Anoxia-induced dopamine release from rat striatal slices: involvement of reverse transport mechanism

Incubation of rat striatal slices in the absence of oxygen (anoxia), glucose (aglycemia), or oxygen plus glucose (ischemia) caused significant increases in dopamine (DA) release. Whereas anoxia decreased extracellular 3,4-dihydroxyphenylacetic acid levels by 50%, aglycemia doubled it, and ischemia returned this aglycemia-induced enhancement to its control level. Although nomifensine, a DA uptake blocker, completely protected the slices against anoxia-induced DA depletion, aglycemia- and ischemia-induced increases were not altered. Moreover, hypothermia differentially affected DA release stimulated by anoxia, aglycemia, and ischemia. Involvement of glutamate in DA release induced by each experimental condition was tested by using MK-801 and also by comparing the glutamate-induced DA release with that during anoxia, aglycemia, or ischemia. MK-801 decreased the anoxia-induced DA depletion in a dose-dependent manner. This treatment, however, showed a partial protection in aglycemic conditions but failed to improve ischemia-induced DA depletion. Like anoxia, DA release induced by exogenous glutamate was also sensitive to nomifensine and hypothermia. These results indicate that anoxia enhances DA release by a mechanism involving both the reversed DA transporter and endogenous glutamate. Partial or complete lack of effect of nomifensine, hypothermia, or MK-801 in the absence of glucose or oxygen plus glucose also suggests that experimental conditions, such as the degree of anoxia/ischemia, may alter the mechanism(s) involved in DA depletion.     

Effects of the aqueous extract of Bryothamnion triquetrum on chemical hypoxia and aglycemia-induced damage in GT1-7 mouse hypothalamic immortalized cells

The neuroprotective ability of the aqueous crude extract of Bryothamnion triquetrum (S. G. Gmelin) Howe and its cinnamic acids was studied in GT1-7 cells exposed to the combination of chemical hypoxia (KCN 3 mM) and aglycemia conditions. These ischemia-like conditions provoked acute and delayed cytotoxicity in GT1-7 cells if extended for more than 90 min. The extract was able to protect from the cell death produced by severe (180 min) chemical hypoxia/aglycemia insult, which cannot be related to its glucose content, and also reduced the cytotoxicity and early production of free radicals produced by mild (105 min) insult. Results showed that some of these protective effects of the extract are partially related to the presence of ferulic acid. The data additionally suggest that neuroprotection exerted by the extract is related to its ability to reduce free-radical generation by mechanisms different from the direct scavenging of the radical entities.     

Studies on the Linkage of Energy Metabolism and Neuronal Activity in the Isolated Perfused Rat Brain

An isolated rat brain preparation was perfused using glucose-free (=aglycemic) media. The high-energy phosphates, substrates of the glycolytic pathway, free atnino acids, acetylcholine as well as the intracellular distribution of hexokinase activity were determined in brain tissues. The EEG was evaluated visually. The levels of glycolytic substrates, glutamate, and glutamine in cortical tissue decreased after aglycemic perfusion, whereas the aspartate level increased and the GABA level remained unchanged. The high-energy phosphate content seemed to be unaffected for about 15 min of aglycemic perfusion and fell significantly after 20 min. The EEG of the isolated brain changed rapidly after starting aglycemic perfusion and became isoelectric after 12–15 min. Hyperglycemic perfusion (35 mmol glucose per liter perfusion medium) did not alter the energy metabolism of the isolated brain. The breakdown of cerebral energy metabolism and of EEG activity was postponed when thiopental was added to the perfusion medium. The soluble hexokinase activity measured in cortical tissue was reduced after aglycemic perfusion and was enhanced after thiopental. Hyperglycemic perfusion did not influence the intracellular hexokinase distribution. The acetylcholine level in the striatum of the isolated rat brain was significantly decreased by aglycemia and was increased in hypothalamus by thiopental. It was suggested that hexokinase bound to the mitochondrial membrane may play an important role in the relationship of energy metabolism and neuronal activity.     

Increased Expression of Calbindin D28k via Herpes Simplex Virus Amplicon Vector Decreases Calcium Ion Mobilization and Enhances Neuronal Survival After Hypoglycemic Challenge

Abstract: Disruption of Ca²⁺ homeostasis often leads to neuron death. Recently, the function of calcium-binding proteins as neuronal Ca²⁺ buffers has been debated. We tested whether calbindin D_28k functions as an intracellular Ca²⁺ buffer by constructing bicistronic herpes simplex virus vectors to deliver rat calbindin cDNA to hippocampal neurons in vitro. Neurons were infected with vectors delivering calbindin or a negative control or were mock-infected. After 12 or 24 h of hypoglycemia, infected cells were made aglycemic during fura-2 calcium ratiometric imaging. In response to this challenge, neuronal overexpressing calbindin had less Ca²⁺ mobilized as compared with negative controls or mock-infected cells. Cells were assayed for survival after 12- or 24-h hypoglycemia or aglycemia. The calbindin vector decreased neuronal death due to hypoglycemia but not aglycemia. Here we demonstrate, in response to hypoglycemic challenge, both decreased Ca²⁺ mobilization and increased survival of cells infected with the calbindin vector.