Claudia Corsi

Striatal Outflow of Adenosine, Excitatory Amino Acids, γ-Aminobutyric Acid, and Taurine in Awake Freely Moving Rats After Middle Cerebral Artery Occlusion Correlations With Neurological Deficit and Histopathological Damage

BACKGROUND AND PURPOSE While a number of studies have investigated transmitter outflow in anesthetized animals after middle cerebral artery occlusion (MCAO) performed by craniectomy, studies have never been performed after MCAO induced by intraluminal filament. In addition, it has been reported that after MCAO, infarct volume correlates with functional outcome and with transmitter outflow, although there are no studies that demonstrate a direct correlation between transmitter outflow and functional outcome. The purpose of the present study was to assess excitatory amino acids, gamma-aminobutyric acid, taurine, and adenosine outflow in awake rats after intraluminal MCAO and to determine whether, in the same animal, outflow was correlated with neurological outcome and histological damage. METHODS Vertical microdialysis probes were placed in the striatum of male Wistar rats. After 24 hours, permanent MCAO was induced by the intraluminal suture technique. The transmitter concentrations in the dialysate were determined by high-performance liquid chromatography. Twenty-four hours after MCAO, neurological deficit and histological outcome were evaluated. RESULTS All transmitters significantly increased after MCAO. Twenty-four hours after MCAO, the rats showed a severe sensorimotor deficit and massive ischemic damage in the striatum and in the cortex (9+/-2% and 25+/-6% of hemispheric volume, respectively). Significant correlations were found between the efflux of all transmitters, neurological score, and striatal infarct volume. CONCLUSIONS In this study, for the first time, amino acid and adenosine extracellular concentrations during MCAO by the intraluminal suture technique were determined in awake and freely moving rats, and a significant correlation was found between transmitter outflow and neurological deficit. The evaluation of neurological deficit, histological damage, and transmitter outflow in the same animal may represent a useful approach for studying neuroprotective properties of new drugs/agents against focal ischemia.Background and Purpose—While a number of studies have investigated transmitter outflow in anesthetized animals after middle cerebral artery occlusion (MCAO) performed by craniectomy, studies have never been performed after MCAO induced by intraluminal filament. In addition, it has been reported that after MCAO, infarct volume correlates with functional outcome and with transmitter outflow, although there are no studies that demonstrate a direct correlation between transmitter outflow and functional outcome. The purpose of the present study was to assess excitatory amino acids, γ-aminobutyric acid, taurine, and adenosine outflow in awake rats after intraluminal MCAO and to determine whether, in the same animal, outflow was correlated with neurological outcome and histological damage. Methods—Vertical microdialysis probes were placed in the striatum of male Wistar rats. After 24 hours, permanent MCAO was induced by the intraluminal suture technique. The transmitter concentrations in the dialysate were deter...

Adenosine Extracellular Brain Concentrations and Role of A2A Receptors in Ischemia

Abstract: Various experimental approaches have been used to determine the concentration of adenosine in extracellular brain fluid. The cortical cup technique or the microdialysis technique, when adenosine concentrations are evaluated 24 hours after implantation of the microdialysis probe, are able to measure adenosine in the nM range under normoxic conditions and in the μM range under ischemia. In vitro estimation of adenosine show that it can reach 30 μM at the receptor level during ischemia, a concentration able to stimulate all adenosine receptor subtypes so far identified. Although the protective role of A1 receptors in ischemia seems consistent, the protective role of A2A receptors appears to be controversial. Both A2A agonists and antagonists have been shown to be neuroprotective in various in vivo ischemia models. Although A2A agonists may be protective, mainly through peripherally mediated effects, A2A antagonists may be protective through local brain mediated effects. It is possible that A2A receptors are tonically activated following a prolonged increase of adenosine concentration, such as occurs during ischemia. A2A receptor activation desensitizes A1 receptors and reduces A1 mediated effects. Under these conditions A2A receptor antagonists may be protective by potentiating all the neuroprotective A1 mediated effects, including decreased neurotoxicity due to reduced ischemia induced glutamate outflow.

Striatal A2A adenosine receptors differentially regulate spontaneous and K+-evoked glutamate release in vivo in young and aged rats

The effect of the adenosine A2A receptor agonist CGS 21680 on glutamate and aspartate release was investigated in the striatum of young and old rats by microdialysis experiments. CGS 21680 (10 microM) significantly increased glutamate and aspartate spontaneous outflow in young but not in old rats. On the contrary, CGS 21680 induced the same decrease in K+-evoked glutamate outflow in both young and aged rats. A lower dose of CGS 21680 (1 microM) failed to modify either spontaneous or K+-evoked outflow. It is suggested that the opposite effects of the A2A agonist on excitatory amino acid outflow may be respectively mediated by striatal A2A adenosine receptors located on glutamatergic terminals and on the striatal indirect output pathway.

Modification of adenosine extracellular levels and adenosine A2A receptor mRNA by dopamine denervation

Adenosine A(2A) receptor antagonists have been proposed as an effective therapy in the treatment of Parkinsons disease. To explore the possibility that dopamine denervation may produce modifications in adenosine A(2A) transmission, we measured the extracellular concentration of adenosine and adenosine A(2A) receptor mRNA in the striatum of rats infused unilaterally with 6-hydroxydopamine in the medial forebrain bundle. Fifteen days after 6-hydroxydopamine infusion, extracellular adenosine levels, measured by in vivo microdialysis, were significantly lower (-35%) in the dopamine-denervated striatum. At the time of the decrease in adenosine levels, an increase in striatal adenosine A(2A) receptor mRNA levels (+20%), measured by in situ hybridization, was observed. Modifications in adenosine A(2A) transmission, following nigrostriatal dopamine neuron degeneration, establish a potential neural basis for the effectiveness of adenosine A(2A) receptor antagonists in the treatment of Parkinsons disease.

The source of brain adenosine outflow during ischemia and electrical stimulation

Adenosine outflow and adenosine and adenine nucleotide content of hippocampal slices were evaluated under two different experimental conditions: ischemia-like conditions and electrical stimulation (10 Hz). Five minutes of ischemia-like conditions brought about an 8-fold increase in adenosine outflow in the following 5 min during reperfusion, and a 2-fold increase in adenosine content, a 43% decrease in ATP, a 72% increase in AMP and a 30% decrease in energy charge (EC) at the end of the ischemic period. After 10 min of reperfusion ATP, AMP and EC returned to control values, while the adenosine content was further increased. Five minutes of electrical stimulation brought about an 8-fold increase in adenosine outflow that peaked 5 min after the end of stimulation, a 4-fold increase in adenosine content and an 18% decrease in tissue EC at the end of stimulation. After 10 min of rest conditions the adenosine content and EC returned to basal values. The origin of extracellular adenosine from S-adenosylhomocysteine (SAH) was examined under the two different experimental conditions. The SAH hydrolase inhibitor, adenosine-2,3-dialdehyde (10 microM), does not significantly modify the adenosine outflow evoked by electrical stimulation or ischemia-like conditions. This finding excludes a significant contribution by the transmethylation pathway to adenosine extracellular accumulation evoked by an electrical or ischemic stimulus, and confirms that the most likely source of adenosine is from AMP dephosphorylation.

Striatal A2A adenosine receptor antagonism differentially modifies striatal glutamate outflow in vivo in young and aged rats.

The effect of the adenosine A2A receptor antagonist SCH 58261 on glutamate release was investigated in the striatum of young and old rats by microdialysis experiments. SCH 58261 (50 nM) significantly decreased the spontaneous and K+-evoked glutamate outflow in young rats. In aged rats, spontaneous glutamate outflow was significantly reduced in comparison to young rats and SCH 58261 significantly increased spontaneous and K+-evoked glutamate outflow. It is suggested that the opposite effects of the A2A antagonist on glutamate outflow in young and aged rats can be respectively attributed to blockade of striatal A2A adenosine receptors located on glutamatergic terminals and on the striatal indirect output pathway.

Regulation of extracellular adenosine levels in the striatum of aging rats

Extracellular adenosine concentrations, evaluated by microdialysis in the striatum of young and aged rats, were 66.8 +/- 0.7 and 71.6 +/- 1.0 nM, respectively. The adenosine deaminase inhibitor EHNA (100 microM) increased the extracellular adenosine levels in young rats only. The adenosine kinase inhibitor iodotubercidin (10 microM) brought about the same increase in young and aged rats. In aged rats the resting adenosine outflow was reduced by the N-methyl-D-aspartate (NMDA) receptor antagonist D-(-)-2-amino-7-phophonoheptanoic acid (D-AP7) (1 mM). It is concluded that extracellular levels of adenosine in the striatum are not affected by age, irrespective of the differences in adenosine deaminase activity and that the release of excitatory amino acids is responsible for much of resting adenosine outflow in aged but not in young rats.

Adenosine A2A receptor antagonism increases striatal glutamate outflow in dopamine-denervated rats.

The objective of the work was to study, by in vivo microdialysis, the effect of the adenosine A(2A) receptor antagonist 7-(2-phenylethyl)-5-amino-2-(2-furyl)-pyrazolo-[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine (SCH 58261) on glutamate outflow in the striata of unilateral 6-hydroxydopamine-infused rats. Two vertical microdialysis probes were implanted bilaterally in both the denervated striatum and in the intact striatum. Glutamate concentrations in the dialysate were determined by high-performance liquid chromatography (HPLC). Infusion of the adenosine A(2A) receptor antagonist SCH 58261 (50 nM), through the microdialysis fiber, significantly increased glutamate outflow from the denervated striatum while it decreased glutamate outflow from the intact striatum. The opposite effects of SCH 58261 on glutamate outflow in the intact and 6-hydroxydopamine-lesioned striatum might be attributed to blockade of striatal adenosine A(2A) receptors located on either striatal indirect output pathways or glutamatergic terminals. These results may be relevant to our understanding of the mechanism of action of adenosine A(2A) receptor antagonists in Parkinsons disease.

In vivo regulation of extracellular adenosine levels in the cerebral cortex by NMDA and muscarinic receptors

The adenosine concentration in samples of perfusate was determined 24 h after implantation of microdialysis fibre in the cortex. High performance liquid chromatography coupled with a fluorometric detector was used. K+ (100 mM) depolarization was followed by a 2- to 4-fold increase in adenosine efflux. The addition of tetrodotoxin (1 microM) to the perfusate was followed by a decrease in spontaneous and K(+)-evoked adenosine efflux. The increase induced by high K+ was markedly inhibited by the NMDA receptor antagonist, D(-)-2-amino-7-phosphonoheptanoic acid (1 mM, D-AP7), but not by the muscarinic receptor antagonist, atropine (1.5 microM). The acetylcholine esterase inhibitor, physostigmine (7 microM), and the muscarinic receptor agonist, oxotremorine (100 microM), significantly enhanced the K(+)-evoked increase in adenosine. The spontaneous efflux of adenosine was not modified by any of the drugs tested. A neurotoxic lesion of the cholinergic pathway innervating the cortex, although inducing a marked decrease in cortical choline acetyltransferase activity, did not significantly modify the cortical adenosine efflux. It is concluded that, under K(+)-depolarizing conditions, adenosine efflux is triggered by excitatory amino acids and enhanced by muscarinic activation.

Effect of N-methyl-d-aspartate on motor activity and in vivo adenosine striatal outflow in the rat

It has been previously found that the systemic administration of low doses of N-methyl-D-aspartate (NMDA) in mice induces motor depression. The effects of the systemic administration of different doses of NMDA (10, 30 and 60 mg/kg s.c.) on the motor activity and on the in vivo extracellular levels of adenosine in the striatum was studied in Sprague-Dawley rats. The adenosine concentration in samples of perfusate was determined 24 h after implantation of a transverse microdialysis probe. At 30 and 60 mg/kg, but not 10 mg/kg, NMDA induced both a significant motor depression (motility and rearing) and a significant increase in the striatal extracellular levels of adenosine. Both the motor depression and the changes in the extracellular levels of adenosine were only evident during the first 30 min after NMDA administration. The non-competitive NMDA receptor antagonist MK-801 (0.1 mg/kg s.c.) completely counteracted the effects of NMDA (30 mg/kg s.c.) on motor activity (motility) and on the striatal extracellular levels of adenosine. The correlation between the behavioural and the biochemical data strongly support the hypothesis that adenosine release in the striatum is a main mechanism responsible for the motor depressant effects produced by the systemic administration of NMDA.