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Repeated amphetamine (AMPH) administration results in behavioral sensitization. To investigate the participation of the opioid system in this phenomenon, we examined the effects of acute and repeated AMPH administration on mu-opioid receptor (MOR) mRNA levels in the nucleus accumbens (NAc) and striatum (STR) of rats, by quantitative non-radioactive in situ hybridization. Five injections of d-AMPH (1.5 mg kg-1, i.p., once every other day), resulted in a sensitization response profile and a significant down-regulation of MOR mRNA levels in the NAc shell, whereas no change was observed in MOR mRNA levels in the NAc core compared to the saline controls. Conversely, MOR mRNA levels were up-regulated in the rostral STR of AMPH-sensitized rats compared to saline controls. No changes in MOR mRNA levels were observed after acute AMPH treatment in any of the brain regions studied. These results suggest that the opioid system participates in the neurobiological underpinnings of behavioral sensitization and that opioid receptor (OR) expression in the STR and NAc shell and core is differentially modulated by repeated AMPH exposure., (Copyright 1999 Elsevier Science B.V.)

The microdialysis technique was used to simultaneously study the in vivo extracellular levels of noradrenaline, glutamate, and gamma aminobutyric acid (GABA) in the bed nucleus of the stria terminalis in order to assess the regulation that noradrenaline may exert upon the release of amino acid neurotransmitters. Perfusion through the probe with UK14304, a selective alpha2-adrenergic agonist, produced a significant decrease of noradrenaline and glutamate extracellular levels. Perfusion through the probe with RX821002, a selective alpha2-adrenergic antagonist, produced a significant increase of noradrenaline and glutamate basal extracellular levels. Perfusion with prazosine, a selective alpha1-adrenergic antagonist, produced a significant decrease of noradrenaline basal extracellular levels without affecting glutamate levels. Under the same conditions, GABA basal extracellular levels were not changed in the presence of any of the alpha-adrenergic ligands studied. The perfusion of high potassium through the probe induced a significant Ca++dependent release of the three neurotransmitters; however, extracellular noradrenaline returned to normal levels even though potassium was still present. In addition, it was observed that alpha-adrenergic receptor ligands exerted differential effects upon K+-induced release of noradrenaline and glutamate. Perfusion with the nonselective alpha-adrenergic antagonist, phenoxybenzamine, presented a biphasic effect upon K+-induced release of noradrenaline; a significant decrease during the first 5 min of stimulation followed by a significant increase in the next 5 min of stimulation. Perfusion with RX821002 produced a significant increase in K+-induced release of noradrenaline that returned to normal basal values before the end of the stimulation period. In contrast, local perfusion with prazosine caused a significant decrease of K+-induced noradrenaline release. In the case of glutamate, perfusion through the probe with phenoxybenzamine produced a significant increase in K+-induced release of glutamate. In addition, RX821002 and prazosine produced a significant increase in K+-induced release of glutamate. Perfusion through the probe with UK14304 produced a significant decrease of both noradrenaline and glutamate K+-induced release. The present results show that noradrenaline in the bed nucleus of stria terminalis exerts a significant inhibition over its own release through alpha2-adrenergic receptors and over glutamate release mainly through alpha2-adrenergic receptors. Thus, the results suggest that noradrenaline in the bed nucleus of the stria terminalis maintains an inhibitory tone over the information flow mediated by glutamate.

Brain-derived neurotrophic factor (BDNF) enhances survival and protects dopaminergic neurons from neurotoxicity. We have examined in primary cultures of fetal mesencephalic neurons the expression of BDNF transcripts and its regulation by glutamate receptor agonists. RT-PCR experiments showed in these cultures the expression of mRNA encoding for neurotrophin receptors TrkA, TrkB and TrkC as well as a differential expression of alternative BDNF gene transcripts derived from exons 1a, 1b, 1c and 1d. In adult rat mesencephalon we only detected BDNF mRNAs originated from exons 1a and 1c. Quantitative RT-PCR analysis of mesencephalic neurons revealed that NMDA, but not kainate treatment, significantly increased BDNF mRNAs derived from exons 1b and 1d. Our data indicate the existence in mesencephalic neurons of different and alternative promoters within the BDNF gene that may be differentially regulated by glutamate receptor stimulation.

The microdialysis technique was used to study the in vivo extracellular levels of norepinephrine in the bed nucleus of the stria terminalis. A basal level of 2.34 +/-0.25 fmol/microl of norepinephrine was observed. Desipramine (2 and 10 microM), a norepinephrine uptake blocker, significantly increased extracellular levels of norepinephrine. Reversed perfusion with high potassium in the presence of 2 microM desipramine induced the release of norepinephrine. Instead, in the presence of 10 microM desipramine, a significant decrease in the induced release of norepinephrine was observed. Clonidine, an alpha2-adrenergic agonist, significantly decreased basal extracellular levels of norepinephrine and the K+-induced release of norepinephrine. In contrast, yohimbine and RX821002, two alpha2-adrenergic antagonists, significantly increased basal extracellular levels of norepinephrine but not the release of norepinephrine induced by 70 mM K+. Perfusion of tetrodotoxin through the probe located in the bed nucleus of the stria terminalis significantly decreased both the basal extracellular level and the K+-induced release of norepinephrine. Furthermore, perfusion of tetrodotoxin through a microdialysis probe implanted in the medial forebrain bundle also decreased basal extracellular levels of norepinephrine in the bed nucleus of the stria terminalis. The results show that in vivo there is a significant noradrenergic tonic activity in the bed nucleus of the stria terminalis. This tonic activity depends on the impulse flow through medial forebrain bundle nerve fibers. Under these conditions, extracellular levels of norepinephrine in the bed nucleus of the stria terminalis are regulated by the magnitude of norepinephrine uptake and by presynaptic alpha2-adrenergic receptors.

1. Lidocaine has been extensively investigated as a potential neuroprotective drug against ischemia-induced neurodegeneration without reaching any satisfactory conclusion. 2. The present work evaluates the effect of lidocaine -17 microM- on the hypoxia-induced release of tritiated D-aspartate from rat striatal slices in superfusion. 3. Hypoxia resulted in a significant increase in the amount of D-aspartate released from striatal slices preloaded with the tritiated excitatory amino acid analog. 4. The addition of lidocaine to the superfusion solution resulted in a drastic reduction in the amount of D-aspartate release evoked by hypoxia, rendering it close to normal values.

1. The effects of acute D-amphetamine administration to rats on the release of endogenous excitatory amino acids from nucleus accumbens slices were studied. 2. D-amphetamine (5 mg/kg and 10 mg/kg; i.p.) significantly increased the spontaneous release of aspartate and glutamate from nucleus accumbens slices. 3. In contrast, D-amphetamine either produced no change or rather decreased K+ (40 mM)-evoked and N-methyl-D-aspartate (100 microM)-evoked release of aspartate and glutamate from the slices, respectively. 4. When D-amphetamine treated rats were pretreated with haloperidol, the effects of D-amphetamine on the spontaneous release of excitatory amino acids were not produced, whereas its effects on N-methyl-D-aspartate-evoked release remained unchanged. 5. These data suggest that amphetamine produces changes in excitatory amino acid-mediated transmission in the nucleus accumbens, that may play a role in amphetamine-induced behavioral or psychotomimetic effects.

In CA1-CA3 hippocampal slices, in vitro ethanol (EtOH) (10-100 mM) evoked, as a function of EtOH concentration, a differential release of aspartate (Asp) and glutamate (Glu). Omission of Ca2+ ions from the superfusion media completely abolished the EtOH-induced release of Asp but not that of Glu. In addition, at 20 mM, EtOH enhanced K(+)-evoked release only of Asp. Finally, delayed changes were observed on NMDA-evoked release of [3H]noradrenaline (NA) in the dentate gyrus (DG) after withdrawal from EtOH for 30 days.

We have evaluated the effects of the novel anti-inflammatory agent florifenine, 2-(1-Pyrrolidinyl)ethyl N-[7-(trifluoromethyl)-4-quinolyl]anthranilate, on topical inflammation in mice, free radical-mediated reactions, arachidonic acid metabolism and some neutrophil functions. Topical administration of florifenine produced dose-related anti-inflammatory activity in 12-O-tetradecanoylphorbol 13-acetate (TPA)-induced ear oedema and with a lower potency, in the response induced by arachidonic acid (AA). Florifenine also inhibited neutrophil migration and PGE2 content in the inflammed ears. In human whole blood, florifenine was a potent and selective inhibitor of TXB2 generation. This anti-inflammatory agent did not exert antioxidant effects but inhibited elastase release in human neutrophils without affecting superoxide anion generation. Florifenine administration to mice dose-dependently inhibited leukocyte migration and PGE2 levels in the air pouch inflammation induced by zymosan. These results demonstrate the topical anti-inflammatory activity of florifenine and provide a basis for understanding the mechanisms involved in the inhibitory effects of this agent on inflammatory responses.

The microdialysis technique was utilized to study the local effects of D1 and D2 family type dopamine (DA) receptor (R) ligands on the in vivo release of endogenous glutamate (GLU) and aspartate (ASP) from rat substantia nigra (SN). Addition to the dialysis perfusion solution of either D1-R and D2-R agonists, such as SKF-38393 (50 and 100 microM) and Quinpirole (5 and 10 microM), resulted in dose-dependent increases in extracellular concentrations of GLU and ASP, respectively. The SKF-38393 and Quinpirole-induced effects were reduced by SCH-23390 (0.5 microM), a D1-R antagonist, and by Spiperone (1.0 microM), a D2-R antagonist, respectively. However, SCH-23390 and Spiperone did increase GLU and ASP extracellular concentrations. Local infusion with Tetrodotoxin (TTX) (1.0 microM), a blocker of voltage-dependent Na+ channels, increased basal extracellular levels of GLU. In addition, co-infusion of TTX and SKF-38393 evoked increases in extracellular GLU levels higher than those observed after SKF-38393 alone. Finally, chemical lesions of nigral DA cells with 6-OH-DA increased the basal extracellular levels of GLU. It is proposed that the release of GLU and ASP from SN may be regulated by D1- and D2-receptors present in this basal ganglia structure. In addition, part of the D1 receptors present in SN might be located presynaptically on GLU-containing nerve endings.

To elucidate the role of mitochondrial oxidative phosphorylation in neuronal aging, we have studied the activity of the respiratory complexes in the brain of young, adult and old mice. In synaptic mitochondria, we found a significant decrease in complexes IV (29%, P < 0.001) and V (21%, P < 0.01) in old as compared with adult mice. Nonsynaptic mitochondria also showed a senescent decrease in complexes I (15%, P < 0.01), II + III (34%, P < 0.01) and IV (17%, P < 0.01) activities. These findings suggest a dysfunction in mitochondrial oxidative phosphorylation in brain aging.

It has been reported previously that N-methyl-D-aspartic acid induces a significant release of [3H]norepinephrine preaccumulated in slices from the hippocampus. In the present study, we investigated whether there are regional differences in the hippocampus regarding this N-methyl-D-aspartate effect. In the absence of Mg2+, N-methyl-D-aspartate (10-200 microM) induced the release of [3H]norepinephrine from superfused minislices containing the dentate gyrus area or the CA1-CA3 region of the hippocampus. Such N-methyl-D-aspartate effects on [3H]norepinephrine release were significantly higher in the dentate gyrus than in the CA1-CA3 area. The N-methyl-D-aspartate effects in both hippocampal areas were also reduced significantly by D-2-amino-5-phosphonovaleric acid (50 microM), an antagonist of the N-methyl-D-aspartate receptor, and by tetrodotoxin, a blocker of the voltage-dependent Na+ channels. The extent of this reduction was the same in the dentate gyrus and the CA1-CA3 area. Further experiments, conducted in the presence of Mg2+, demonstrated that N-methyl-D-aspartic acid increased K(+)-induced release of [3H]norepinephrine from dentate gyrus minislices but not from the CA1-CA3 area. The results are consistent with the existence of a higher density and/or different subtypes of N-methyl-D-aspartate receptors modulating [3H]norepinephrine release in the dentate gyrus as compared with the CA1-CA3 hippocampal area.

After 5 weeks of haloperidol, positive symptoms in drug-naive schizophrenic patients substantially subsided. Negative symptoms, although with a different temporal pattern, decreased after the fifth week of haloperidol treatment; specifically, a decrease was seen in anhedonia and affective flattening, whereas avolition-apathy and attentional impairment presented no changes. Alogia showed a decrease during the third week and a trend to return to placebo scores during weeks 4 and 5. Changes in affective flattening, alogia and attentional impairment correlated with changes in positive symptoms. During placebo, plasma homovanillic acid (HVA) correlated with negative symptoms and with changes presented by negative symptoms between the first and the fifth treatment week. These data show that negative symptoms respond differentially to neuroleptics and suggest that avolition-apathy may represent a different behavioral component of the schizophrenia process.

The microdialysis technique was utilized to study the effects of N-methyl-D-aspartate (NMDA) receptor ligands on the in vivo release of endogenous glutamate (Glu) and aspartate (Asp) from the rat striatum. Addition of NMDA (250 and 500 microM) to the dialysis perfusion solution resulted in a striking dose-dependent increase in extracellular concentrations of Glu and Asp in the striatum. The NMDA-induced effects were reduced in a dose-related way by prior perfusion with 75 microM dizocilpine (MK-801), a non-competitive NMDA receptor antagonist. MK-801, at 75 microM, produced no changes on basal levels of Glu and Asp. However, 100 microM MK-801 did increase Glu and Asp extracellular concentrations. Local infusion with 500 microM D-serine, an agonist at the glycine site associated to the NMDA receptor, significantly increased basal level of Glu, but not Asp. Such D-serine-induced effects were reduced by 7-Cl-kynurenic acid (200 microM), a selective blocker of the glycine site present in the NMDA receptor. It is proposed that activation of NMDA receptors by endogenous Glu and Asp enhances the subsequent release of these excitatory amino acids in the striatum. Part of these NMDA receptors might be located presynaptically on cortico-striatal nerve endings. In addition, postsynaptic NMDA receptors present in the striatum may also indirectly modulate the release of Glu and Asp, through trans-synaptic mechanism.

Slices of hippocampal area CA1 were employed to test the hypothesis that the release of glutamate and aspartate is regulated by the activation of excitatory amino acid autoreceptors. In the absence of added Mg2+, N-methyl-D-aspartate (NMDA)-receptor antagonists depressed the release of glutamate, aspartate, and gamma-aminobutyrate evoked by 50 mM K+. Conversely, the agonist NMDA selectively enhanced the release of aspartate. The latter action was observed, however, only when the K+ stimulus was reduced to 30 mM. Actions of the competitive antagonists 3-[(+/- )-2-carboxypiperazin-4-yl]-propyl-l-phosphonic acid (CPP) and D-2-amino-5-phosphonovalerate (D-AP5) differed, in that the addition of either 1.2 mM Mg2+ or 0.1 microM tetrodotoxin to the superfusion medium abolished the depressant effect of CPP without diminishing the effect of D-AP5. These results suggest that the activation of NMDA receptors by endogenous glutamate and aspartate enhances the subsequent release of these amino acids. The cellular mechanism may involve Ca2+ influx through presynaptic NMDA receptor channels or liberation of a diffusible neuromodulator linked to the activation of postsynaptic NMDA receptors. (RS)-alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, a selective quisqualate receptor agonist, and kainate, an agonist active at both kainate and quisqualate receptors, selectively depressed the K(+)-evoked release of aspartate. Conversely, 6-cyano-7-nitro-quinoxaline-2,3-dione, an antagonist active at both quisqualate and kainate receptors, selectively enhanced aspartate release. These results suggest that glutamate can negatively modulate the release of aspartate by activating autoreceptors of the quisqualate, and possibly also of the kainate, type. Thus, the activation of excitatory amino acid receptors has both presynaptic and postsynaptic effects.

A brain slice superfusion system and HPLC coupled to fluorometric detection were used to study depolarization-evoked release of endogenous glutamate (Glu) and aspartate (Asp) from substantia nigra (SN) slices. Veratridine (15-25 microM) and K+ (50 mM) evoked both a Ca2(+)-dependent and Ca2(+)-dependent release of Glu and Asp. However, the pattern of Glu release was different from that of Asp. In addition, cortical ablation significantly reduced the evoked release of Glu but not that of Asp. There results indicate the existence in SN of functional Glu- and Asp-releasing nerve terminals. Some of the Glu-enriched nerve terminals might belong to a cortico-nigral neuronal pathway.

1. The effects of excitatory amino acid agonists on the release of newly-synthesized [3H]dopamine was examined in slices of nucleus accumbens of the rat. 2. L-glutamate and N-methyl-D-aspartate stimulated the release of newly-synthesized [3H]dopamine, which was completely inhibited by physiological concentrations of magnesium and by the selective and non-competitive antagonist MK-801. 3. Other ligands for excitatory amino acids subtype receptors, such as AMPA and kainic acid, had no effect of newly-synthesized [3H]dopamine release. 4. Frontal cortical ablation produced a significant increase on the N-methyl-D-aspartate-stimulated release of [3H]dopamine. 5. These data suggest that dopaminergic function in the rat nucleus accumbens is modulated by N-methyl-D-aspartate receptors, the sensitivity of which is determined, at least in part, by glutamatergic and/or aspartergic afferents from the frontal cortex.

The clinical features of eight patients, four females, aged 4 to 15 years under chronic hemodialysis for terminal renal failure (creatinine clearance 10 ml.min.1,73 m2 or less) are reported. Initial diseases were Alport syndrome, systemic lupus erythematosus, chronic glomerulonephritis (n = 2), bilateral polycystic kidney, prune belly syndrome and reflux nephropathy (n = 2). Distal vascular approach by means of arteriovenous fistulas was preferred for these patients and the kinetic urea model was used to evaluate the performance of the procedure. Patients required nine to twelve hours of hemodialysis per week for optimal results. Mean weight decreases of 1 to 3 kg and reductions in blood urea nitrogen and serum potassium of 40 mg.dl and 2,5 mEq.1, respectively, were observed. The main complications of hemodialysis were the disequilibrium syndrome, infections at the site of insertion of the arteriovenous fistulae and congestive heart failure. Three patients were submitted to renal transplantation with live donors homografts: one died and the other two remain alive but under chronic hemodialysis. Five children are attending school regularly, and two of them are waiting a kidney donor for transplantation. Despite encouraging results chronic hemodialysis in children constitutes only primary supportive therapy prior to renal transplantation.

Excitatory synaptic transmission in the CNS can be modulated by endogenous substances and metabolic states that alter release of the transmitter, usually glutamate and/or aspartate. To explore this issue, we have studied the release of endogenous glutamate and aspartate from synaptic terminals of the CA3-derived Schaffer collateral, commissural and ipsilateral associational fibers in slices of hippocampal area CA1. These terminals release glutamate and aspartate in about a 5:1 ratio. The release process is modulated by adenosine, by the transmitters themselves and by nerve terminal metabolism. Adenosine inhibits the release of both amino acids by acting upon an A1 receptor. The transmitters, once released, can regulate their further release by acting upon both an NMDA and a non-NMDA (quisqualate/kainate) receptor. Activation of the NMDA receptor enhances the release of both glutamate and aspartate, whereas activation of the non-NMDA receptor depresses the release of aspartate only. Superfusion of CA1 slices with a glucose-deficient medium increases the release of both amino acids and reduces the glutamate/aspartate ratio. These results have implications for the regulation of excitatory synaptic transmission in the CA1 area and for the mechanism of hypoglycemic damage to CA1 pyramidal cells.

Recent studies have demonstrated the existence of cortico-nigral fibers in the brain which may use L-glutamic or L-aspartic acid as their transmitters. Such cortico-nigral neuronal pathway seems to functionally interact with intrinsic neuronal systems in substantia nigra (SN) by first activating different subtypes of excitatory amino acid (EAA) receptors. Particularly interesting are results showing that activation of N-methyl-D-aspartate (NMDA) subtype receptor may evoke the Ca(2+)-dependent release of dopamine from dendrites of nigro-striatal neurons. In addition, NMDA and other EAA receptors may influence the functional activity of gabaergic, glycinergic and peptidergic neurons located in SN. Acidic aminoacid-containing neurons in SN may thus play a pivotal role in determining the outflow information from SN to caudal motor-related as well as rostral limbic-related areas in the brain.

The spontaneous and depolarization-evoked release of radiolabeled D-aspartic acid, previously taken up by rat striatal slices, was studied by using a superfusion system. Veratridine (10-50 microM), electrical field stimulation (20 Hz, 1.0 V, 60 sec), and potassium (53 mM) markedly potentiated the release of D-[3H]aspartate from striatal slices. The release of L-[3H]glutamate was also increased by veratridine, according to a pattern and time course of release similar to that of D-[3H]aspartate. However, the ratio of D-[3H]aspartic acid release evoked by veratridine over spontaneous levels of release was much higher when compared to that of radiolabeled L-glutamate. Omission of calcium from the superfusion medium almost completely suppressed D-[3H]aspartate release evoked by veratridine or by electrical stimulation whereas high K+-evoked release of the [3H]amino acid was only slightly reduced. However, increasing Mg2+ concentration to 12 mM in the superfusion medium did substantially block D-[3H]aspartate release induced by K+-depolarization. Additional experiments showed that tetrodotoxin (1 microM), a blocker of voltage-dependent Na+ channels, totally abolished veratridine-evoked release of D-[3H]aspartate from striatal slices. Finally, lesion studies showed that unilateral ablation of the frontoparietal cortex was accompanied by a significant decrease in the high-affinity uptake of striatal D-[3H]aspartate and by a large and parallel loss from striatal slices in D-[3H]aspartate release evoked by either veratridine or high K+. In contrast, unilateral injection of kainic acid into the striatum did not influence depolarization-evoked release of D-[3H]aspartate from striatal slices. The findings reported suggest that D-[3H]aspartic acid may be taken up preferentially and then released, in a Ca2+-dependent manner, by veratridine and electrical stimulation from nerve terminals belonging to the cortico-striatal pathway. In addition, the results provide further support for the view that excitatory amino acids may act as neurotransmitters at the cortico-striatal nerve fibers.

The specific binding of L-[3H]-glutamic acid (GLU) was investigated in synaptic membranes from rat substantia nigra. L-[3H]-GLU binding to the membrane preparations occurred in a reversible and saturable way. The specific binding was stimulated by the presence of CaCl2 and was reduced by freezing and thawing the membranes. Scatchard analysis of the saturation isotherms yielded a non-linear plot suggesting that the binding reaction does not occur through a simple bimolecular association. Assuming non-interacting binding sites, a high (KD1, 139 nM; Bmax1, 3.5 pmoles/mg protein) and a low (KD2, 667 nM; Bmax2, 15.1 pmoles/mg protein) affinity L-[3H]-GLU binding site were obtained. The kinetics of dissociation of bound L-[3H]-GLU was biphasic; the respective dissociation rate constant (k-1) being 0.20 min-1 and 0.013 min-1. A series of amino acid receptor agonists and antagonists were tested as inhibitors of L-[3H]-GLU specific binding. Quisqualic acid, L-GLU and D-alpha-aminoadipate (D-alpha-AA) were the most potent inhibitors. DL-2-amino-4-phosphonobutyrate (APB), N-Methyl-D-aspartate (NMDA) and D-GLU were moderate inhibitors, whereas diaminopimelic acid (DAPA) and glutamate diethyl ester (GDEE) exhibited the lowest relative potency. Kainic acid (KA), gamma-aminobutyric acid (GABA) and bicuculline were not able to modify at any concentration used the specific binding of L-[3H]-GLU. These data demonstrate the presence of specific GLU binding sites in synaptic structures at substantia nigra level and support the idea that excitatory amino acids may play a role in synaptic transmission in this brain region.

The possibility was investigated that ethanol, chronically administered, might produce changes in the opioid receptors located on the sympathetic nerves of the rat vas deferens. Administration of ethanol, 3-9 g/kg per day for six days, resulted in tolerance to the hypnotic effects induced by ethanol (4 g/kg i.p.) and pentobarbital (35 mg/kg i.p.). Ethanol administration also resulted in tolerance to the effects of morphine and beta-endorphin on the muscular twitch evoked in the rat vas deferens by neuronal stimulation. In contrast, chronic ethanol did not significantly change the effects on vas deferens contractions induced by exogenous bioamines such as noradrenaline and dopamine and by acetylcholine. These results suggest that ethanol and opioids have certain biochemical mechanisms in common which might express themselves at the level of prejunctional components involved in the modulation of neurotransmitter action in the rat vas deferens.

Excitatory amino acids such as L-glutamate (Glu) and quisqualate (QUIS) markedly potentiated K+-evoked release of exogenous [3H]dopamine (DA) from rat striatal slices. Intrastriatal kainic acid injections resulted in a total disappearance of the stimulatory effects of Glu on evoked-release of [3H]DA as well as in a parallel reduction in the maximal number (Bmax) of a D-aspartate-insensitive L-[3H]Glu binding site in striatal particulate fractions. Following cortical ablation, the potentiating effect of Glu on [3H]DA release in decorticated striatal slices lasted longer, compared to normal slices, and occurred during the 2nd min following K+-depolarization. However, the extent (%) of Glu stimulation on [3H]DA release remained the same in decorticated and normal striatal slices. Cortical ablation produced also a significant decrease in the Bmax and in the KD' of the D-aspartate-insensitive binding site towards L-[3H]Glu. These results support the proposal that the D-aspartate-insensitive Glu binding site is somehow related to an amino acid receptor-mediated modulation of dopaminergic transmission in the rat corpus striatum.

The specific binding of l-[(3)H]glutamic acid to crude membrane preparations obtained from rat striatum was studied with the aid of radioligand binding techniques. Saturation isotherms and binding of 40 nM l-[(3)H]glutamate in the presence of increasing concentrations of unlabeled l-glutamate revealed an anomalous kinetic pattern which suggested the presence of three populations of l-[(3)H]glutamate affinity binding sites in fresh striatal membranes. The presence of the three binding sites became more evident in frozen membranes and in membranes prepared from striatal slices previously subjected to K(+)-depolarization; in addition, these experimental manipulations increased the specific binding of l-[(3)H]glutamate. A number of glutamate analogs were tested with the aim to discriminate among the three binding sites. Of the analogs tested, d-aspartate displaced membrane-bound l-[(3)H]glutamate from only two of the three binding sites. This finding permitted to investigate the characteristics of the d-aspartate-insensitive affinity site, independently to the other sites, by just including 1 mM d-aspartate in the binding assays. Specific binding of l-[(3)H]glutamate to the d-aspartate-insensitive site occurred in a reversible manner. A saturation curve with sigmoidal characteristic was generated when fresh, frozen and depolarized membranes were incubated with increasing concentrations of l-[(3)H]glutamate. Hill plots revealed slopes close or higher than 2 in all these membrane preparations. K(D), and Hill slopes values were found not to differ significantly among the different membrane preparations used. However, frozen membranes as well as membranes obtained from striatal slices previously depolarized by high K(+) (55 mM), showed a significant increase in B(max) as compared to fresh and non-depolarized membranes, respectively. Only l-glutamate, quisqualate and l-glutamate diethylester were relatively potent in inhibiting the specific binding of l-[(3)H]glutamate. l-Aspartate and N- methyl- d,l -aspartate competed partially and with relatively less potency whereas d-glutamate and kainate, even when tested at 0.5 mM, produced only a slight displacement of membrane-bound radioligand. The characteristics and pharmacological specificity of this binding site correlates very well with previous observations from us (Rudolph and Bustos, 1983; Rudolph et al., 1983) when studying the effects that l-glutamate and structural analogs produce upon depolarization-evoked release of [(3)H]dopamine from rat striatal slices. The proposal is made that the d-aspartate-insensitive binding site, reported in this work, might be related to a quisqualate-type receptor which becomes predominantly exposed following depolarization and might exert then a modulation of neuronal transmission in the rat striatum.

The spontaneous and veratridine-evoked release of radioactive d-aspartic acid, previously taken up by rat substantia nigra slices, was studied by using a superfusion system. Veratridine (25 ?M, 1 min) markedly produced a 14-fold increase in d-[(3)H]aspartic acid release from nigral slices. Omission of Ca(2+) and increasing Mg(2+) concentration to 12 mM in the superfusion medium did substantially block d-[(3)H]aspartate release induced by veratridine depolarization. Nevertheless, veratridine was able to evoke [(3)H]amino acid release which seemed to be, at least, 30% Ca(2+)-independent. Additional experiments showed that tetrodotoxin (0.01-0.1 ?M), a blocker of voltage-dependent Na(+) channels, totally abolished veratridine-evoked release of d-[(3)H]aspartate from nigral slices. Lesion studies were performed in order to learn about the nature of the neuronal compartment in the substantia nigra upon which veratridine-depolarization acted to induce d-[(3)H]aspartate release. Unilateral ablation of the fronto-parietal cortex was accompanied by a significant decrease in the accumulation of nigral d-[(3)H]aspartate and by a large loss from ipsilateral nigral slices in d-[(3)H]aspartate release evoked by veratridine. In contrast, both the accumulation and veratridine-evoked release of [(3)H]dopamine, remained unchanged in the ipsilateral substantia nigra slices to the lesion. The findings reported suggest that d-[(3)H]aspartic acid may be taken up and then released, in a Ca(2+)-dependent manner, by nerve terminals located in the substantia nigra. In addition, the results shown provide support to the view that l-glutamate and/or l-aspartate may act as neurotransmitters at the cortico-nigral neuronal pathway.

The specific binding of L-[3H]-glutamic acid (GLU) was examined in membrane preparations derived from heads of Drosophila melanogaster. L-[3H]-GLU bound to the membrane preparation with biphasic kinetics in a reversible and saturable way. The specific binding was inhibited by Cl- and Ca2+ ions. Saturation analysis of the data fit a model of two independent binding sites with dissociation constants (KD) of 29 nM and 249 nM and corresponding binding site densities (Bmax) of 5.7 and 24.6 pmol/mg protein. A series of excitatory amino acid receptor agonists and antagonists were tested as inhibitors of L-[3H]-GLU specific binding. Some compounds almost totally abolished the specific binding, whereas others reduced the binding to 45% over the concentration range used. Among the first type of inhibitors, L-GLU was the most potent, D-aspartate (D-ASP) and D-GLU were 10 times less effective than L-GLU, whereas quisqualic acid had the lowest potency. Among the second type of inhibitors, glutamate diethyl ester, D-alpha-amino adipate, and N-methyl-D-aspartate showed the highest potency, and DL-2-amino-4-phosphonobutyrate was the less effective. Kainic acid and gamma-amino butyric acid were not able to modify at any concentration used the specific binding of L-[3H]-GLU. These data suggest the presence of putative GLU receptors in the brain of Drosophila.

A superfusion system was used to study the effects of excitatory amino acids (EAA) on release of [3H]dopamine ([3H]DA) previously taken up by rat substantia nigra (SN) slices. The EAA tested (20-250 microM), with the exception of quisqualate and kainate, markedly evoked [3H]DA release from nigral slices when Mg2+ ions were omitted from the superfusion medium. The EAA receptor agonists exhibited the following relative potency in stimulating [3H]DA release: L-glutamate (L-Glu) greater than N-methyl-D-aspartate (NMDA) greater than NM(D,L)A greater than D-Glu much greater than quisqualate = kainate. D-2-Amino-5-phosphonovalerate (100-200 microM), an antagonist for NMDA receptors, substantially reduced [3H]DA release evoked by L-Glu or NMDA. In contrast, L-Glu diethyl ester (100-200 microM) produced a lesser blocking effect on [3H]DA release evoked by the EAA. Further experiments showed that the NMDA-mediated release of [3H]DA was totally suppressed by the omission of Ca2+ or by the addition of tetrodotoxin (0.1 microM) to the superfusion medium. In addition, strychnine, an antagonist for glycine (Gly) receptors, significantly decreased NMDA (100 microM)-evoked as well as glycine (100 microM)-evoked release of [3H]DA from nigral slices. The results shown support the idea that activation of NMDA subtype receptors in SN may trigger a Ca2+-dependent release of DA from dendrites of nigro-striatal DA-containing neurons. Furthermore, a transsynaptic mechanism that may partially involve Gly-containing interneurons is proposed to account for some of the events mediating NMDA receptor activation and DA release in SN.

A superfusion system was used to study the effects of neuroexcitatory amino acids upon spontaneous and depolarization-evoked release of exogenously taken up and newly synthesized [(3)H]dopamine by rat striatal slices. Neither l-glutamate nor other aminoacids such as l-aspartate and d-glutamate (5 x 10(?5) M) modified the spontaneous release of exogenous [(3)H]dopamine from rat striatal slices. In contrast, these neuroexcitatory aminoacids did potentiate spontaneous release of striatal [(3)H]dopamine newly synthesized from [(3)H]tyrosine. A different pattern of effects emerged when depolarization-evoked release of dopamine was studied. Only l-glutamate (5 x 10(?6)-1 x 10(?4) M) potentiated dopamine release under these experimental conditions in a rather specific and stereoselective manner. In addition, similar results were obtained regardless of whether depolarization-induced release of exogenous or newly synthesized [(3)H]dopamine was studied. The effect of l-glutamate on depolarization-induced release depended both upon the degree of neuronal depolarization and upon the presence of external Ca(2+) in the superfusion medium and it was blocked by l-glutamate diethylester. Furthermore, this effect of l-glutamate seemed quite specific with regard to regional localization within the brain as it was only demonstrated in slices from striatum and not in slices from olfactory tubercle or hippocampus. It is suggested that during depolarization a Ca(2+)-dependent event occurs at the striatal membrane level which changes the sensitivity of the dopamine release process to neuroexcitatory aminoacids in such a way as to render it relatively more specific and stereoselective towards l-glutamate stimulation. The findings reported have led us to propose that l-glutamic acid could play a role as a neuromodulator of dopaminergic transmission in the rat corpus striatum.

In an attempt to determine if alterations in intraneuronal Ca2+ may regulate tyrosine hydroxylase activity, brain slices were subjected to experimental manipulations known to increase the intraneuronal concentration of free Ca2+ ions. Incubation of either striatal or olfactory tubercle slices in a Na+-free medium for 15 min at 37 degrees resulted in a marked increase in the activity of tyrosine hydroxylase present in the 20,000 g supernatant fraction of homogenates prepared from the slices. Tyrosine hydroxylase isolated from slices previously incubated in a Na+-free, choline-enriched medium or in a Na+-free, sucrose-enriched medium exhibited maximal activities when assayed at pH 6.0 and 7.0, respectively. However, the percentage stimulation of enzyme activity induced by incubation of the slices in a Na+-free medium was maximal when the enzyme assays were performed at pH 7.0. The observed increase in enzyme activity seems to be mediated by a decrease in the apparent Km of the enzyme for pteridine cofactor, regardless of whether the kinetic enzyme analyses were conducted at pH 6.0 or 7.0, and by an increase in the Ki of the enzyme for end-product inhibitor dopamine. The apparent kinetic changes in the enzyme do not seem to result from alterations in the endogenous dopamine content of the slices, and they are independent of any increase in dopamine release that might have occurred as a response to the augmented intraneuronal Ca2+ concentration. Furthermore, the activation of tyrosine hydroxylase produced by incubating slices in a Na+-free medium is observed even in slices depleted of dopamine by pretreatment of rats with reserpine 90 min before preparation of brain slices. The activation of tyrosine hydroxylase observed under these experimental conditions does not seem to be mediated by cAMP or by a cAMP-dependent phosphorylation process. It is suggested that the changes in tyrosine hydroxylase reported are mediated primarily by a rise in the free Ca2+ concentration within the nerve tissue. These observations are consistent with the hypothesis that the kinetic activation of tyrosine hydroxylase produced after depolarization of central dopaminergic neurons may occur through a Ca2+-dependent even other than transmitter release.

In this study the authors investigated the relationship between secretory IgA levels and the appearance of a compensatory mechanism, through IgM action in allergic children.

Authors studied the importance of occult bacteremia among children, under 24 months of age, presenting fever without localizing signs. 113 patients were studied, six had positive blood cultures (5.4%). Agents identified were: N. meningitidis twice, E. coli, Staphylococcus aureus, Streptococcus viridians and group B Streptococcus. Some clinical and laboratory data (body temperature, WBC count, neutrophil count and band count), were studied as predictors of bacteremia. None of these were statistically related to presence of bacteremia.