Jorge Abarca

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.

Changes in extracellular levels of glutamate and aspartate in rat substantia nigra induced by dopamine receptor ligands: In vivo microdialysis studies

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 μM) and Quinpirole (5 and 10 μM), 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 μM), a D1-R antagonist, and by Spiperone (1.0 μM), a D2-R antagonist, respectively. However, SCH-23390 and Spiperone did increase GLU and ASP extracellular concentrations. Local infusion with Tetrodotoxin (TTX) (1.0 μM), 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.

Transient increase of brain derived neurotrophic factor mRNA expression in substantia nigra reticulata after partial lesion of the nigrostriatal dopaminergic pathway.

By using non-isostopic in situ hybridization we have demonstrated a transient increase of BDNF mRNA in the lateral subregion of the substantia nigra pars reticulata 1 week after intrastriatal application of 6-OH-DA. These changes correlate with a partial reduction of dopamine (DA) content in the striatum but with a normal tyrosine hydroxylase immunoreactivity in substantia nigra pars compacta. Our data suggest that non-DA, BDNF expressing cells in substantia nigra pars reticulata may play a role in neuronal protection after partial lesions of the DA nigrostriatal pathway.

NMDA receptors mediate an early up-regulation of brain-derived neurotrophic factor expression in substantia nigra in a rat model of presymptomatic Parkinson's disease

The clinical symptoms of Parkinsons disease (PD) appear late and only when the degenerative process at the level of the nigrostriatal dopamine (DA) pathway is quite advanced. An increase in brain‐derived neurotrophic factor (BDNF) expression may be one of the molecular signals associated to compensatory and plastic responses occurring in basal ganglia during presymptomatic PD. In the present study, we used in vivo microdialysis, semiquantitative reverse transcriptase–polymerase chain reaction, and immunohistochemistry to study N‐methyl‐D‐aspartic acid (NMDA) receptor regulation of BDNF expression in substantia nigra (SN) of adult rats after partial lesioning of the nigrostriatal DA pathway with unilateral striatal injections of 6‐hydroxydopamine (6‐OHDA). A time‐dependent partial decrease of striatal DA tissue content as well as parallel and gradual increases in extracellular glutamate and aspartate levels in SN were found 1 to 7 days after unilateral 6‐OHDA intrastriatal injection. Instead, the number of tyrosine hydroxylase–immunoreactive (IR) cells in the ipsilateral SN pars compacta remained statistically unchanged after neurotoxin injection. Intrastriatal administration of 6‐OHDA also produced an early and transient augmentation of pan‐BDNF, exon II–BDNF, and exon III–BDNF transcripts in the ipsilateral SN. The pan‐BDNF and exon II–BDNF transcript increases were completely abolished by the prior systemic administration of MK‐801, a selective antagonist of NMDA receptors. MK‐801 also blocked the increase in BDNF‐IR cells in SN observed 7 days after unilateral 6‐OHDA intrastriatal injections. Our findings suggest that a coupling between glutamate release, NMDA receptor activation, and BDNF expression may exist in the adult SN and represent an important signal in this midbrain nucleus triggered in response to partial DA loss occurring in striatal nerve endings during presymptomatic PD.

Release of d-[3H]aspartic acid from the rat substantia nigra: effect of veratridine-evoked depolarization and cortical ablation

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.

Release of d-[3H]aspartic acid from the rat striatum: Effect of veratridine-evoked depolarization, fronto-parietal cortex ablation, and striatal lesions with kainic acid

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.

An NR2B-Dependent Decrease in the Expression of trkB Receptors Precedes the Disappearance of Dopaminergic Cells in Substantia Nigra in a Rat Model of Presymptomatic Parkinson's Disease

Compensatory changes occurring during presymptomatic stages of Parkinsons disease (PD) would explain that the clinical symptoms of the disease appear late, when the degenerative process is quite advanced. Several data support the proposition that brain-derived neurotrophic factor (BDNF) could play a role in these plastic changes. In the present study, we evaluated the expression of the specific BDNF receptor, trkB, in a rat model of presymptomatic PD generated by intrastriatal injection of the neurotoxin 6-OHDA. Immunohistochemical studies revealed a decrease in trkB expression in SN pars compacta (SNc) seven days after 6-OHDA injection. At this time point, no change in the number of tyrosine hydroxylase (TH) immunoreactive (TH-IR) cells is detected, although a decrease is evident 14 days after neurotoxin injection. The decrease in TH-positive cells and trkB expression in SNc was significantly prevented by systemic administration of Ifenprodil, a specific antagonist of NR2B-containing NMDA receptors. Therefore, an NR2B-NMDA receptor-dependent decrease in trkB expression precedes the disappearance of TH-IR cells in SNc in response to 6-OHDA injection. These results support the idea that a functional coupling between NMDA receptors and BDNF/trkB signalling may be important for the maintenance of the dopaminergic phenotype in SNc during presymptomatic stages of PD.

Uptake and release of manganese by rat striatal slices

Abstract Accumulation of manganese in rat corpus striatum slices was found to be non-saturable, although relatively strongly temperature dependent, and inhibited by 2,4-dinitrophenol (2,4-DNP). Once incorporated, the metal ion was released by K + (55 mM) depolarization in the presence of calcium ions, following a time course of efflux parallel to that of [ 3 H]dopamine ([ 3 H]DA). The release of the metal ion, however, was not induced by tyramine. [ 3 H]DA release was also induced by low concentrations of manganese chloride. The possibility exists that these findings may be related in some way to the functional deficiency of the nigro-striatal dopaminergic system found after manganese poisoning.

Exposure to repeated immobilization stress inhibits cocaine-induced increase in dopamine extracellular levels in the rat ventral tegmental area

A higher vulnerability to drug abuse has been observed in human studies of individuals exposed to chronic or persistent stress, as well as in animal models of drug abuse. Here, we explored the effect of repeated immobilization stress on cocaine-induced increase in dopamine extracellular levels in VTA and its regulation by corticotropin-releasing factor (CRF) and GABA systems. Cocaine (10mg/Kg i.p.) induced an increase of VTA DA extracellular levels in control rats. However, this effect was not observed in repeated stress rats. Considering the evidence relating stress with CRF, we decided to perfuse CRF and CP-154526 (selective antagonist of CRF1 receptor) in the VTA of control and repeated stress rats, respectively. We observed that perfusion of 20μM CRF inhibited the increase of VTA DA extracellular levels induced by cocaine in control rats. Interestingly, we observed that in the presence of 10μM CP-154526, cocaine induced a significant increase of VTA DA extracellular levels in repeated stress rats. Regarding the role of VTA GABA neurotransmission, cocaine administration induced a significant increase in VTA GABA extracellular levels only in repeated stress rats. Consistently, cocaine was able to increase VTA DA extracellular levels in repeated stress rats when 100μM bicuculline, an antagonist of GABAA receptor, was perfused intra VTA. Thus, both CRF and GABA systems are involved in the lack of response to cocaine in the VTA of repeated stress rats. It is tempting to suggest that the loss of response in VTA dopaminergic neurons to cocaine, after repeated stress, is due to an interaction between CRF and GABA systems.

4-Methylthioamphetamine Increases Dopamine in the Rat Striatum and has Rewarding Effects In Vivo

4‐Methylthioamphetamine (MTA) is a phenylisopropylamine derivative whose use has been associated with severe intoxications. MTA is usually regarded as a selective serotonin‐releasing agent. Nevertheless, previous data have suggested that its mechanism of action probably involves a catecholaminergic component. As little is known about dopaminergic effects of this drug, in this work the actions of MTA upon the dopamine (DA) transporter (DAT) were studied in vitro, in vivo and in silico. Also, the possible abuse liability of MTA was behaviourally assessed. MTA exhibited an in vitro affinity for the rat DAT in the low micromolar range (6.01 μM) and induced a significant, dose‐dependent increase in striatal DA. MTA significantly increased c‐Fos‐positive cells in striatum and nucleus accumbens, induced conditioned place preference and increased locomotor activity. Docking experiments were performed in a homology model of the DAT. In conclusion, our results show that MTA is able to increase extracellular striatal DA levels and that its administration has rewarding properties. These effects were observed at concentrations or doses that can be relevant to its use in human beings.