Gonzalo Bustos

Regulation of excitatory amino acid release by N-methyl-d-aspartate receptors in rat striatum: in vivo microdialysis studies

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.

Modulation of Dendritic Release of Dopamine by N‐Methyl‐D‐Aspartate Receptors in Rat Substantia Nigra

Abstract: 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 μM), 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) > N‐methyl‐d‐aspartate (NMDA) > NM(D,L)A > d‐Glu ≫ quisqualate = kainate. d‐2‐Amino‐5‐phosphonovalerate (100–200 μ.M), an antagonist for NMDA receptors, substantially reduced [3H]DA release evoked by l‐Glu or NMDA. In contrast, l‐Glu diethyl ester (100–200 μM) 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 μM) to the superfusion medium. In addition, strychnine, an antagonist for glycine (Gly) receptors, significantly decreased NMDA (100 μM)‐evoked as well as glycine (100 μM‐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.

Noradrenaline inhibits glutamate release in the rat bed nucleus of the stria terminalis: in vivo microdialysis studies.

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 α2‐adrenergic agonist, produced a significant decrease of noradrenaline and glutamate extracellular levels. Perfusion through the probe with RX821002, a selective α2‐adrenergic antagonist, produced a significant increase of noradrenaline and glutamate basal extracellular levels. Perfusion with prazosine, a selective α1‐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 α‐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 α‐adrenergic receptor ligands exerted differential effects upon K+‐induced release of noradrenaline and glutamate. Perfusion with the nonselective α‐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 α2‐adrenergic receptors and over glutamate release mainly through α2‐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. J. Neurosci. Res. 55:311–320, 1999. 

Medullary noradrenergic neurons projecting to the bed nucleus of the stria terminalis express mRNA for the NMDA-NR1 receptor

The bed nucleus of the stria terminalis pars ventralis (vBNST) receives dense noradrenergic terminals and contains the highest concentration of noradrenaline (NA) in the brain. We used autoradiography following retrograde axonal transport of [(3)H]-NA to identify selectively whether noradrenergic neurons innervating the vBNST originate in the medulla oblongata and/or the locus coeruleus. In combination with this technique, non-isotopic in situ hybridization for the NMDA-NR1 receptor subunit mRNA was used to examine, on the same brain sections, its expression in noradrenergic neurons that innervate the vBNST. The results showed that 60 +/- 6% and 35 +/- 7% of the total number of radiolabeled cells detected after injection of [(3)H]-NA in the vBNST were located in brainstems A1 and A2 noradrenergic cell groups, respectively. In addition, 18.5 +/- 4.2% of radiolabeled cells in A1 and 15.7 +/- 5% in A2 also expressed the mRNA for the NMDA-NR1 receptor subunit. In contrast, only 4 +/- 3% of the radiolabeled cells were present in the locus coeruleus, and none of these cells was positive to NMDA-NR1 receptor subunit mRNA. The present results provide evidence that BNST noradrenergic fibers and terminals originate predominantly from A1 and A2 noradrenergic cell groups, and that a significant number of these noradrenergic neurons also express the mRNA for the NMDA-NR1 receptor subunit. The observation that brainstem noradrenergic neurons innervating the vBNST express NMDA receptor mRNA gives anatomical support to the regulation of NA release by NMDA presynaptic receptors.

Regulation of norepinephrine release from the rat bed nucleus of the stria terminalis: In vivo microdialysis studies

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/μl of norepinephrine was observed. Desipramine (2 and 10 μM), a norepinephrine uptake blocker, significantly increased extracellular levels of norepinephrine. Reversed perfusion with high potassium in the presence of 2 μM desipramine induced the release of norepinephrine. Instead, in the presence of 10 μM desipramine, a significant decrease in the induced release of norepinephrine was observed. Clonidine, an α2‐adrenergic agonist, significantly decreased basal extracellular levels of norepinephrine and the K+‐induced release of norepinephrine. In contrast, yohimbine and RX821002, two α2‐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 α2‐adrenergic receptors. J. Neurosci. Res. 50:1040–1046, 1997. © 1997 Wiley‐Liss, Inc.

Differential regulation of glutamate, aspartate and γ-amino-butyrate release by N-methyl-d-aspartate receptors in rat striatum after partial and extensive lesions to the nigro-striatal dopamine pathway

The in vivo microdialysis methodology was used to assess the effect of N-methyl-D-aspartate (NMDA) receptor ligands on glutamate (GLU), aspartate (ASP) and gamma-aminobutyrate (GABA) extracellular levels in the striatum of anaesthetized rats, after damage to the dopamine (DA) nigrostriatal pathway by injections of different doses of 6-hydroxydopamine (6-OH-DA) seven days earlier. The 6-OH-DA treated rats were divided into two groups, corresponding to animals with 20-80% (partial) and 85-99% (extensive) striatal DA tissue depletion, respectively. In rats with partial DA depletion, the striatal extracellular ASP levels significantly increased after intrastriatal dialysis perfusion with MK-801 (100 microM), an antagonist of NMDA receptors. In addition, a change in the pattern of local NMDA (500 microM)- induced efflux of ASP was observed in the striatum of these rats. However, in these partially DA-depleted striata no changes were found in basal extracellular levels of GLU, ASP and GABA or in NMDA- and MK-801-mediated effluxes of GLU and GABA relative to striata from sham rats. In contrast, rats with extensive striatal DA depletion exhibited a significant increase in ASP and GABA extracellular striatal levels, after intrastriatal dialysis perfusion with NMDA. In addition, the MK-801-mediated stimulation of extracellular ASP levels was accentuated along with the appearance of a MK-801 mediated increase in extracellular striatal GLU. Finally, basal extracellular levels of ASP, but not of GLU and GABA, were found to increase in extensive DA-depleted striata when compared to sham and partially DA-depleted striata. Thus, a differential regulation of basal and NMDA receptor-mediated release of transmitter amino acids occur seven days after partial and extensive DA-depleted striatum by 6-OH-DA-induced lesions of the nigrostriatal DA pathway. These findings may have implications as regards the participation of NMDA receptors in the compensatory mechanisms associated with the progress of Parkinsons disease, as well as in the treatment of this neurological disorder.

Regulation of endogenous noradrenaline release from the bed nucleus of stria terminalis

The bed nucleus of stria terminalis (BNST) contains the highest concentration of noradrenaline (NA) in the brain. Minislices of the ventral portion of the bed nucleus of stria terminalis (vBNST) were used to study the release of endogenous NA. High K+ induced a Ca(2+)-dependent and reserpine-sensitive release of NA. Clonidine (1 microM), an alpha 2-noradrenergic receptor agonist, significantly decreased K(+)-induced release of NA, whereas yohimbine (1 microM), an alpha 2-noradrenergic antagonist, increased this release. N-Methyl-D-aspartate (NMDA), a specific agonist of NMDA-type glutamate receptors, evoked the release of NA from vBNST minislices. In the presence of D-serine (10 microM), an agonist at the glycine site associated with the NMDA receptor, the NMDA effect was significantly higher. Glycine (1 microM) also increased NA release evoked by NMDA. However, glycine exhibited a significant effect by itself, suggesting the existence of strychnine-sensitive glycine receptors in vBNST. Endogenous NA release induced by 40 mM K+ and NMDA was not additive. Thus, vBNST minislices seem to be a good model to study the release of endogenous NA in the CNS. Such NA release in the vBNST is regulated by alpha 2-noradrenergic receptors and by glutamate through NMDA receptors.

NMDA-NR1 receptor subunit mRNA expression in rat brain after 6-OH-dopamine induced lesions: A non-isotopic in situ hybridization study

Antisense digoxigenin‐labeled deoxyoligonucleotides probes and non‐isotopic in situ hybridization (HIS) techniques have been used to explore the NMDA‐NR1 receptor subunit mRNA distribution in different brain areas of rats which had their dopaminergic nigrostriatal pathway previously lesioned with intracerebral administration of 6‐OH‐dopamine (6‐OH‐DA). Intense and significant hybridization signals for NR1 mRNA were found in dentate gyrus and regions CA1‐CA2‐CA3 of the hippocampus, in layers II‐III and V‐VI of the cerebral cortex, and in the cerebellum of sham‐treated rats. Basal ganglia structures such as the striatum exhibited few NR1 mRNA hybridization signals as compared to the hippocampus and cerebral cortex. In contrast, both zona compacta and reticulata of substantia nigra (SN) showed a reduced number of cells with nevertheless intense NR1 mRNA HIS signals. The NR1 mRNA distribution in the brain was affected in a brain regional selective manner by 6‐OH‐DA induced lesions of DA neuronal systems. A striking increase in NR1 mRNA HIS signals was observed in both striata after unilateral lesioning with 6‐OH‐DA. Instead, in SN compacta but not in reticulata, a moderate but significant bilateral reduction of NR1 mRNA was observed after unilateral 6‐OH‐DA injection. No significant changes in NR1 mRNA were detected in cerebral cortex and other brain regions after 6‐OH‐DA treatment. These studies, and others reported in the literature, support the view that extensive lesions of nigrostriatal DA‐containing neurons in the brain may trigger compensatory or adaptative responses in basal ganglia structures such as striatum and substantia nigra which involve glutamatergic neurons and the genic expression of NMDA receptors.

Effects of d-amphetamine administration on the release of endogenous excitatory amino acids in the rat nucleus accumbens

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.

Effects of in vitro ethanol and chronic ethanol consumption on the release of excitatory amino acids in the rat hippocampus

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.