Search

N-Methyl-D-aspartate (NMDA) stimulated the release of endogenous dopamine from striatal slices prepared from adult Sprague-Dawley rats. A mixture of sodium fluoride and aluminum chloride (AlF4-) added to the slices significantly potentiated the NMDA-stimulated release of dopamine in a concentration- and time-dependent manner. The AlF4- mixture had no effect on the nonstimulated basal efflux of dopamine, and no increases in NMDA-stimulated release were observed when NaF was replaced with NaCl. Similarly, AlCl3 or a mixture of NaCl and AlCl3 had no effect on NMDA-stimulated release. The AlF(4-)-induced increase in NMDA-stimulated dopamine release was totally blocked by magnesium or the selective NMDA glycine antagonist 7-chlorokynurenic acid. Striatal slices depolarized with KCl (15 mM) also released dopamine and this release was similarly potentiated by AlF4-. However, KCl-stimulated dopamine release from striatal synaptosomes was not potentiated by concentrations of AlF4- that greatly increased release from striatal slices. NMDA did not stimulate the release of dopamine from striatal synaptosomes in the absence or presence of aluminum fluoride. Modulators of adenylate cyclase (forskolin) and protein kinase C (phorbol esters) did not enhance NMDA-stimulated dopamine release. The protein kinase C inhibitor H-7 also did not reduce the potentiating effects of AlF4-. The mixed cholinergic agonist carbachol and the calcium ionophore A23187 mimicked the AlF4- effect although the increase in NMDA-stimulated dopamine release produced by these agents was less than that seen with AlF4-.(ABSTRACT TRUNCATED AT 250 WORDS)

Rat brain cortical slices released tritiated norepinephrine ([3H]NA) during a 2-min stimulation with N-methyl-D-aspartate (NMDA). Dithiothreitol (DTT; 0.1-5 mM), present for 6 min prior to stimulation, dose-dependently increased the release of [3H]NA from cortical slices stimulated with a maximally effective concentration of NMDA (500 microM). Similar results were observed for [3H]NA release from hippocampal slices and tritiated and endogenous dopamine release from striatal slices. DTT treatment also markedly shifted the dose-response curve of NMDA to the left. Cortical slices released approximately the same amount of [3H]NA with 10 microM NMDA following DTT treatment (about 5%) as non-DTT-treated control slices did with 500 microM NMDA. The effects of DTT were fully reversed by subsequent treatment with 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB; 0.5 mM). DTT treatment did not significantly alter the ability of magnesium (1.3 mM) or the polyamine antagonist arcaine to block the NMDA-stimulated release of [3H]NA. In contrast, DTT treatment significantly attenuated the antagonist effects of the competitive glycine antagonist, 7-chlorokynurenic acid, and the competitive NMDA antagonist, 2-aminophosphonopentanoic acid. These results suggest that oxidation and reduction of disulfide bonds located within the NMDA receptor complex might regulate the activation of the NMDA receptor. This could have important consequences in vivo if endogenous oxidizing/reducing systems are found to have similar effects on NMDA-stimulated responses.

Recent studies have demonstrated the presence of a novel cocaine metabolite, cocaethylene, present in post-mortem tissue and blood samples from accident victims who tested positive for both cocaine and ethanol (Hearn et al., 1991). We report in this study that cocaethylene potently inhibits presynaptic dopamine uptake in vitro and effectively substitutes for cocaine in a drug discrimination protocol in rats

Subchronic treatment with the dopamine agonist apomorphine produces a sensitization to the stereotypic effects of subsequent apomorphine challenge. The present study investigated the effects of this subchronic treatment on apomorphine induced stereotypic behavior and striatal dopamine synthesis, release, metabolism, and D2 receptor binding. The pretreatment, which enhanced the behavioral response to apomorphine challenge, also elevated basal dopamine synthesis and metabolism, but left the ability of a challenge dose of apomorphine to inhibit dopamine synthesis and metabolism unaltered. Thus, ongoing dopamine synthesis and extracellular levels of metabolites would be higher following apomorphine challenge in animals treated subchronically with the agonist. In contrast, neither synaptosomal dopamine release in response to depolarizing stimuli nor the density of D2 dopamine receptors was altered by the treatment. Overall, the results suggest that, while we did not find evidence of autoreceptor desensitization per se, apomorphine treatment may result in enhanced extracellular dopamine levels following dopamine agonist challenge to provide a greater stimulation of an intact dopamine receptor system.

Two groups of adult Sprague-Dawley rats were maintained on a nutritionally complete liquid diet. In one group, 37% of the calories normally provided by dextrin were replaced with ethanol. Animals were maintained on this diet for eight weeks. The addition of ethanol (200 mM) in vitro significantly inhibited both calcium influx and dopamine release from control synaptosomes but did not alter calcium influx or dopamine release from synaptosomes isolated from ethanol-treated rats. The dihydropyridine calcium channel agonist Bay K 8644 (1 nM) significantly increased both calcium entry and dopamine release from control synaptosomes depolarized with 15 mM KCl. Bay K 8644 (1 nM) had no significant effect on either calcium entry or dopamine release in synaptosomes isolated from ethanol-treated animals. This loss of functional effect was accompanied by a slight (15%) but statistically insignificant increase in the binding of 3 H-nitrendipine to striatal membranes from ethanol-treated rats as compared to control. The calcium-channel blocker, ω-conotoxin (500 nM) had no effect on voltage-dependent calcium uptake into synaptosomes prepared from control or ethanol-treated rats. Conotoxin (500 nM) inhibited the voltage-dependent release of endogenous dopamine from synaptosomes isolated from both groups by 36–44%. Ethanol (200 mM) added in vitro to control synaptosomes did not alter conotoxin's inhibition of dopamine release but completely abolished the ω-conotoxin-induced inhibition of dopamine release in synaptosomes isolated from ethanol-treated animals. These results suggest that DHP-sensitive and ω-conotoxin-sensitive calcium channels in rat brain respond differentially to chronic exposure to ethanol.

Glutamate and GABA Receptors and Transporters: Structure, Function and Pharmacologyedited by Jan Egeberg, Arne Shousboe and Povl Krogsgaard-Larsen, Taylor & Francis, 2002. £99 (hardback) (xii + 427 pages) ISBN 074840819The processes that regulate the major excitatory and inhibitory neurotransmitter systems in the brain are the subject of this new book. The amino acids glutamate and GABA are inextricably linked in the control of neuronal excitability and this volume provides an up-to-date review of the basic physiology and pharmacology of these two important neurotransmitters. The editors have assembled an impressive list of contributing authors who have provided 18 chapters covering everything from the structure and function of glutamate and GABA receptors to the use of transgenic models to study the role of these transmitter systems in disease.The book begins with an excellent chapter reviewing the function and pharmacology of ionotropic glutamate receptors. Additional chapters in this first section are devoted to detailed reviews of the structure of these receptors and of processes that regulate their cellular trafficking. Particularly useful are tables summarizing the results of the extensive mutagenesis experiments that have helped identify the modular components of these receptors. The results of these mutagenesis studies were elegantly complemented and verified by the recent determination of the crystal structure of a kainate-bound GluR2 subunit in which the extracellular domains had been linked via short spacers. The extensive pharmacology of NMDA and non-NMDA receptors are covered in separate chapters and provide a chemist's eye-view of how receptor-selective compounds are developed. These sections are followed by two chapters devoted to the metabotropic glutamate receptors that have received a great deal of attention recently as important modulators of synaptic plasticity.The section covering the physiology and pharmacology of ionotropic GABA receptors begins with a review of studies performed with cerebellar granule cells as a model of how GABAA receptors provide inhibitory control within the golgi-cell–granule-cell loop of the cerebellum. This chapter, although well written, seems somewhat out of place as an introduction to GABAA receptors because it is followed by a section than takes a more global approach of reviewing what is known of GABAA receptor structure and function. As with the glutamate receptors, this section also contains individual chapters devoted to metabotropic GABAB receptors. Although the existence of GABAB receptors was first proposed in the 1980s, based on the ability of l-baclofen to activate a bicuculline-insensitive response, the cDNAs for members of this family were not isolated until 1997. As noted in these chapters, the subsequent use of molecular tools has facilitated our understanding of these enigmatic receptors.Rounding out the discussion are two chapters focused on the transporters that regulate the extracellular availability of glutamate and GABA. The inclusion of these chapters in this single volume is particularly appreciated because the function of these important proteins is often over-shadowed by their more well known ionotropic relatives. In fact, amino acid transporters are surprisingly complex and display a range of electrophysiological behaviors in addition to their transmitter transport activities. As with other neurotransmitters, pharmacological agents that modulate transporter activity are under active development.The final chapters of the book focus on taking what is known of the structure and function of glutamate and GABA receptors and transporters and applying it to studies of behavior and pathophysiology. Particularly interesting are two chapters devoted to the use of experimental animals that lack all or part of specific receptor subunits. These animals are often the only way that the role of specific subunits in various behaviors can be addressed because subunit-selective antagonists are not available for all receptor combinations. The final chapters of this volume offer insights into the role of the glutamate and GABA systems in disease and how receptor-selective compounds might be useful in their treatment. Much of this is speculative at present but there is growing evidence that both hyper- and hypo-functioning of either the glutamate or GABA receptor systems can lead to neuronal pathology and might contribute to such diverse illnesses as schizophrenia and Alzheimer's disease.A few minor issues need to be addressed by the editors in future versions of this book. In particular, there is a considerable amount of redundancy with respect to the presentation of the basic molecular and structural organization of the glutamate and GABA receptors. In this respect, the book resembles that of a proceedings rather than a smoothly edited textbook. Changes are also needed in the organization and integration of the figures because all of the color figures are contained in one continuous section rather than being included in their respective chapters.These shortcomings notwithstanding, this text does serve a useful role as a central repository of relatively advanced information pertaining to the major excitatory and inhibitory neurotransmitter systems of the brain. It is likely that it would do well as the source book for a graduate-level course on excitatory and inhibitory transmitter systems, and students and teachers of neuroscience and neuropharmacology would find it a welcome addition to their library.

The presynaptic neurotoxin omega-conotoxin (omega-CgTx) was tested for its ability to inhibit voltage-dependent calcium flux and transmitter release in rat brain synaptosomes. Conotoxin (0.001-10 microM) had no effect on calcium uptake or endogenous dopamine release from rat striatal synaptosomes in the absence of potassium depolarization. Fast-phase potassium stimulated calcium influx was only partially (20-30%) inhibited by conotoxin at concentrations between 1 nM and 10 microM. The fast-phase release of endogenous dopamine from the same synaptosomal preparation was inhibited by approximately 25% at 0.01 microM and by 60% at 10 microM. These results suggest that a subgroup of high affinity omega-CgTx-sensitive calcium channels may be involved in regulating the release of endogenous dopamine from brain synaptosomes.

Plasma norepinephrine and epinephrine from cannulated trout were measured by high pressure liquid chromatography with electrochemical detection. The catecholamines were extracted with acid-washed alumina using a microfilter assembly which permitted analysis of small volumes of plasma. Mean (± S.D.) values for plasma norepinephrine and epinephrine were 1.83 (0.97) pmol ml−1 and 8.95 (4.94) pmol ml−1, respectively. These values are compared with catecholamine levels from other vertebrate species.

The effects of ethanol on ATP-dependent Ca2+ uptake and inositol 1,4,5 trisphosphate (Ins-P3)-induced Ca2+ release were assessed in rat cerebellar and cortical microsomes. Ins-P3, 0.2–10 μM, released 7–14% of ATP loaded microsomal Ca2+ stores after a 15-sec exposure. Ethanol, 250–750 mM, added after ATP-dependent loading, significantly released microsomal Ca2+ stores, and the amount released was additive to that seen with Ins-P3 alone. The presence of ethanol, 250–750 mM, during the ATP-dependent loading period, resulted in decreased Ca2+ uptake that correlated with decreases in Ins-P3-induced Ca2+ release. Chronic ethanol treatment failed to produce any alterations in ethanol's ability to promote Ca2+ release or to inhibit ATP-dependent Ca2+ uptake. Furthermore, Ins.-P3-induced Ca2+ release was not altered by chronic ethanol treatment. These results suggest that Ins-P3 sensitive Ca2+ stores are resistant to pharmacologically relevant concentrations of ethanol, and do not appear to be involved in the chronic cellular effects of ethanol.

The effects of ethanol on resting and KCl-depolarized cytosolic calcium concentrations ([Ca 2+ ] i ) were measured in purified rat cortical synaptosomes using the calcium indicator fura-2. Ethanol (500–700 mM) significantly elevated the resting [Ca 2+ ] i by 13–25% in the presence of 100 μM external calcium. In the absence of external Ca 2+ , ethanol (50–200 mM) significantly increased [Ca 2+ ] i by 17–23%. Ethanol (200–700 mM) also significantly decreased KCl-induced rise in [Ca 2+ ] i (ΔK) by 40–82% in fura-2 loaded preparations incubated in the absence or presence of external calcium. Ethanol did not produce a significant change in ΔK at 50 and 100 mM concentrations. These results suggest that ethanol may cause an elevation in [Ca 2+ ] i via mobilization of intracellular calcium stores which may be linked to a calcium-dependent inactivation of voltage-sensitive calcium channels.

The time course of simultaneous Ca2+ entry and endogenous dopamine release was examined in mouse striatal synaptosomes depolarized by 30 mM KCl. Ca2+ entry and endogenous dopamine release exhibited fast and slow phase processes. The fastest rates occurred between 0 and 1 s. Ca2+ uptake and dopamine release dropped off quickly with 5-15 s rates at 13 and 10%, respectively, of the 0-1 s rate. Both processes were maintained at relatively high rates at the 1-3 and 3-5 s intervals suggesting mixed fast and slow phase processes. Uptake of Ca2+ and release of dopamine occurred in parallel over the entire 30 s measurement period; however, approximately 70% of the Ca2+ uptake and dopamine release occurred within the first 5 s following depolarization. A calculated ratio of Ca2+ entry versus dopamine release showed that approximately 1-2 Ca2+ ions were required to cause the release of one dopamine molecule. This ratio remained constant from 1 to 15 s following depolarization. Our results suggest that Ca2+ entry is coupled to endogenous dopamine release for both the fast and slow phase process.

[3H]Dopamine (DA) uptake and endogenous DA release were measured in mouse striatal P2 synaptosomes over a 1 30-s period in the presence and absence of KCl depolarization. Approximately 70% of the total DA release over the 30-s period occurred within the first 5 s of depolarization. [3H]DA uptake over this same period was less than 5% of the corresponding release value. The magnitudes and rates of uptake were moderately inhibited by 30 mM KCl depolarization. These decreases were not fully explained by the reduction in sodium present during depolarization or by the dilution of labelled DA by released endogenous DA. Non-linear regression analysis of the uptake data revealed fast (less than 1 s) and slow (greater than 1 s) components of uptake which were differentially affected by KCl depolarization. The results of this study indicate the presence of a rapid, multi-component synaptosomal DA uptake process which appears to play a small role in regulating in vitro endogenous DA release during the initial seconds of KCl depolarization. If synaptosomal uptake reflects the in vivo situation, presynaptic neuronal uptake may be more important in the long-term regulation of transmitter release by maintaining intracellular dopamine storage pools which are linked to synthesis and release processes.

The calcium channel agonist Bay K 8644 (0.1-100 nM) significantly increased the fast-phase entry of calcium and release of endogenous dopamine from rat striatal synaptosomes partially depolarized with 15 mM KCl. This increase was completely blocked by 10 nM nimodipine which had no inhibitory effect on calcium influx and dopamine release in the absence of Bay K 8644. Bay K 8644's agonist effect was attenuated with higher KCl concentrations. These findings suggest that Bay K 8644, in combination with partial KCl depolarization, may expose brain synaptosomal calcium channels which are sensitive to nanomolar concentrations of dihydropyridine calcium channel blockers.

We examined the effects of dihydropyridine Ca2+-channel agonists on synaptosomal voltage-dependent Ca2+ entry and endogenous dopamine release. The (-) isomer of Bay K 8644 and the (+) isomer of Sandoz compound 202-791 were 100-1000 times more potent than their respective opposite enantiomers in enhancing Ca2+ uptake and dopamine release from striatal synaptosomes. The active isomer of each of these compounds increased Ca2+ entry and dopamine release to the same extent at a concentration of 1 nM. Fast-phase Ca2+ entry into synaptosomes isolated from cerebellum, cortex, and hippocampus was sensitive to nanomolar concentrations of Bay K 8644. No effect of Bay K 8644 was observed in synaptosomes isolated from brainstem. Bay K 8644 increased synaptosomal Ca2+ uptake and endogenous dopamine release from striatal synaptosomes only during the initial seconds of KCl-induced depolarization. The greatest increase was observed during the first second of depolarization. No effect was observed after greater than or equal to 5 sec of depolarization. Bay K 8644 did not alter Ca2+ uptake or dopamine release under resting conditions (5 mM KCl) or in response to KCl at greater than 15 mM. The activity of Bay K 8644 was also attenuated by lowering the concentrations of divalent cations in the incubation medium. Agonist activity was observed at Mg2+ concentrations greater than 500 microM (Ca2+ held at 100 microM) and Ca2+ concentrations greater than 100 microM (Mg2+ held at 1000 microM). These results suggest that the Ca2+ channels present in synaptosomes are sensitive to nanomolar concentrations of dihydropyridine agonists under a narrow range of experimental conditions.

Plasma levels of catecholamines, cortisol, and glucose were monitored in rainbow trout during a 6-week forced swimming exercise programme. Compared to resting non-exercised controls, resting trained fish had lower levels of epinephrine, norephinephrine, cortisol, and glucose during the last 3 weeks of training. Initially, trained fish that were swimming had higher levels of epinephrine than resting trained fish. After 2 weeks of exercise, swimming did not significantly elevate epinephrine levels in trained fish. Glucose levels were consistently greater in swimming fish than in resting fish. At the end of the training period, exercised trout had lower (15–20%) oxygen consumption rates while resting or swimming than unexercised fish. After a 5-month forced swimming exercise programme plasma levels of catecholamines and glucose were monitored in trained and untrained cannulated rainbow trout after 2 min of mild agitation. Trained fish showed an immediate (within 1 min) increase in the levels of epinephrine, but not norepinephrine and a delayed (within 15 min) increase in the levels of plasma glucose. Epinephrine levels returned to pre-stress levels within 15 min. Untrained fish had no significant increase in the plasma levels of norepinephrine, epinephrine, or glucose.

We examined the role of calcium in the stimulus-secretion coupling process of brain neurons by measuring the potassium-stimulated release of endogenous dopamine from striatal synaptosomes in the presence and absence of extracellular calcium. Intracellular free calcium levels were also monitored under these conditions using the fluorescent calcium chelator, fura-2. The fast-phase (less than 3 s) of potassium-stimulated dopamine release was completely blocked by removing calcium from the external medium. Elimination of calcium from the medium with EGTA only partially blocked the slow phase (60 s) of K+-stimulated dopamine release. Depolarization of synaptosomes in the presence of extracellular calcium significantly increased intracellular calcium levels as measured by fura-2. No changes in intracellular calcium were observed during depolarization in calcium free-medium. Reductions in the sodium concentration of the extracellular medium produced a significant increase in the basal release of dopamine under calcium-free conditions. Depolarization of synaptosomes under these conditions markedly enhanced the release of dopamine. These results suggest that the slow-phase of dopamine release from synaptosomes does not require calcium but may be mediated via the reversal of the sodium-linked dopamine transport system.

Rapid decompression of fingerling coho salmon (Oncorhynchus kisutch) after simulated dives of 30.5- and 40.5-msw (metres seawater) increased the activity of the fibrinolytic system. Anticoagulated blood samples were analyzed for fibrinolytic activity by measuring the rate of lysis after clotting the blood with a thrombin solution. Immediately and at 1 h postdecompression from 30.5 msw the lysing rate was above normal but returned to normal within 3 h and remained stable for ~24 h, but at 48 h it was depressed. The activity of the fibrinolytic system appeared to follow that of the coagulation system, indicating that the lytic system of fishes is sensitive and responsive to stress-induced changes in hemostatic parameters. Key words: Oncorhynchus kisutch, fibrinolysis, decompression stress, bubbles, coagulation

Chronic ethanol treatment resulted in a marked reduction in the release of dopamine from striatal synaptosomes in response to depolarization. Calcium entry into the same synaptosomal preparations was not altered. Calculation of the ratio of calcium entry vs dopamine release showed that, under control conditions, approximately 15 calcium ions were required to cause the release of 1 dopamine molecule. Chronic ethanol treatment increased this ratio to more than 80:1, suggesting that chronic ethanol administration altered the coupling between calcium entry and dopamine release. Addition of ethanol in vitro to synaptosomes isolated from chronic ethanol-treated rats returned this ratio to approximately 20:1. These results suggest that chronic ethanol treatment results in dependence which is reflected biochemically in striatum through changes in the coupling between voltage-dependent calcium entry into nerve endings and subsequent neurotransmitter release.

Striatal synaptosomes isolated from 3-, 12-, and 24-month-old C57B1/6J mice were exposed to low (5 mM) and high (30 mM) concentrations of potassium of 1, 3, 5, 15 and 30 s. There were no age-related differences in the total intrasynaptosomal content of dopamine among the three age groups of mice. The basal release of endogenous dopamine from synaptosomes isolated from 24-month-old animals was significantly greater than that from synaptosomes isolated from 3-month-old animals at all time points tested. Similarly the potassium stimulated release of dopamine from 24-month-old animals was also higher than that from the 3-month-old group, but this increase was not statistically significant. The net voltage-dependent release of dopamine (stimulated minus basal) was similar for all groups at all time points measured. The increase in basal dopamine release in the 24-month-old animals may reflect an increased fragility or leakiness of the synaptosomal preparation.