The glutamate-glutamine cycle is thought to be of paramount importance in the mature brain; however, its significance is likely to vary with regional differences in distance between astrocyte and synapse. The present study is aimed at evaluating the role of this cycle in cultures of cerebellar neurons, mainly consisting of glutamatergic granule cells. Cells were incubated in medium containing [U-13 C]glutamate or [U-13 C]glutamine in the presence and absence of unlabeled glutamine and glutamate, respectively. Cell extracts and media were analyzed using highperformance liquid chromatography (HPLC) and gas chromatography combined with mass spectrometry (GC/MS). Both [U-13 C]glutamate and [U-13 C]glutamine were shown to be excellent precursors for synthesis of neuroactive amino acids and tricarboxylic acid (TCA) cycle intermediates. Labeling from [U-13 C]glutamate was higher than that from [U-13 C]glutamine in all metabolites measured. The presence of [U-13 C]glutamate plus unlabeled glutamine in the experimental medium led to labeling very similar to that from [U-13 C]glutamate alone. However, incubation in medium containing [U-13 C]glutamine in the presence of unlabeled glutamate almost abolished labeling of metabolites. Thus, it could be shown that glutamate is the preferred substrate for intermediary metabolism in cerebellar neurons. Label distribution indicating TCA cycle activity showed more prominent cycling from [U-13 C]glutamine than from [U-13 C]glutamate. Labeling of succinate was lower than that of the other TCA cycle intermediates, indicating an active role of the c-amino butyric acid shunt in these cultures. It can be concluded that the cerebellar neurons rely more on reuptake of glutamate than supply of glutamine from astrocytes for glutamate homeostasis.

Culturing mouse cerebellar neurones (predominantly glutamatergic) in the presence of [1) 13 C]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 lM 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.

Pyruvate recycling is a pathway for complete oxidation of glutamate. The cellular location and the physiological significance of such recycling has been debated during the last decade. The present study was aimed at elucidating whether recycling takes place in neuron-enriched cultures of dissociated cerebella, consisting mainly of glutamatergic granule cells, some GABAergic neurons, and few astrocytes. These cultures and cultures of astrocytes from cerebellum were incubated in medium containing [U-(13)C]glutamate, and cell extracts were analyzed by gas chromatography and mass spectrometry. Additionally, in the case of the neuron-enriched cultures, a magnetic resonance (MR) spectrum was obtained. It could be shown that the atom percentage excess of the isotopomer representing pyruvate recycling in glutamate (M + 4) was similar for astrocytes and neuron-enriched cultures. However, the latter showed more recycling in glutamine (synthesized in the small fraction of astrocytes) than the pure astrocyte cultures, whereas the reverse was the case for aspartate. In fact, the atom percentage excess of the isotopomer representing pyruvate recycling in glutamine was slightly but significantly higher than that in glutamate in the neuron-enriched cultures. It can be concluded that pyruvate recycling is clearly present in neurons, and this was verified by MR spectroscopy.

Vulnerable plaques constitute a risk for serious heart problems, and are difficult to identify using existing methods. Hyperspectral imaging combines spectral- and spatial information, providing new possibilities for precise optical characterization of atherosclerotic lesions. Hyperspectral data were collected from excised aorta samples (n = 11) using both white-light and ultraviolet illumination. Single lesions (n = 42) were chosen for further investigation, and classified according to histological findings. The corresponding hyperspectral images were characterized using statistical image analysis tools (minimum noise fraction, K-means clustering, principal component analysis) and evaluation of reflectance/fluorescence spectra. Image analysis combined with histology revealed the complexity and heterogeneity of aortic plaques. Plaque features such as lipids and calcifications could be identified from the hyperspectral images. Most of the advanced lesions had a central region surrounded by an outer rim or shoulder-region of the plaque, which is considered a weak spot in vulnerable lesions. These features could be identified in both the white-light and fluorescence data. Hyperspectral imaging was shown to be a promising tool for detection and characterization of advanced atherosclerotic plaques in vitro. Hyperspectral imaging provides more diagnostic information about the heterogeneity of the lesions than conventional single point spectroscopic measurements.