Effect of ischemia and reperfusion on lambda of the lumped constant of the [14C]deoxyglucose technique.

We measured the parameter lambda, which is the ratio of the distribution spaces of 2-deoxy-D-glucose (DG) and glucose in the brain, in a model of focal cerebral ischemia in the cat. lambda is the parameter in the lumped constant of the [14C]DG technique most susceptible to changes in ischemia. Cats were subjected to occlusion of the middle cerebral artery for a period of 2 h. During the last 60 min of occlusion, [14C]DG was infused in a programmed fashion so as to obtain a stable arterial blood [14C]DG concentration. The brain was funnel-frozen to preserve tissue metabolites and the frozen brain was sampled regionally (4 to 7-mg samples) for local concentrations of glucose, ATP, phosphocreatine (PCr), and lactate. In a separate series of cats, the infusion of [14C]DG was started after 2 h of occlusion and 3 h of recirculation. In both series, lambda declined slightly for increased levels of tissue glucose and increased appreciably as tissue glucose decreased. A similar relationship was observed between lambda and ATP and PCr, although the correlation was not as clear. Since lambda, and hence the lumped constant, increases in ischemia as well as in postischemic tissue, it is important to measure tissue glucose concentration if quantitative values of local cerebral glucose metabolism are desired in this condition.     

Effect of nitric oxide on L-[3H]glutamate binding to rat brain synaptic membranes.

Nitric oxide (NO), which is spontaneously generated from sodium nitroprusside, was shown to inhibit L-[3H]glutamate binding to rat brain synaptic membranes in a concentration-dependent manner. The L-glutamate binding inhibited by NO, was largely recoverable by the addition of hemoglobin, a scavenger of NO, to the assay medium. These results suggest that NO may play an important role in the modulation of excitatory neurotransmission through direct interaction of L-glutamate binding to its physiological synaptic membrane receptors.     

Regional localization of [14C] methylphenidate in rabbit brain

1. Neuroanatomical distribution of [14C] methylphenidate has been examined in rabbit brain following intracerebroventricular administration. 2. After about a week of implantation of cannula, in lateral ventricle, in albino rabbits, [14C] methylphenidate was injected through the cannula. The distribution of [14C] methylphenidate was examined at 15, 60 and 180 min after the injection in twelve brain regions. 3. The highest levels were observed at the first sampling time (15 min), in medulla and cervical spinal cord. Pons, caudate, tegmentum and hypothalamus also showed significant uptake of [14C] methylphenidate. Cerebral cortex and thalamus showed very low uptake. 4. The uneven distribution suggests a special affinity of methylphenidate for certain brain regions. The implications of this finding for the central actions of methylphenidate are discussed.     

Non-NMDA glutamate receptor binding in canine brain after global cerebral ischemia and reperfusion

We employed a canine model to test the effects of global cerebral ischemia and reperfusion on binding to α-amino-3-hydroxy-5-methyl-4-isoxazole proprionate (AMPA), kainate (KA), and metabotropic glutamate receptors. Ischemia was induced by 10 min of cardiac arrest, followed by restoration of spontaneous circulation for periods of 0, 0.5, 2, 4, and 24 h. Frozen sections were prepared from parietal and temporal cortex, hippocampus, and striatum, and in vitro autoradiography was performed with one of three radioligands: [³H]AMPA, [³H]KA, or [³H]glutamate (using conditions allowing specific labeling of the metabotropic binding site). In striatum, metabotropic binding was unchanged, whereas AMPA and KA binding decreased by 20–30% at 30 min postischemia, remaining depressed through 24h. In cortex, AMPA and metabotropic binding were decreased at several timepoints after ischemia and recirculation, particularly in parietal cortex, whereas KA binding was unaffected in this tissue. Binding to hippocampal regions was largely unchanged, except for a decrease in KA binding at 2 and 4 h postischemia. These findings contrast with results from parallel studies showing increased striatal binding to NMDA receptors following ischemia. Decreased binding to non-NMDA glutamate receptors in striatum and parietal cortex may serve to protect against damage mediated through these receptors.     

Age-related changes in uptake and release on L-[3H]noradrenaline in brain slices of senescence accelerated mouse

High K+ and N-methyl-D-aspartate (NMDA) evoked L-[3H]noradrenaline (NA) release to a similar degree in the brain slices of 1-month-old senescence-accelerated resistant mice (SAM-R/1) and senescence-accelerated prone mice (SAM-P/8). However, 30 mM KCl-induced L-[3H]NA release significantly diminished in SAM-P/8 from 3 to 12 months without changing in SAM-R/1. In addition, NMDA-induced L-[3H]NA release was also reduced at 3 months and lowered to a level of spontaneous release at 12 months in SAM-P/8, but no age-related changes in SAM-R/1 were observed. It is suggested that NA release from NA nerve terminals responsive to depolarization is reduced in SAM-P/8 at an earlier stage than in SAM-R/1. Furthermore, NMDA receptors which could be localized in the soma and/or nerve terminals, seem to be involved in NA release and to be decreased with advancing age in SAM-P/8.     

Effect of aspartame on N-methyl-D-aspartate-sensitive L-[3H]glutamate binding sites in rat brain synaptic membranes

Aspartame (L-aspartyl-L-phenylalanine methyl ester), an artificial low-calorie sweetener, was shown to dose-dependently inhibit L-[3H]glutamate binding to its N-methyl-D-aspartate-specific receptors. L-Aspartic acid, a major endogenous metabolite of aspartame, inhibited the binding more stronger than aspartame, while the other metabolites, L-phenylalanine and methanol, had no effect at the same concentration. Aspartame caused a significant change in the affinities of L-[3H]glutamate binding without altering the Vmax values of the binding, suggesting the inhibition is competitive. These in vitro findings suggested that aspartame may act directly on the N-methyl-D-aspartate-sensitive glutamate recognition sites in the brain synaptic membranes.     

GABA alters the metabolic fate of [U-13C]glutamate in cultured cortical astrocytes

The effect of gamma-aminobutyric acid (GABA) on glutamate metabolism was studied by (13)C-nuclear magnetic resonance (NMR) spectroscopy. Cerebral cortical astrocytes were incubated with 0.5 mM [U-(13)C]glutamate and 5 mM glucose in the presence or absence of 0.2 mM GABA for 2 hr. (13)C-labeled glutamate, glutamine, and aspartate were observed in cell extracts, and (13)C-labeled glutamine and lactate were present in the media. Both uniformly labeled glutamate and [1,2,3-(13)C]glutamate derived from the tricarboxylic acid (TCA) cycle were present in the cells. The consumption of [U-(13)C]glutamate and glucose was unchanged in the presence of GABA; however, the formation of [U-(13)C]lactate and [U-(13)C]aspartate from metabolism of [U-(13)C]glutamate was increased in cells incubated with GABA. The total concentration of aspartate was increased to the same extent as the (13)C-labeled aspartate, suggesting increased entry of [U-(13)C]glutamate into the TCA cycle to allow for the transamination of GABA. Although the concentrations of unlabeled glucose and lactate in the media were unchanged in the presence of GABA, the concentration of alanine was decreased, indicating that there was decreased transamination of the unlabeled pyruvate from glucose metabolism. The amount of [U-(13)C]glutamate converted to [U-(13)C]glutamine and [U-(13)C]lactate was increased in the presence of GABA. However, since the overall consumption of [U-(13)C]glutamate was not different, it can be concluded that the amount of [U-(13)C]glutamate used for energy was decreased. This suggests that exogenous GABA could substitute for glutamate as an energy source for astrocytes. The results indicate that the presence of GABA influences the metabolic fate of both glutamate and glucose in astrocytes, suggesting that fluctuations in the concentration of GABA in normal and pathological conditions can alter the compartmentation of glial metabolism in brain.     

The effect of isoflurane on the release of [H]-acetylcholine from rat brain cortical slices

Volatile general anaesthetics are believed to affect synaptic transmission, but their actions in the central nervous system (CNS) remains unclear. Acetylcholine (ACh) is one of the most important neurotransmitter in the CNS and thus, it is possible that its release could be one of the targets for volatile anaesthetic action. However, the effects of these agents on the release of ACh are not yet fully understood. Rat brain cortical slices were loaded with [³H]-choline in order to study the effect of isoflurane on the release of [³H]-ACh from this preparation. Isoflurane (28, 43, 54, 95 and 182 nM) significantly increased the basal release of [³H]-ACh. This effect was independent of the extracellular sodium and calcium concentration but was decreased by tetracaine and dantrolene, inhibitors of Ca^2+-release from intracellular stores. These findings indicate that isoflurane may cause a Ca^2+-release from internal stores that increases [³H]-ACh release in rat brain cortical slices.     

On the mechanism of enhanced release of [C]glutamate in hippocampal long-term potentiation

K⁺-induced release of [¹⁴C]glutamate was studied in slices of dentate gyrus prepared from control rats and rats in which long-term potentiation (LTP) had been induced in vivo. At all concentrations of Ca²⁺ studied, release from potentiated slices was greater than from control slices. In the same preparations both Ruthenium Red and caffeine enhanced basal release but in potentiated tissue the Ruthenium Red-induced release was significantly greater than in control tissue. These results are discussed in the light of our recent finding that enhanced transmitter release is associated with LTP.     

Regulation of NMDA-stimulated [14C]GABA and [3H]acetylcholine release by striatal glutamate and dopamine receptors.

Striatal function is heavily influenced by glutamatergic and dopaminergic afferent input. To ultimately better understand how the N-methyl-D-aspartate (NMDA) antagonist, phencyclidine (PCP), alters striatal function, we sought to determine how NMDA receptor function is influenced by activation of other glutamatergic receptors and by dopaminergic receptors. To this end, we used NMDA-stimulated efflux of [14C]GABA and [3H]acetylcholine (ACh) from striatal slices to assess the influence of these receptors on NMDA function. NMDA-stimulated [14C]GABA release was more sensitive to NMDA and glycine antagonists than was [3H]ACh release, suggesting that different NMDA receptors regulate the release of these neurotransmitters. Furthermore, NMDA-stimulated [3H]ACh release was inhibited by a D2 receptor mechanism whereas NMDA-stimulated [14C]GABA release was enhanced by D1 receptor activation. NMDA and (+/-)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid hydrobromide (AMPA) interact additively to evoke [3H]ACh release, and synergistically to evoke [14C]GABA release. An additive effect of NMDA and kainate (KA) was found on [14C]GABA release, but NMDA and KA acted in a less than additive manner in evoking [3H]ACh release. KA-stimulated [3H]ACh release was largely blocked by NMDA antagonists, suggesting mediation through activation of NMDA receptors, probably secondary to KA-induced glutamate release. A selective group II metabotropic receptor agonist inhibited NMDA-stimulated [14C]GABA and [3H]ACh release. On the other hand, NMDA-stimulated [14C]GABA release was potentiated by activation of group I metabotropic receptors. Thus, in addition to the differential modulation by D1- and D2-like receptors, the release of striatal neurotransmitters by NMDA receptor activation depends on the extent to which the other glutamate receptors, both ionotropic and metabotropic, are activated.     

Evidence for a presynaptic adenylate cyclase system facilitating [3H]norepinephrine release from rat brain neocortex slices and synaptosomes

The effects of drugs known to enhance intracellular cyclic AMP levels on depolarization-induced [3H]norepinephrine release from superfused rat neocortical slices and synaptosomes were investigated. The adenylate cyclase activator forskolin, the membrane-permeating cyclic AMP analogues 8-bromo-cyclic AMP and dibutyryl cyclic AMP, as well as the phosphodiesterase inhibitors isobutylmethylxanthine and 4-(3-cyclopentyloxy-4-methoxyphenyl)-2-pyrolidone (ZK 62771) enhanced the electrically evoked release of [3H]norepinephrine from superfused rat brain neocortex slices. 8-Bromo-cyclic GMP was without effect on the electrically evoked release. When [3H]norepinephrine release was enhanced by prolonging the electrical pulse duration from 2 msec to 10 msec, the relative inhibitory effect of the Ca2+ channel blocker Cd2+ and the relative facilitatory effect of the K+ channel blocker 4-aminopyridine remained unaffected. In striking contrast, the relative facilitatory effects of forskolin and 8-bromo-cyclic AMP were strongly reduced, whereas the effect of ZK 62771 was almost doubled. When veratrine-induced release of [3H]norepinephrine from cortex synaptosomes was examined, the facilitatory effects of forskolin, 8-bromo-cyclic AMP, and ZK 62771 were even more pronounced than in brain slices. The data strongly support the hypothesis that a presynaptic adenylate cyclase system plays a facilitatory role in the stimulus-secretion coupling process in central noradrenergic nerve terminals.     

Accumulation of glutamate is regulated by calcium and protein kinase C in rat hippocampal slices exposed to ischemic states

There is now convincing evidence that excessive accumulation of the excitatory amino acid glutamate (Glu) in the extracellular space is toxic to neurons. However, the regulation of the release and uptake of Glu in producing this toxic concentration has not been adequately ascertained. The authors report that in hippocampal slices, the output of Glu significantly increased under in vitro ischemic states. Glu in the extracellular space increased fivefold. Since daurisoline, a drug that blocks N-type Ca²⁺ channels, or Ca²⁺ -free solution potently and effectively lowered this stimulated output, it was hypothesized that the Glu output is mediated by Ca²⁺ influx in nerve terminals. When the slices were incubated for 30 minutes under ischemic state, daurisoline caused only small alterations in the postischemic accumulation of Glu. However, Glu accumulation was markedly attenuated by H-7, but not by calmidazolium, facilitated by PDB whereas 8-bromo-cAMP was without effect. It appears therefore that during a 30-minute ischemic insult, protein kinase C (PKC) was involved in the Glu accumulation of supernatant. A direct demonstration of this concept was obtained by showing significant increases in PKC activation in presynaptic nerve terminals (from 1.34 ± 0.1 to 9.34 ± 0.89 U) following 30 minutes of ischemia. DNQX, a non-NMDA receptor antagonist, potently reduced PKC activities and decreased extra Glu accumulation. Also observed was the inhibition of 1-[³H]-Glu uptake into synaptosomes by PDB. These results provide direct evidence that Ca²⁺ influx enhances Glu release, which in turn leads to inhibition of its reuptake, and is coupled with PKC activities in presynaptic nerve terminals.     

GABA and benzodiazepine-induced modification of [14C]L-glutamic acid release from rat spinal cord slices

The spontaneous and potassium-evoked release of [14C]-label from rat spinal cord slices preloaded with [14C]L-glutamic acid and its modification by GABA and related drugs, such as flurazepam, was studied as a possible indirect measure of presynaptic inhibition and of the ability of benzodiazepines to augment it. GABA (100 microM) reduced the spontaneous release of [14C]-label (glutamate) provided that GABA metabolism was blocked by amino-oxyacetic acid (AOAA), but failed to reduce the potassium-evoked release of glutamate, although muscimol (10 microM) had some effect. In contrast, flurazepam (1-100 microM) did not affect spontaneous release but produced some inhibition of the evoked release (through a system insensitive to 10 microM bicuculline). This inhibition became more marked in the presence of both GABA and AOAA, and was then overcome by bicuculline. It is concluded that either some benzodiazepine receptors must be occupied for GABA to produce an effect on evoked release and/or, that the benzodiazepines can only augment GABA function once a certain amount has been released. Studies of the rapid distribution of [14C]-label from glutamate, to GABA, glutamine and other amino acids, using high voltage electrophoresis, showed the importance of blocking metabolic pathways in studies of this kind.     

NMDA-sensitive L-[3H]glutamate binding in cerebral cortex of El mice

NMDA-sensitive L-[3H]glutamate binding was examined in the brains of El mice, a genetic animal model of epilepsy, and in ddY mice. In whole brain, Scatchard analysis showed that both stimulated and unstimulated El mice had significantly lower Bmax values for binding than did ddY mice. In regional studies, the binding of NMDA-sensitive L-[3H]glutamate was significantly less in the cerebral cortex of both stimulated and unstimulated El mice than in that of ddY mice. These data suggest that NMDA receptors may be involved in the genetic susceptibility of El mice to seizures.     

The effect of sevoflurane on the release of [3H]dopamine from rat brain cortical slices

Dopamine is a neurotransmitter that exerts major control on important brain functions and some lines of studies suggest that dopaminergic neurotransmission may be a potential target for volatile anesthetics. In the present study, rat brain cortical slices were labeled with [(3)H]dopamine to investigate the effects of sevoflurane on the release of this neurotransmitter. [(3)H]dopamine release was significantly increased in the presence of sevoflurane (0.46 mM) and this effect was independent of extracellular or intracellular calcium. In addition, [(3)H]dopamine release evoked by sevoflurane was not affected by TTX (blocker of voltage-dependent sodium channels) or reserpine (a blocker of the vesicular monoamine transporter). These data suggest that the dopamine release induced by sevoflurane is non-vesicular, independent of exocytosis and, would be mediated by the dopamine transporter (DAT). GBR12909 and nomifensine, inhibitors of DAT, decreased the release of [(3)H]dopamine evoked by sevoflurane. The same effect was also observed when the brain cortical slices were incubated at low temperature and low extracellular sodium. Ouabain, a Na(+)/K(+) ATPase pump inhibitor, which is known to induce dopamine release through reverse transport, decreased [(3)H]dopamine release induced by sevoflurane. In conclusion, the present study suggests that sevoflurane increases [(3)H]dopamine release in brain cortical slices that is mediated by DAT located at the plasma membrane.     

Sodium ion movements and the spontaneous and electrically stimulated release of [H]GABA and [C]glutamic acid from rat cortical slices

The effect on spontaneous and electrically stimulated release of [³H]GABA and [¹⁴C]glutamic acid from rat cortical slices of agents and ionic conditions that affect sodium ion movements, was studied in anin vitro superfusion system. Those conditions that elevated non-inulin Na⁺ content of the slice accelerated spontaneous efflux and inhibited electrically stimulated release of the labeled amino acids. Agents which blocked the entry of Na⁺ with electrical field stimulation failed to diminish release of the amino acids. Therefore, the entry of Na⁺ into the slice with membrane depolarization is not a requirement for amino acid neurotransmitter release from cortical slices in response to electrical field stimulation. The acceleration of spontaneous efflux and inhibition of electrically stimulated release in the presence of an elevated non-inulin Na⁺ content was discussed in relation to sodium coupled transport of amino acids.