María Inés Forray

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.

Chronic Morphine Treatment and Withdrawal Increase Extracellular Levels of Norepinephrine in the Rat Bed Nucleus of the Stria Terminalis

Extracellular levels of norepinephrine (NE) and glutamate (Glu) in the ventral bed nucleus of the stria terminalis (vBNST) of saline‐ and chronic morphine‐treated rats, with or without withdrawal, were studied by means of the in vivo microdialysis technique in anesthetized rats. In addition, the tissue concentration of NE was studied at different rostrocaudal levels of the vBNST. Chronic morphine treatment significantly increased extracellular levels of NE, but not Glu, in vBNST. At 48 h after naloxone‐induced morphine withdrawal there was a further significant increase in the extracellular levels of NE, but not Glu, in vBNST. The presence of UK 14304, an α2‐adrenergic agonist, induced a significant decrease in NE extracellular levels in all experimental groups. In contrast, UK 14304 induced a significant decrease in Glu extracellular levels only in saline‐treated rats. The results also show that the vBNST presents a rostrocaudal gradient of NE and contains 9.4% of total brain NE. The increase in NE extracellular levels in vBNST induced by chronic morphine treatment and the further increase in NE levels 48 h after naloxone‐induced morphine withdrawal suggest that NE in vBNST may be involved in the pharmacological effects of chronic morphine and withdrawal.

Research reportRegulation 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.

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.

Acute morphine administration increases extracellular DA levels in the rat lateral septum by decreasing the GABAergic inhibitory tone in the ventral tegmental area

We studied the effect of an acute systemic administration of morphine and of a local intra‐ventral tegmental area (VTA) infusion of the same drug on extracellular levels of dopamine (DA) in the lateral septum (LS) by in vivo microdialysis in anesthetized rats. The extracellular levels of 5‐hydroxytryptamine (5‐HT) were also measured in all dialysate samples. The acute systemic administration of morphine dose‐dependently increased extracellular levels of DA but not of 5‐HT in the LS, in the absence or presence of fluoxetine. This morphine effect was antagonized by the previous administration of naloxone, a specific opioid antagonist. The local infusion of morphine in the VTA also induced a significant increase of the extracellular levels of DA in the LS, concomitantly with a decrease of γ‐aminobutyric acid (GABA) extracellular levels in the VTA itself. Intriguingly, the LS extracellular levels of DA returned to basal values before the VTA GABA extracellular levels recovered. Our results show for the first time that an acute administration of morphine increases DA extracellular levels in the LS. The results also suggest that DA cells in the VTA and innervating the LS are under an inhibitory GABAergic tone sensitive to morphine. Taken together, our neurochemical data and previous studies involving LS DA in stress‐related behavior support the hypothesis that DA in the LS plays a significant role in addictive behavior. The participation of LS DA and 5‐HT systems in stress‐induced relapse to drug seeking should be studied further.

Estrogen Receptors α and β Differentially Regulate the Transcriptional Activity of the Urocortin Gene

Urocortin (Ucn), a highly conserved metazoan gene, is related to stress and feeding, behaviors with significant gender differences. We investigated whether estrogens regulate the expression of the Ucn gene using transient transfection in PC12 cells with the human Ucn (hUcn) promoter coupled to luciferase and either α or β estrogen receptors (ERα or ERβ, respectively). The results demonstrate that estradiol (E2) increases the activity of the hUcn promoter via ERα, and decreases hUcn promoter activity through ERβ. Deletions of the hUcn promoter show that the increase in promoter activity mediated by E2-ERα depends on a promoter region containing a half-estrogen response element and an Sp1 site, and the decrease mediated by E2-ERβ depends on a proximal promoter region containing a cAMP response element. Ucn and ERs coexist in neurons of rat hypothalamic nuclei, giving anatomical support for a direct effect of estrogen receptors on the Ucn gene. By in situ hybridization, we observed that cycling female rats have a higher number of cells expressing Ucn mRNA than males in the paraventricular nucleus of the hypothalamus (PVN) and the septum. Both of these brain nuclei are related to stress behaviors and express moderate levels of Ucn. Furthermore, Ucn mRNA was significantly decreased in the PVN and increased in the septum 30 d after ovariectomy. Acute E2 administration to ovariectomized rats significantly increased Ucn mRNA expression in the PVN and septum. In conclusion, our in vitro and in vivo evidence suggests that estrogens exert a direct and differential transcriptional regulation of the Ucn gene.

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.

Adrenalectomy decreases corticotropin-releasing hormone gene expression and increases noradrenaline and dopamine extracellular levels in the rat lateral bed nucleus of the stria terminalis

The bed nucleus of the stria terminalis (BNST) has a high density of corticotropin‐releasing hormone (CRH)‐containing neurons that are significantly innervated by noradrenergic and dopaminergic nerve terminals. This limbic structure is involved in the extrahypothalamic response to stress. The purpose of the present work is to study whether the absence of glucocorticoids, induced by a long‐term adrenalectomy, regulates CRH gene expression and noradrenaline and dopamine extracellular levels in the rat BNST. The results showed that adrenalectomy decreases CRH mRNA in the dorsal lateral BNST but not in the ventral lateral BNST. Adrenalectomy also decreases CRH‐like immunoreactivity both in BNST subnuclei and in the central nucleus of the amygdala. In addition, adrenalectomy significantly increases noradrenaline and dopamine extracellular levels in the lateral BNST. The present results suggest that adrenalectomy regulates CRH gene expression and noradrenaline and dopamine extracellular levels in the BNST in an opposite way. Thus, the present study adds novel evidence further supporting that the BNST and the central nucleus of the amygdala form part of an adrenal steroid‐sensitive extrahypothalamic circuit that has been involved in fear and anxiety responses and in clinical syndromes such as melancholic depression, posttraumatic stress disorders, and addiction.