Jorgen Drejer

Elevation of the Extracellular Concentrations of Glutamate and Aspartate in Rat Hippocampus During Transient Cerebral Ischemia Monitored by Intracerebral Microdialysis

Abstract: Rats were implanted with 0.3‐mm‐diameter dialysis tubing through the hippocampus and subsequently perfused with Ringers solution at a flow rate of 2 (μ1/min. Samples of the perfusate representing the extracellular fluid were collected over 5‐min periods and subsequently analyzed for contents of the amino acids glutamate, as‐partate, glutamine, taurine, alanine, and serine. Samples were collected before, during, and after a 10‐min period of transient complete cerebral ischemia. The extracellular contents of glutamate and aspartate were increased, respectively, eight‐and threefold during the ischemic period; the taurine concentration also was increased 2.6‐fold. During the same period the extracellular content of glutamine was significantly decreased (to 68% of the control value), whereas the concentrations of alanine and serine did not change significantly during the ischemic period. The concentrations of γ‐aminobutyric acid (GABA) were too low to be measured reliably. It is suggested that the large increase in the content of extracellular glutamate and aspartate in the hippocampus induced by the ischemia may be one of the causal factors in the damage to certain neurons observed after ischemia.

Cellular Origin of Ischemia-Induced Glutamate Release from Brain Tissue In Vivo and In Vitro

Abstract: The uptake and release of d‐[3H]aspartate (used as a tracer for endogenous glutamate and aspartate) were studied in cultured glutamatergic neurons (cere‐bellar granule cells) and astrocytes at normal (5 mM) or high (55 mM) potassium and under conditions of hypo‐glycemia, anoxia or “ischemia” (combined hypoglycemia and anoxia). In glutamatergic neurons it was found that “ischemic” conditions led to a 2.4‐fold increase in the potassium‐induced release of d‐[3H]aspartate as compared to normal conditions. Hypoglycemia or anoxia alone affected the release only marginally. The ischemia‐induced increase in the evoked d‐[3H]aspartate release was shown to be calcium‐dependent. In astrocytes no difference was found in the potassium‐induced release between the four conditions and the K+‐induced release was not calcium‐dependent. The uptake of d‐[3H]‐aspartate was found to be stimulated at high potassium in both glutamatergic neurons (98%) and in astrocytes (70%). This stimulation of d‐aspartate uptake, however, was significantly reduced under conditions of anoxia or “ischemia” in both cell types. In glutamatergic neurons (but not in astrocytes) hypoglycemia also decreased the potassium stimulation of d‐aspartate uptake. In a previous report it was shown, using the microdialysis technique, that during transient cerebral ischemia in vivo the extracellular glutamate content in hippocampus was increased eightfold. In the present paper it is shown that essentially no increase in extracellular glutamate is seen under ischemia when the perfusion is performed using calcium‐free, cobalt‐containing perfusion media. The results from the in vitro and in vivo experiments indicate that the glutamate accumulated extracellularly under ischemia in vivo originates from transmitter pools in glutamatergic neurons. Moreover, the released glutamate cannot be efficiently disposed of due to a lack of activation by potassium of the high‐affinity glutamate uptake system in neurons and astrocytes under ischemic conditions.

Pyruvate Carboxylase Activity in Primary Cultures of Astrocytes and Neurons

Abstract: The activity of the pyruvate carboxylase was determined in brains of newborn and adult mice as well as primary cultures of astrocytes, of cerebral cortex neurons, and of cerebellar granule cells. The activity was found to be 0.25 ± 0.14, 1.24 ± 0.07, and 1.75 ± 0.13 nmol min−1 mg−1 protein in, respectively, neonatal brain, adult brain, and astrocytes. Neither of the two types of neurons showed any detectable enzyme activity (i.e., < 0.05 nmol min−1 mg−1). It is therefore concluded that pyruvate carboxylase is an astrocytic enzyme.

Regional Cerebral Glucose Phosphorylation and Blood Flow After Insertion of a Microdialysis Fiber Through the Dorsal Hippocampus in the Rat

Abstract: Local cerebral glucose metabolism (LCMRglc) and local cerebral blood flow (LCBF) were studied following implantation of a microdialysis fiber in rat dorsal hippocampus. Recovery time after implantation varied from 0 to 24 h. All rats showed pronounced disturbances in LCMRglc and LCBF during the first 2 h of implantation. The changes consisted of (a) a general decrease in blood flow and glucose phosphorylation and (b) small areas (spots) around the fiber with increased glucose phosphorylation and decreased blood flow. Animals allowed to recover for 24 h demonstrated a near normalization of LCMRglu and LCBF, and the focal disturbances (spots) of glucose phosphorylation and blood flow disappeared. The slight reduction in blood flow and glucose metabolism at this time must be accepted, because extension of the recovery period beyond 24 h may give interpretation problems due to the developing gliosis.

Direct Evidence That Excitotoxicity in Cultured Neurons Is Mediated via N-Methyl-D-Aspartate (NMDA) as well as Non-NMDA Receptors

Abstract: Cultured GABAergic cerebral cortex neurons were exposed to the excitatory amino acid (EAA) L‐glutamate, kainate (KA), N‐methyl‐D‐aspartate (NMDA), or RS‐α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolopropionate (AMPA). To ensure a constant glutamate concentration in the culture media during the exposure periods, the glutamate uptake inhibitor L‐aspartic acid β‐hydroxamate was added at 500 μM to the cultures that were exposed to glutamate. Each of these EAAs was able to induce neurotoxicity. It was not possible to reduce or prevent glutamate‐induced cytotoxicity by blocking only one of the glutamate receptor subtpes with either the NMDA receptor antagonist D‐(‐)‐2‐amino‐5‐phosphonopentanoate (APV) or with one of the specific non‐NMDA antagonists 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX) and 6,7‐dinitroquinoxaline‐2,3‐dione (DNQX). However, if the cultures were exposed simultaneously to glutamate and the antagonists in combination, i.e., APV plus CNQX or APV plus DNQX, the toxicity was completely prevented. Furthermore, CNQX and DNQX were shown to be selective blockers of cytotoxic phenomena induced by non‐NMDA glutamate agonists with no effect on NMDA‐induced cell death. Likewise, APV prevented NMDA‐induced cell death without affecting the KA‐ or AMPA‐induced neurotoxicity. It is concluded that EAA‐dependent neurotoxicity is induced by NMDA as well as non‐NMDA receptors.

Characterization of uptake and release processes ford- andl-aspartate in primary cultures of astrocytes and cerebellar granule cells

The uptake ofl-andd-aspartate was studied in astrocytes cultured from prefrontal cortex and in granule cells cultured from cerebellum. A high affinity uptake system forl- andd-aspartate was found in both cell types, and the two stereoisomers exhibited essentially the sameKm- andVmax-values in bouth astrocytes (l-aspartate:Km 77 μM;Vmax 11.8 nmol×min−1×mg−1;d-aspartate:Km 83 μM;Vmax 14.0 nmol×min−1×mg−1) and granule cells (l-aspartate:Km 32 μM;Vmax 2.8 nmol ×min−1×mg−1;d-aspartate:Km 26 μM;Vmax 3.0 nmol×min−1×mg−1). To investigate whetherl-glutamate,l-aspartate andd-aspartate use the same uptake system a detailed kenetic analysis was performed. The uptake kinetics of each one of the three amino acids was studied in the presence of the two other amino acids, and no essential differences between the uptake characteristics of the amino acids were found. In addition to the uptake studies the release ofD-aspartate from cerebellar granule cells was investigated and compared withl-glutamate release. A Ca2+-dependent, K+-induced release was found for both amino acids.

GABA Induces Functionally Active Low-Affinity GABA Receptors on Cultured Cerebellar Granule Cells

The effect of γ‐aminobutyric acid (GABA) and its agonists muscimol and 4,5,6,7‐tetrahydroisoxazolo[5‐4‐c]pyridin‐3‐ol (THIP) on the development of GABA receptors on cerebellar granule cells was studied by cultivation of the cells in media containing these substances. It was found that the presence of 50 μM GABA in the culture media led to the induction of low‐affinity GABA receptors (KD 546 ± 117 nM) in addition to the high‐affinity receptors (KD 7 ± 0.5 nM) which were present regardless of the presence of GABA in the culture media. The functional activity of the GABA receptors was tested by investigating the ability of GABA to modulate evoked glutamate release from the cells. It was found that GABA could inhibit evoked glutamate release (ED50 10 ± 3 (μM) only when the cells had been cultured in the presence of 50 νM GABA, 50 μM muscimol, or 150 μM THIP, i.e., under conditions where low‐affinity GABA receptors were present on the cells. This inhibitory effect of GABA could be blocked by 120 μM bicuculline and mimicked by 50 μM muscimol or 150 μM THIP whereas 150 μM (‐)‐baclofen had no effect. It is concluded that GABA acting extracellularly induces formation of low‐affinity receptors on cerebellar granule cells and that these receptors are necessary for mediating an inhibitory effect of GABA on evoked glutamate release. The pharmacological properties of these GABA receptors indicate that they belong to the so‐called GABAA receptors.

Energy metabolism in glutamatergic neurons, GABAergic neurons and astrocytes in primary cultures.

Several aspects of energy metabolism (glucose utilization, lactate production,14CO2 production from labeled glucose, glutamate or pyruvate, oxygen consumption and contents of ATP and phosphocreatine) were measured in cerebellar granule cells (glutamatergic) in primary cultures and compared with corresponding data for cerebral cortical neurons (mainly GABA-ergic) and astrocytes. Cerebellar granule cells and astrocytes were metabolically more active than cerebral cortical neurons. Glutamate which is utilized as a major metabolic fuel as astrocytes and, to a lesser extent, in cerebral cortical neurons, was virtually not oxidized in cerebellar granule cells.

Chaotropic Ions Affect the Conformation of Quisqualate Receptors in Rat Cortical Membranes

Abstract: Binding of [3H](R,S)‐α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid ([3H]AMPA) to quisqualate receptors in the presence of SCN ions produced curvilinear Scatchard plots. Kinetic investigations of [3H]‐AMPA binding showed that the curvilinearity cannot be explained by assuming binding to two separate binding sites or by considering it due to cooperative interaction. A more likely explanation is that the quisqualate receptors exist in two states, one with high and one with low affinity for [3H]AMPA. Chaotropic ions change the relaxation constant between the two states.

Ontogenetic development of glutamate and gaba metabolizing enzymes in cultured cerebral cortex interneurons and in cerebral cortex in vivo

The development of the enzymes phosphate activated glutaminase (PAG), glutamate dehydrogenase (GLDH), glutamic‐oxaloacetic‐transaminase (GOT), glutamine synthetase (GS), GABA‐transaminase (GABA‐T) and ornithine‐δ‐aminotransferase (Orn‐T) was followed in mouse cerebral cortex in vivo and in cultured mouse cerebral cortex interneurons. It was found that GLDH, GOT and Orn‐T exhibited an enhanced developmental pattern in the cultured neurons compared to cerebral cortex. The activities of PAG and GABA‐T developed in parallel in vivo and in culture but the activity of GS remained low in the cultured neurons compared to the increasing activity of this enzyme found in vivo. Compared to cerebral cortex the cultured neurons exhibited higher activities of PAG, GLDH and Orn‐T, whereas the activities of GABA‐T and GOT were lower in the cultured cells. The activity of GS in the cultured neurons was only 5–10% of the activity in cerebral cortex in vivo. It is concluded that neurons from cerebral cortex represent a reliable model system by which the metabolism and function of GABAergic neurons can be conveniently studied in a physiologically meaningful way.