992) that vesicular GABA release from cerebellar neuronal cultures could essentially be eliminated by culturing the cells in the presence of KA. These studies indicated that KA is a potent toxin for GABAergic neurones in cerebellar cultures, in keeping with previous studies of Seil et al. (1979) using organotypic cerebellar cultures. In order to investigate whether exposure of the granule neurones to KA might affect the expression of GABAergic characteristics in analogy to the investigations of rat hippocampus (Schwarzer and Sperk 1995; Sperk et al. 2003), cultures were exposed to a higher concentration of KA (0.5 mm), which has no toxic action on the granule cells (Frandsen and Schousboe 1990). In order to assess whether GABA synthesis in the cerebellar cultures might be related to the presence of GAD (Martin and Rimvall 1993; Waagepetersen et al. 2001), these cultures were immunolabelled for expression of GAD using a GAD67-specific antibody. Moreover, to relate the immunohistochemical GAD staining and the 13C GABA labelling to GABA synthesis, additional experiments concerning GABA labelling and GAD-like immunoreactivity were performed in neocortical neurones in culture, a preparation known primarily to represent GABAergic neurones (Yu et al. 1984; Hertz and Schousboe 1987).

Materials

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Cerebral cortical and cerebellar granule neurones
  6. NMR spectroscopy and HPLC
  7. GAD immunostaining
  8. Data analysis
  9. Results
  10. Immunocytochemistry of GAD
  11. Discussion
  12. Acknowledgements
  13. References

Plastic tissue culture dishes were purchased from Nunc A/S (Roskilde, Denmark), fetal calf serum from Seralab Ltd (Sussex, UK) and Gibco (Invitrogen, Barcelona, Spain), and culture medium from Gibco-BRL, Life Technologies A/S (Roskilde, Denmark) and Biochrom KG (Berlin, Germany). NMRI mice were purchased from Møllegaard Breeding Center (Copenhagen, Denmark) and Iffa Credo (St Germain-sur-l'Arbreste, France). [1−13C]glucose (98% + enriched) and D2O were from Cambridge Isotopes Laboratories (Woburn, MA, USA). KA was from Sigma Chemical Co. (St Louis, MO, USA) and from Tocris Cookson Inc. (Ellisville, MO, USA). Rabbit anti-GAD67 polyclonal antibody was from Chemicon International Inc. (Temecula, CA, USA) and goat anti-rabbit IgG fluorescein conjugate and Alexa 488 were from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA) and Molecular Probes (Leiden, the Netherlands), respectively. All other chemicals were of the purest grade available from regular commercial sources.

Cerebral cortical and cerebellar granule neurones

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Cerebral cortical and cerebellar granule neurones
  6. NMR spectroscopy and HPLC
  7. GAD immunostaining
  8. Data analysis
  9. Results
  10. Immunocytochemistry of GAD
  11. Discussion
  12. Acknowledgements
  13. References

All animal procedures were conducted according to national regulations.

Cerebral cortical neurones (GABAergic neurones, Yu et al. 1984; Drejer et al. 1987) were isolated from 15-day-old mouse fetuses and cultured essentially as described by Hertz et al. (1989). Cerebellar granule cells were isolated from cerebellum of 7-day-old mice (Schousboe et al. 1989). The brain tissue was trypsinized followed by trituration in a DNase solution containing a trypsin inhibitor from soybeans. Cells were suspended (3 × 106 cells/mL) in a slightly modified Dulbecco's minimum essential medium (DMEM) containing 28 mm[1−13C]glucose and 10% (v/v) fetal calf serum and seeded in poly-d-lysine-coated, 15-cm diameter Petri dishes. Some cultures were exposed to medium containing 50 µm or 0.5 mm KA from day 0. Cytosine arabinoside (20 µm) was added after 24–48 h to prevent astrocyte proliferation. At day 7, KA was added to a final concentration of 0.5 mm for 2 h in some cultures maintained in 50 µm KA for 7 days. After 7 days in culture, medium was removed and cells were washed with 0.9% saline and extracted with 70% (v/v) ethanol, followed by centrifugation at 4000 g for 5 min. The supernatants were lyophilized and stored at − 20°C. Cellular protein in the ethanol pellets was determined after re-dissolving in 1 m KOH at 37°C for 30 min and dilution 1 : 4, using the Pierce BCA (Pierce, Rockford, IL, USA) protein assay with bovine serum albumin as standard.

NMR spectroscopy and HPLC

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Cerebral cortical and cerebellar granule neurones
  6. NMR spectroscopy and HPLC
  7. GAD immunostaining
  8. Data analysis
  9. Results
  10. Immunocytochemistry of GAD
  11. Discussion
  12. Acknowledgements
  13. References

Proton-decoupled 125.5 MHz 13C NMR spectra were obtained on a Bruker DRX-500 spectrometer (Bruker AG, Fällanden, Switzerland). Samples were re-dissolved in D2O containing 0.10% ethylene glycol as an internal standard. Spectra were accumulated using a 30° pulse angle, 25 kHz spectral width with 64-K data points. The acquisition time was 1.3 s, and a 2.5 s relaxation delay was used. The number of scans was typically 10 000 for each cell extract. Some spectra were also broadband-decoupled only during acquisition to avoid nuclear Overhauser effects. From several sets of spectra, factors for this effect were obtained and applied to all spectra.

Amino acids and glutathione in the cell extracts were quantified by high-performance liquid chromatography HPLC analysis on a Hewlett Packard 1100 system (Agilent Technologies, Palo Alto, CA, USA) with fluorescence detection, after derivatization with o-phthaldialdehyde (Geddes and Wood 1984).

GAD immunostaining

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Cerebral cortical and cerebellar granule neurones
  6. NMR spectroscopy and HPLC
  7. GAD immunostaining
  8. Data analysis
  9. Results
  10. Immunocytochemistry of GAD
  11. Discussion
  12. Acknowledgements
  13. References

For GAD immunostaining, cells (neocortical or cerebellar) were seeded at a density of 1.6 × 106 cells/mL in Permanox chamber slides (Nunc A/S, Roskilde, Denmark) and maintained in medium containing 50 µm KA (only cerebellar cultures) for 7 days. Cultures were rinsed with phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde in PBS [30 min at room temperature (22–24°C)] and permeabilized with a solution of 0.1% saponin and 3% goat serum in PBS. After this, the cultures were incubated overnight at 4°C with the primary anti-GAD67 antibody (1 : 750) in a solution containing 1% goat serum in PBS, rinsed with PBS and incubated for 1 h at room temperature with the secondary antibody, anti-rabbit IgG fluorescein conjugate (1 : 200) and Alexa 488 (1 : 1500). After rinsing with PBS, the slides were coverslipped with Mowiol (Calbiochem Ltd, Nottingham, UK). The cells were examined using a fluorescence microscope equipped with phase contrast (Nikon E1000; Nikon, Tokyo, Japan) using 40× and 60× objectives and digitally photographed with a ColorView camera (Soft Imaging Systems, Stuttgart, Germany). Control of the secondary antibody, performed by omitting the primary antibody, showed no fluorescence. GAD67-immunopositive cells were counted in five to nine fields of the culture and referred to the total number of cells as visualized in matched phase-contrast microphotographs. Results from three independent cultures were averaged.

Data analysis

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Cerebral cortical and cerebellar granule neurones
  6. NMR spectroscopy and HPLC
  7. GAD immunostaining
  8. Data analysis
  9. Results
  10. Immunocytochemistry of GAD
  11. Discussion
  12. Acknowledgements
  13. References

Relevant peaks in the 13C NMR spectra were identified and integrated. The amounts of 13C were quantified from the integrals of the peak areas, using ethylene glycol as internal standard.

Results are presented as mean ± SEM. Differences between groups were analysed statistically with one-way anova followed by the LSD (least significant difference) post-hoc test, and p < 0.05 was considered statistically significant.

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Cerebral cortical and cerebellar granule neurones
  6. NMR spectroscopy and HPLC
  7. GAD immunostaining
  8. Data analysis
  9. Results
  10. Immunocytochemistry of GAD
  11. Discussion
  12. Acknowledgements
  13. References

Typical spectra of cell extracts from cerebellar neurones maintained in the presence of 50 µm KA (Fig. 1, top) and neocortical neurones (Fig. 1, bottom) cultured in medium containing [1−13C]glucose for 7 days are shown in Fig. 1. Labelling of GABA is clearly present in extracts obtained from both neocortical and cerebellar cultures. Additionally, glutamate, alanine and lactate were labelled. In order to understand the results obtained from the NMR spectra, it is necessary to know the relevant metabolic conversions of [1−13C]glucose. Through glycolysis, [1−13C]glucose is metabolized to [3−13C]pyruvate which can be converted to [3−13C]lactate or [3−13C]alanine. Alternatively, [3−13C]pyruvate may enter the tricarboxylic acid (TCA) cycle via pyruvate dehydrogenase (PDH) as [2−13C]acetylCoA. [4−13C]Glutamate and, subsequently, [2−13C]GABA may be formed from labelled 2-oxoglutarate, leaving the TCA cycle during the first turn. In case 2-oxoglutarate does not leave the cycle till the next turn, glutamate and GABA will be labelled in the 2- or 3- and 3- or 4-positions, respectively. For a detailed labelling scheme, see Hassel et al. (1995a).

image

Figure 1. NMR spectra of cell extracts of cerebellar granule neurones cultured in the presence of 50 µm KA (top) and cultured neocortical neurones (bottom). Neurones were cultured as detailed in Materials and methods in a medium containing 28 mm[1−13C]glucose for 7 days. Abbreviations: 1, alanine C-3; 2, lactate C-3; 3, GABA C-3; 4, glutamate C-3; 5, glutamate C-4; 6, GABA C-2; 7, GABA C-4; 8, glutamate C-2.

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It was next examined how exposure of the cerebellar neuronal cultures to different concentrations of KA influenced the labelling of metabolites from [1−13C]glucose. Thus, cerebellar neurones were cultured with or without 50 µm or 0.5 mm KA for 7 days, or with 50 µm KA for 7 days and 0.5 mm KA for the last 2 h. Neocortical neurones maintained in [1−13C]glucose containing medium were used for comparison. Figures 2(a and b) show the cellular contents of glutamate and GABA, and the incorporation of label in glutamate C-4 and GABA C-2 in the different groups of cultures. The content of glutamate was decreased in the cerebellar neurones cultured in the presence of 0.5 mm KA for 7 days compared to all other groups, except the group exposed to 0.5 mm KA for 2 h following the 7-day culture period in 50 µm KA. However, labelling was only reduced in the 0.5 mm KA group compared to neocortical neurones and cerebellar neurones cultured in the absence of KA. Compared to the cerebellar cells cultured in the absence of KA, the glutamate content was lower in the cells cultured in the presence of 50 µm KA for 7 days and additionally incubated for 2 h in 0.5 mm KA. The content and labelling of GABA were lower in cerebellar cultures compared to neocortical neurones. Maintenance of the cerebellar cultures in medium containing 50 µm KA during the entire culture period had no effect on GABA content and labelling. However, exposure to 0.5 mm KA reduced the GABA level. Culturing with 50 µm KA and subsequent treatment with 0.5 mm KA for the last 2 h led to an even more pronounced decrease in both the content and labelling of GABA.

image

Figure 2. Cellular contents (nmol/mg protein) of glutamate (a, □), GABA (b, □) and of [4−13C]glutamate (a, ▪) and [2−13C]GABA (b, ▪) in neocortical and cerebellar neuronal cultures prepared as described in Materials and methods. The culture medium contained 28 mm[1−13C]glucose throughout the culture period (7 days). In case of the cerebellar cultures, the medium contained KA at different concentrations and time periods as specified under the bars. The bars represent averages of three to five experiments, with SEM values indicated by vertical lines. Statistically significant differences between groups were analysed by one-way anova followed by the LSD post-hoc test. p-values of < 0.05 were considered significant. a, significantly different from neocortical neurones; b, significantly different from cerebellar neurones, 0 mm KA; c, significantly different from cerebellar neurones, 50 µm KA; d, significantly different from cerebellar neurones, 0.5 mm KA.

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The cellular contents of [3−13C]lactate and [3−13C]alanine, both derived from [3−13C]pyruvate, are shown in Figs 3(a and b). Lactate labelling was similar when comparing neocortical and cerebellar neurones. Culturing in the presence of 0.5 mm KA for 7 days resulted in an increase in the content of [3−13C]lactate. However, if the KA concentration was raised to 0.5 mm only during 2 h following culturing in 50 µm KA, this increase was not observed (Fig. 3a). The GABAergic neurones and cerebellar cultures had similar contents of [3−13C]alanine, yet [3−13C]alanine was decreased in cerebellar cultures exposed to 0.5 mm KA for 2 h (Fig. 3b). The glutathione content in the two types of neurones was approximately 15 nmol/mg protein. Exposure of the cerebellar cultures to KA had no effect on the glutathione content regardless of the KA concentration. The content of aspartate in the cerebellar cultures maintained either in plain culture medium or in the presence of 50 µm KA was similar to that in the GABAergic neurones (results not shown). Exposure of the cerebellar cultures chronically or acutely to 0.5 mm KA reduced the aspartate content compared to GABAergic neurones or cerebellar cultures maintained in plain culture medium (results not shown).

image

Figure 3. Cellular contents (nmol/mg protein) of [3−13C]lactate (a) and [3−13C]alanine (b) in neocortical and cerebellar neuronal cultures prepared as described in Materials and methods. The culture medium contained 28 mm[1−13C]glucose throughout the culture period (7 days). In case of the cerebellar cultures, the medium contained KA at different concentrations and time periods as specified under the bars. The bars represent averages of three to five experiments, with SEM values indicated by vertical lines. Statistically significant differences between groups were analysed by one-way anova followed by the LSD post-hoc test. p-values of < 0.05 were considered significant. a, significantly different from neocortical neurones; b, significantly different from cerebellar neurones, 0 mm KA; c, significantly different from cerebellar neurones, 50 µm KA; d, significantly different from cerebellar neurones, 0.5 mm KA.

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Immunocytochemistry of GAD

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Cerebral cortical and cerebellar granule neurones
  6. NMR spectroscopy and HPLC
  7. GAD immunostaining
  8. Data analysis
  9. Results
  10. Immunocytochemistry of GAD
  11. Discussion
  12. Acknowledgements
  13. References

Cerebellar neuronal cultures grown for 7 days in plain culture medium, or in medium containing 50 µm KA, were immunostained using a GAD67-specific antibody. Figure 4 shows phase-contrast photomicrographs (Figs 4a and c) and immunofluorescence micrographs (Figs 4b and d) of these cultures. A subpopulation of cells that were immunopositive for GAD were identified based on the co-localization of GAD-immunopositive structures (arrowheads in Figs 4b and d) and cell somas (arrowheads in Figs 4a and c). In keeping with this, quantification of the immunostaining in the cell bodies (arrowheads in Fig. 4) showed that 6.0 ± 1.3% (mean ± SEM, n = 3) of the neurones cultured in plain media exhibited GAD-like immunofluorescence. In KA-treated (50 µm) cultures, the corresponding value was 5.4 ± 0.5% (n = 3). KA exposure had no major effect on the overall morphological appearance of the neuronal cultures (Figs 4a and c). On the other hand, KA treatment caused a reduction in the GAD-like immunofluorescence, particularly in the cell processes (arrows, Figs 4b and d). This is demonstrated at higher magnification in Fig. 5. A different pattern of GAD immunostaining was observed in the processes of control and KA-treated cultures. Control cells showed an overall punctate immunostaining in the processes (Figs 5a and b), whereas in KA-treated cultures there were GAD-immunopositive cells with non-punctate GAD immunostaining (Figs 5c and d). However, in spite of the reduced immunostaining, cell cultures treated with KA still exhibited GAD-positive cell bodies (5.4 ± 0.5%, arrowheads, Figs 4c and d). For comparison, phase-contrast and GAD immunostaining in cortical neuronal cultures is shown in Figs 4(e and f), respectively. In these cultures, neurites were profusely stained for GAD showing a dense network of GABAergic neurones.

image

Figure 4. Phase-contrast (a, c and e) and GAD67 green fluorescence (b, d and f) photomicrographs of cultured cerebellar granule cells (a–d) and neocortical neurones (e, f). The neuronal cultures were prepared as detailed in Materials and methods. After culturing under the different conditions, the cultures were used for phase-contrast microscopy and GAD67 immunostaining. (a and b) The same field from cultures grown in plain medium; (c and d) the same field from cultures grown in 50 µm KA; (e and f) the same field from neocortical neurones. Arrows and arrowheads indicate cell processes and cell soma, respectively. Bar = 20 µm. The photomicrographs are representative of visual fields from eight different wells of two independent culture preparations.

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image

Figure 5. GAD67 green fluorescence photomicrographs of cultured cerebellar granule cells showing details of the processes in control (a and b) and 50 µm KA-treated (c and d) cultures (for details, see Materials and methods). Photomicrographs correspond to different fields of the same culture. Bar = 20 µm.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Cerebral cortical and cerebellar granule neurones
  6. NMR spectroscopy and HPLC
  7. GAD immunostaining
  8. Data analysis
  9. Results
  10. Immunocytochemistry of GAD
  11. Discussion
  12. Acknowledgements
  13. References

Primary cultures prepared from dissociated early post-natal cerebellum of rats or mice consist predominantly (95%) of neurones (Messer 1977). The vast majority of these neurones (∼90%) are granule cells known to require a depolarizing signal for survival and differentiation (Lasher and Zagon 1972). This signal can be provided by inclusion in the culture medium of either a high concentration of K+ or excitatory amino acid receptor agonists such as N-methyl-d-aspartate or kainate (Lasher and Zagon 1972; Thangnipon et al. 1983; Gallo et al. 1987; Schousboe et al. 1989; Damgaard et al. 1996; Balázs et al. 1988a,b,c).

Such cultures have been shown to release both glutamate and GABA upon depolarization in a Ca2+-dependent manner, i.e. vesicular release (Pearce et al. 1981; Drejer et al. 1982; Palaiologos et al. 1988, 1989; Belhage et al. 1992; Damgaard et al. 1996), suggesting the presence of both GABAergic and glutamatergic neurones (e.g. Damgaard et al. 1996). This is in keeping with the finding that 6% of the cell bodies exhibited GAD-like immunostaining and with the pronounced punctate staining in the cell processes. This association between vesicular release and punctate GAD staining may be supported by the finding in this and previous studies that both of these parameters can be eliminated by exposure of the cultures to an excitatory insult (Drejer and Schousboe 1989; Damgaard et al. 1996; Kovacs et al. 2003). It should be noted that, although the cultures lose the ability to release GABA from vesicles as well as the vesicular transporter (Drejer and Schousboe 1989; Kovacs et al. 2003), the GAD-like immunostaining in the cell bodies was found to be unaffected by exposure to 50 µm KA. This is in keeping with the interpretation of Kovacs et al. (2003) that exposure to an excitotoxic insult does not eliminate the GABAergic cells but changes their phenotype.

GABA labelling from 13C glucose has been reported both in vivo (Chhina et al. 2001; Oz et al. 2003) and in neocortical neuronal cultures (Sonnewald et al. 1991, 1993; Waagepetersen et al. 1998). Furthermore, incubation with [U-13C]glutamate has been shown to result in labelling of GABA in neocortical neurones (Westergaard et al. 1995). Using short-term (2–4 h) incubation conditions with 13C-labelled glutamate or glucose in cultures of cerebellar neurones, no NMR detectable labelling of GABA could be demonstrated (Qu et al. 2000; Waagepetersen et al. 2000). Employing a more sensitive method, mass spectrometry, labelled GABA was detected in these cultures after a 2 h incubation with [U-13C]glutamate, the immediate precursor for GABA (Qu et al. 2000). However, upon culturing the cerebellar neurones in the presence of [1−13C]glucose (> 98% enriched) for 7 days, pronounced GABA labelling was observed in the present study using NMR spectroscopy. Regardless of the presence of 50 µm KA, both the content and the labelling of GABA were only approximately 40% lower than that in the GABAergic neocortical neurones. In a previous study, Hassel et al. (1995b) reported an intracellular GABA content of 20 nmol/mg protein in cerebellar neuronal cultures not exposed to KA, which is similar to the value found in the present study. Thus, it is clear that GABA is present in cerebellar cultures, although in lower amounts than that found in neocortical neurones representing predominantly GABAergic cells expressing the GABA-synthesizing enzyme GAD as well as vesicular release (Yu et al. 1984; Drejer et al. 1987; Belhage et al. 1993; Waagepetersen et al. 2001). In this context it should be noted that cerebellar and neocortical neuronal cultures had essentially identical glutamate contents and labelling indicating similar rates of utilization of glucose for glycolysis and subsequent metabolism in the TCA cycle. This is compatible with the findings that lactate and alanine labelling was identical in the two types of cultures. Comparable percentage labelling of GABA from [U-13C]glutamate after short-term incubation in the two types of cultures has been reported (Qu et al. 2000; Waagepetersen et al. 2002). The present study shows that GABA was extensively labelled from [1−13C]glucose, which unequivocally proves that GABA was synthesized endogenously, a finding compatible with the immunostaining for GAD67. It should be noted that in spite of the high content of GABA the percentage enrichment was only approximately 20% of its theoretical value, which is 50%. This low enrichment was also found for glutamate, the immediate precursor for GABA. It may thus be assumed that GABA synthesis involves precursors other than glucose, e.g. amino acids in the culture medium. It appears enigmatic that 6% of the neuronal cell bodies in the cultures expressed GAD-like immunoreactivity and yet the GABA content and rate of synthesis was as high as two-thirds of that observed in cultured neocortical GABAergic neurones. It can be calculated based on volume information given in Schousboe et al. (1975) that the 20 nmol/mg cellular protein of GABA measured in the present study is equivalent to approximately 2 mm concentration. If this resides in 6% of the cells the concentration would be approximately 30 mm, which would likely impose a serious osmotic problem. This value would also be 10-fold higher than the average GABA concentration in the cerebral cortical neurones calculated in the same way assuming that 80% of the neurones in these cultures are GABAergic.

Therefore, there must be a redistribution of GABA from these neurones to the glutamatergic neurones not expressing GAD. Alternatively, the latter neurones might be responsible for GABA synthesis, although such synthesis seems unlikely as the GABA synthetic pathway involving GAD is by far the most prominent in the brain (Martin and Rimvall 1993).

Using the present experimental paradigm, exposure to a high concentration of KA (0.5 mm) for 7 days caused a small but significant decrease in both content and labelling of GABA and glutamate compared to that in cells cultured in the absence of KA. In this context it should be noted that the glutathione content of the cells was not affected by exposure to KA, indicating a high degree of integrity of the cultures. The reduction in GABA and glutamate may be the result of a reversal of the amino acid carriers caused by the KA-induced depolarization (Belhage et al. 1993; Schousboe et al. 2004). Other possibilities include increased degradation or decreased synthesis of GABA and glutamate. The observation that in cultures chronically exposed to 0.5 mm KA the labelling of lactate was increased but that of alanine was not affected, may be an indication of a compartmentalized metabolism. This may be related to the fact that alanine aminotransferase exists in a mitochondrial and a cytosolic isoform (Balázs 1965) being involved in net production of pyruvate and alanine, respectively, as pointed out by Cooper (1988). Contrary to this, lactate dehydrogenase is only present in the cytosol.

It is of interest that exposure for a brief period of time to a high concentration of KA at the end of the culture period in a medium containing 50 µm KA led to a selective reduction in a pool of non-labelled GABA as the amount of GABA in this group was lower than in all other groups, whereas labelling was the same as in the group receiving 0.5 mm KA for the entire culture period.

The fact that cerebellar neurones contain a considerable amount of GABA raises the question of a possible functional role of GABA in these cells. Based on its neurotrophic action (Belhage et al. 1998), GABA might serve a protective role especially in an acute excitotoxic situation. Alternatively, non-synaptic release of GABA could activate extrasynaptic GABA receptors, leading to a modulatory tonic inhibition (Rossi et al. 2003). Therefore studies aimed at demonstrating the release mechanisms and the distribution of GABA in the cerebellar culture will be of interest.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Cerebral cortical and cerebellar granule neurones
  6. NMR spectroscopy and HPLC
  7. GAD immunostaining
  8. Data analysis
  9. Results
  10. Immunocytochemistry of GAD
  11. Discussion
  12. Acknowledgements
  13. References

This research was supported by the Danish MRC (52-00-1011 and 52-00-1747), the Norwegian Epilepsy, Blix, Lundbeck and NOVO Nordisk Foundations and the Spanish grants FIS 01/1318 and SAF-FEDER 2003–04930. Zoila Babot is the recipient of a fellowship from MEC. The excellent technical and secretarial assistance of Bente Urfjell and Hanne Danø, respectively, is greatly appreciated.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Cerebral cortical and cerebellar granule neurones
  6. NMR spectroscopy and HPLC
  7. GAD immunostaining
  8. Data analysis
  9. Results
  10. Immunocytochemistry of GAD
  11. Discussion
  12. Acknowledgements
  13. References
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