Impact of inflammation on energy metabolism in astrocytes. Cytokines such as IL-1a and TNFa stimulate in a time-, concentration-, and transcription-dependent manner glucose utilization in astrocytes. This stimulation of glucose utilization by astrocytes upon exposure to IL-1a and TNFa does not lead to enhanced release of glycolytically derived lactate. Another potential fate of the additional glucose taken up under the action of cytokines could be storage as glycogen. However, this is clearly not the case since glycogen levels are decreased as a consequence of exposure to the cytokines. Oxidative metabolism could represent yet another route of glucose metabolism that could be modulated by cytokines. Using the MTT colorimetric technique (the colorimetric reaction of MTT is proportional to the activity of mitochondrial dehydrogenases, thus representing an indirect measure of mitochondrial respiration) we have observed that additional glucose imported by astrocytes upon exposure to cytokines is not processed through the Krebs cycle. These data suggest that activation of another pathway (e.g. pentose phosphate pathway) is necessary to explain the effect of the cytokines. The effect of IL-1a and TNFa on [3H]2DG uptake which is observed after 24 hours requires RNA and protein synthesis since actinomycin D and cycloheximide prevent the effect of the cytokines. We also showed that cytokines have no effect when applied briefly (e.g., 15 min) followed by [3H]2DG measurement 24 hours later suggesting that continued application of the cytokine is required for the observed metabolic effect. Interestingly, treatment of astrocytes with a combination of IL-1a and TNFa or with a combination of either IL-1a or TNFa together with glutamate was considerably more effective in stimulating [3H]2DG uptake than either cytokines or glutamate alone.
To examine the implication of the IL-1 receptor in the effect of IL-1a, astrocytes were cultured for 24 hours in the absence or presence of IL-1a, with and without the addition of a naturally occurring IL-1 receptor antagonist (IL-1ra). Treatment of astrocyte cultures with IL-1ra (50 ng/ml) blocked the effect of IL-1a on the 2DG uptake, indicating the involvement of IL-1 receptors. Since astrocytes produce significant amounts of the free radical nitric oxide (NO) in response to cytokines, we tested the hypothesis that cytokine-stimulated 2DG uptake in astrocytes could be mediated via generation of NO. However, the nitric oxide synthase inhibitor L-NAME (300µM) was without effect on cytokine-evoked 2DG uptake. Since the activity of the Na+/K+ATPase is one of the major energy-consuming cellular processes, we tested the effect of the Na+/K+ATPase inhibitor ouabain on IL-1a- and TNF?-stimulated 2DG uptake. Ouabain application (100 µM) failed to reverse the cytokine-mediated increase of 2DG uptake, indicating that the increase in glucose consumption induced by cytokines is not mediated by an activation of the Na+/K+ ATPase. We next investigated the signaling pathway underlying the effect of cytokines on energy metabolism using inhibitors of phospholipase C (U73122) or of PI3kinase (LY 294002). IL-1a- and TNFa- induced increase in 2DG uptake was markedly blocked by LY294002 (10µM) and not by U73122 (10µM), suggesting that the effect of cytokines involves predominantly an activation of the PI3K signaling pathway. In contrast, these inhibitors have no effect on the previously described short-term effect of glutamate on glucose utilisation.
Assessment of deficits in cognitive functions at different ages in transgenic mice. A new task design was developed to assess more accurately several aspects of spatial memory by mice in the water maze and to control for the poor performance of some mice due to possible stress effects (rats in a nearby room, large size and color of the pool). To improve the animal's performance in the Morris maze, we have therefore reduced the swimming pool (123cm in diameter and 30cm in height) and increased the number of visual cues in the testing room to allow better spatial orientation. We have also installed a new testing room reserved to mice. Twenty female mice (C57/bl6 aged 20 weeks were trained in two different experiments: Animals received four learning trials on each of five consecutive days. The pool was filled with water kept at a temperature of 27°C.The position of the hidden platform remained fixed for the entire period of the acquisition. Each trial consisted of the mice being placed into the water, at one of four starting points : N, E, S, W. The starting positions for each trial were randomly determined. A trial ended when the mouse reached the hidden platform and remained there for 20s. During the 30s inter-trial interval, the animals were maintained in a dry container. At the end of the 20 training trials, each mice received a probe trial. For this trial, the platform was removed from the pool and the mouse was allowed to swim for 60s, before being removed from the pool.
From working memory tests we can assess both short and long term retention of different places in the pool. We were also able to assess what appears as an indication of a short duration inhibitory response towards the most recently learned position during the acquisition of each new position. This provides the basis for dissociating three different memory stages possibly affected in the mutant mice.