Quinolinic acid induced neurodegeneration in the striatum: a combined in vivo and in vitro analysis of receptor changes and microglia activation.

PURPOSE: Huntington's disease (HD) is a progressive neurodegenerative disorder, which is characterised by prominent neuronal cell loss in the basal ganglia with motor and cognitive disturbances. One of the most well-studied pharmacological models of HD is produced by local injection in the rat brain striatum of the excitotoxin quinolinic acid (QA), which produces many of the distinctive features of this human neurodegenerative disorder. Here, we report a detailed analysis, obtained both in vivo and in vitro of this pharmacological model of HD. MATERIALS AND METHODS: By combining emission tomography (PET) with autoradiographic and immunocytochemical confocal laser techniques, we quantified in the QA-injected striatum the temporal behavior (from 1 to 60 days from the excitotoxic insult) of neuronal cell density and receptor availability (adenosine A(2A) and dopamine D(2) receptors) together with the degree of microglia activation. RESULTS: Both approaches showed a loss of adenosine A(2A) and dopamine D(2) receptors paralleled by an increase of microglial activation. CONCLUSION: This combined longitudinal analysis of the disease progression, which suggested an impairment of neurotransmission, neuronal integrity and a reversible activation of brain inflammatory processes, might represent a more quantitative approach to compare the differential effects of treatments in slowing down or reversing HD in rodent models with potential applications to human patients.

Adenosine A(2A) receptors modulate BDNF both in normal conditions and in experimental models of Huntington's disease.

Brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family, enhances synaptic transmission and regulates neuronal proliferation and survival. Functional interactions between adenosine A(2A) receptors (A(2A)Rs) and BDNF have been recently reported. In this article, we report some recent findings from our group showing that A(2A)Rs regulate both BDNF functions and levels in the brain. Whereas BDNF (10 ng/ml) increased the slope of excitatory postsynaptic field potentials (fEPSPs) in hippocampal slices from wild-type (WT) mice, it was completely ineffective in slices taken from A(2A)R knock-out (KO) mice. Furthermore, enzyme immunoassay studies showed a significant reduction in hippocampal BDNF levels in A(2A)R KO vs. WT mice. Having found an even marked reduction in the striatum of A(2A)R KO mice, and as both BDNF and A(2A)Rs have been implicated in the pathogenesis of Huntington's disease (HD), an inherited striatal neurodegenerative disease, we then evaluated whether the pharmacological blockade of A(2A)Rs could influence striatal levels of BDNF in an experimental model of HD-like striatal degeneration (quinolinic acid-lesioned rats) and in a transgenic mice model of HD (R6/2 mice). In both QA-lesioned rats and early symptomatic R6/2 mice (8 weeks), the systemic administration of the A(2A)R antagonist SCH58261 significantly reduced striatal BDNF levels. These results indicate that the presence and the tonic activation of A(2A)Rs are necessary to allow BDNF-induced potentiation of synaptic transmission and to sustain a normal BDNF tone. The possible functional consequences of reducing striatal BDNF levels in HD models need further investigation.

The cannabinoid receptor agonist WIN 55,212-2 attenuates the effects induced by quinolinic acid in the rat striatum.

The ability of CB(1) receptors to regulate the release of glutamate in the striatum, together with the finding that, in experimental models of Huntington disease (HD), both endocannabinoid levels and CB(1) receptor densities are reduced, has prompted the investigation on the neuroprotective role of the cannabinoids in HD. Quinolinic acid (QA) is an excitotoxin that, when injected in the rat striatum reproduces many features of HD and that acts by stimulating glutamate outflow. The aim of the present study was to test the ability of the cannabinoid receptor agonist WIN 55,212-2 to prevent the effects induced by QA in the rat striatum. In microdialysis experiments, probe perfusion with WIN 55,212-2 significantly and dose-dependently prevented the increase in extracellular glutamate induced by QA. In electrophysiological recordings in corticostriatal slices, the application of WIN 55,212-2 prevented QA-induced reduction of the field potential amplitude. Both effects of WIN 55,212-2 were prevented by the CB(1) receptor antagonist AM 251. In in vivo experiments, intrastriatal WIN 55,212-2 significantly attenuated the striatal damage induced by QA, although no significant effects were observed on a behavioural ground. These data demonstrate that the stimulation of CB(1) receptors might lead to neuroprotective effects against excitotoxic striatal toxicity.

Adenosine A 2A receptor antagonists prevent the increase in striatal glutamate levels induced by glutamate uptake inhibitors.

Active uptake by neurons and glial cells is the main mechanism for maintaining extracellular glutamate at low, non-toxic concentrations. Activation of adenosine A(2A) receptors increases extracellular glutamate levels, while A(2A) receptor antagonists reduce stimulated glutamate outflow. Whether a modulation of the glutamate uptake system is involved in the effects elicited by A(2A) receptor blockers has never been investigated. This study examined the ability of adenosine A(2A) receptor antagonists to prevent the increase in glutamate levels induced by blockade of the glutamate uptake. In rats implanted with a microdialysis probe in the dorsal striatum, perfusion with 4 mm l-trans-pyrrolidine-2,4-dicarboxylic acid (PDC, a transportable competitive inhibitor of glutamate uptake), or 10 mm dihydrokainic acid (DHK, a non-transportable competitive inhibitor that mainly blocks the glial glutamate transporter GLT-1), significantly increased extracellular glutamate levels. The effects of PDC and DHK were completely prevented by the adenosine A(2A) receptor antagonists SCH 58261 (0.01 mg/kg i.p.) and/or ZM 241385 (5 nm via probe). Since an impairment in glutamate transporter function is thought to play a major role in neurodegenerative disorders, the regulation of glutamate uptake may be one of the mechanisms of the neuroprotective effects of A(2A) receptor antagonists.

Modulation of glutamate release and excitotoxicity by adenosine A2A receptors.

Because an increased glutamate outflow is thought to play a crucial role in triggering excitotoxic neuronal death, drugs able to regulate glutamate release could be effective for the management of neurodegenerative diseases. In this article, the authors discuss the hypothesis that adenosine A2A receptor antagonists (A2A antagonists) may belong to the aforementioned category. In rats bilaterally lesioned with the excitotoxin quinolinic acid (QA) in the striatum, the A2A antagonist SCH 58261 significantly reduced the motor, EEG, and neuropathologic changes induced by the lesion. Such effects of SCH 58261 occurred only at low doses and were paralleled by an inhibition of QA-stimulated glutamate release. The role played by A2A antagonists in the regulation of glutamate outflow was also confirmed by preliminary results obtained in the model of paired-pulse stimulation in corticostriatal slices. Conversely, based on data obtained in cultured striatal neurons, A2A antagonists appear unable to directly inhibit NMDA effects. In conclusion, A2A antagonists show clear neuroprotective effects in models of brain injury, although their actual therapeutic potential needs to be confirmed in a wider range of doses and in models of neurodegenerative diseases in which presynaptic and postsynaptic effects play different relative roles.

The selective mGlu(5) receptor agonist CHPG inhibits quinpirole-induced turning in 6-hydroxydopamine-lesioned rats and modulates the binding characteristics of dopamine D(2) receptors in the rat striatum: interactions with adenosine A(2a) receptors.

In 6-hydroxydopamine-lesioned rats, the selective mGlu(5) receptor agonist (RS)-2-Cholro-5-Hydroxyphenylglycine (CHPG, 1-6 microg/10 microl intracerebroventricularly) significantly inhibited contralateral turning induced by quinpirole and, to a lesser extent, that induced by SKF 38393. The inhibitory effects of CHPG on quinpirole-induced turning were significantly potentiated by an adenosine A(2A) receptor agonist (CGS 21680, 0.2 mg/kg IP) and attenuated by an A(2A) receptor antagonist (SCH 58261, 1 mg/kg IP). In rat striatal membranes, CHPG (100-1,000 nM) significantly reduced the affinity of the high-affinity state of D(2) receptors for the agonist, an effect potentiated by CGS 21680 (30 nM). These results show the occurrence of functional interactions among mGlu(5), adenosine A(2A), and dopamine D(2) receptors in the regulation of striatal functioning, and suggest that mGlu(5) receptors may be regarded as alternative/integrative targets for the development of therapeutic strategies in the treatment of Parkinson's disease.

SCH 58261 (an adenosine A(2A) receptor antagonist) reduces, only at low doses, K(+)-evoked glutamate release in the striatum.

The aim of the present work was to determine whether systemic administration of the adenosine A(2A) receptor antagonist, SCH 58261 (7-(2-phenylethyl)-5-amino-2-(2-furyl)-pyrazolo-[4,3-e]-1,2,4,triazolo[1,5-c]pyrimidine), could modulate striatal glutamate outflow in the rat. Microdialysis experiments were performed in male Wistar rats implanted with microdialysis probes in the striatum. Pretreatment (15 min before) with SCH 58261 (0.01 and 0.1, but not 1 mg/kg intraperitoneally) significantly prevented K(+)-stimulated glutamate release. These results suggest that SCH 58261 could possess neuroprotective effects in the low dose range, while, at higher doses, the occurrence of additional mechanisms may limit the neuroprotective potential of this drug.

The mGlu5 receptor agonist CHPG stimulates striatal glutamate release: possible involvement of A2A receptors.

The intrastriatal perfusion of the selective metabotropic glutamate (mGlu)5 receptor agonist (RS)-2-chloro-5-hydroxy-phenylglycine (CHPG, 1000 microM) significantly increased (approximately + 100%, p < 0.05) glutamate extracellular levels with respect to basal values. The potentiating effect of CHPG was prevented by the selective mGlu5 receptor antagonist 2-methyl-6(phenyl-ethynyl)-pyridine (MPEP, 250 microM)) and by the adenosine A2A receptor antagonist SCH 58261 (2 mg/kg, i.p.). The results show that mGlu5 receptors are involved in the regulation of striatal glutamate release and suggest an involvement of adenosine A2A receptors in mGlu5 receptor-mediated effects.

Age-related decline in the functional response of striatal group I mGlu receptors.

In order to verify whether striatal group I metabotropic glutamate (mGlu) receptors undergo functional alteration in ageing, the effects induced by the selective agonist 3,5-dihydroxyphenylglycine (DHPG) in the striatum of young (3 months) and aged (24-25 months old) rats were compared. The ability of DHPG to stimulate phosphoinositide (PI) hydrolysis (striatal slices), to influence striatal dopamine release (in vivo microdialysis) and to potentiate the effects of NMDA on extracellular field potential amplitude (extracellular recordings on striatal slices) was reduced in the striatum of old vs young rats. These results show an age-dependent reduction in the functional response of striatal group I mGlu receptors, which may be one of the factors underlying the reduced ability aged striatum to integrate information.

Effects of SCH 58261, an adenosine A(2A) receptor antagonist, on quinpirole-induced turning in 6-hydroxydopamine-lesioned rats. Lack of tolerance after chronic caffeine intake.

In unilaterally 6-hydroxydopamine (6-OHDA)-lesioned rats, a rodent model of Parkinson's disease (PD), the adenosine A(2A) receptor antagonist SCH 58261 significantly increased (+180%, p <.01) the number of rotations induced by a low dose of quinpirole (a dopamine D(2) receptor agonist), while it did not significantly modify the effects of a comparably low dose of SKF 38393 (a dopamine D(1) receptor agonist). The dose-dependent potentiating effects of SCH 58261 on quinpirole-induced turning were similar in caffeine-treated rats (1 g/l in drinking water over 14 days) and control animals (tap water). The selective potentiating effects of SCH 58261 on D(2)-dependent turning confirm the existence of a potent and specific A(2A)/D(2) receptor-receptor interaction. The persistence of the potentiating effects of SCH 58261 after chronic caffeine intake suggests that no tolerance should develop towards the antiparkinsonian effects of adenosine A(2A) receptor antagonists with chronic treatment.

Stimulation of adenosine A1 receptors attenuates dopamine D1 receptor-mediated increase of NGFI-A, c-fos and jun-B mRNA levels in the dopamine-denervated striatum and dopamine D1 receptor-mediated turning behaviour.

Adenosine A1 receptors antagonistically and specifically modulate the binding and functional characteristics of dopamine D1 receptors. In the striatum this interaction seems to take place in the GABAergic strionigro-strioentopeduncular neurons, where both receptors are colocalized. D1 receptors in the strionigro-strioentopeduncular neurons are involved in the increased striatal expression of immediate-early genes induced by the systemic administration of psychostimulants and D1 receptor agonists. Previous results suggest that a basal expression of the immediate-early gene c-fos tonically facilitates the functioning of strionigro-strioentopeduncular neurons and facilitates D1 receptor-mediated motor activation. The role of A1 receptors in the modulation of the expression of striatal D1 receptor-regulated immediate-early genes and the D1 receptor-mediated motor activation was investigated in rats with a unilateral lesion of the ascending dopaminergic pathways. The systemic administration of the A1 agonist N6-cyclopentyladenosine (CPA, 0.1 mg/kg) significantly decreased the number of contralateral turns induced by the D1 agonist SKF 38393 (3 mg/kg). Higher doses of CPA (0.5 mg/kg) were necessary to inhibit the turning behaviour induced by the D2 agonist quinpirole (0.1 mg/kg). By using in situ hybridization it was found that CPA (0.1 mg/kg) significantly inhibited the SKF 38393-induced increase in the expression of NGFI-A and c-fos mRNA levels in the dopamine-denervated striatum. The increase in jun-B mRNA expression could only be inhibited with the high dose of CPA (0.5 mg/kg). A stronger effect of the A1 agonist was found in the ventral striatum (nucleus accumbens) compared with the dorsal striatum (dorsolateral caudate-putamen). The results indicate the existence of antagonistic A1-D1 receptor-receptor interactions in the dopamine-denervated striatum controlling D1 receptor transduction at supersensitive D1 receptors.

The intrastratial injection of an adenosine A(2) receptor antagonist prevents frontal cortex EEG abnormalities in a rat model of Huntington's disease.

The influence of 3,7-dimethyl-1-propargylxanthine (DMPX) an adenosine A(2) receptor antagonist, was studied in the quinolinic acid (QA) model of Huntington's disease. Male Wistar rats received bilateral intrastriatal injections of QA (300 nmol) alone or plus DMPX (0.02, 0.2 and 2 microg). At the dose of 0.2 microg, DMPX completely prevented QA-induced EEG abnormalities at the level of frontal cortex. The results support the hypothesis of a neuroprotective role of adenosine A(2) receptor antagonists.

Selective agonists of metabotropic glutamate receptors elicit significant EEG effects when infused in the nucleus accumbens of rats.

The effect of intra-accumbens infusion of selective group I ((S)-3,5-dihydroxyphenylglycine, DHPG), group II ((2S,3S,4S)-CCG/(2S,1'S,2'S)-2-(carboxycyclopropyl)glycine, L-CCG-I) and group III ((L-(+)-2-amino-4-phosphonobutyric acid, L-AP4) metabotropic glutamate (mGlu) receptor agonists was studied in male Wistar rats. A computerised electroencephalographic (EEG) power spectral analysis was performed. While DHPG (400 nmoles) induced EEG and behavioural limbic seizures, L-CCG-I (400 nmoles) and L-AP4 (800 nmoles) induced a 'depressant' EEG with an increase in relative power in the slow-frequency bands and a decrease in relative power in the high-frequency bands) and behavioural effects. These results show for the first time that the stimulation of groups I, II and III mGlu receptors located in the nucleus accumbens significantly influences the EEG tracing in rats.

Metabotropic glutamate receptor agonist (1S,3R-ACPD) increased frontal cortex dopamine release in aged but not in young rats.

The effects of the metabotropic glutamate (mGlu) receptor agonist (1S,3R)-1-Amino cyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD) infusion on frontal cortex dopamine extracellular levels were studied by microdialysis in young (3 months) and aged (24 months) rats. Basal dopamine levels were significantly higher in young than in aged rats. (1S,3R)-ACPD (1 mM) significantly increased dopamine efflux in aged rats, an effect which was antagonized by the mGlu receptor antagonist, (S)-alpha-methyl-4-carboxypheniylglycine (MCPG) (2 mM). On the contrary, (IS,3R)-ACPD up to the concentration of 2 mM failed to influence dopamine extracellular levels in young rats. These results suggest that the agonist of mGlu receptor group I and/or II can improve dopamine release under conditions of deficiency of extracellular dopamine concentration as observed in aging.

Adenosine A1 and A2A receptor antagonists stimulate motor activity: evidence for an increased effectiveness in aged rats.

The motor effects of selective adenosine A1 and A2A receptor antagonists were tested in young (2 months) and aged (24 months) Wistar rats. In young rats, both the selective A2A receptor antagonist 5-amino-7-(2-phenylethyl)-2-2(2-furyl)-pyrazolo[4,3-e]-1,2,4-triazo++ + lo[1,5-c]pyrimidine (SCH 58261, minimal effective dose 2 mg/kg intraperitoneally (i.p.)) and the selective A1 receptor antagonist 8-cyclopentyl-1,3-dimethylxanthine (CPT, minimal effective dose 1.2 mg/kg i.p.) stimulated motor activity. In old rats, both compounds induced significant motor activation starting from doses lower than those required in young animals. Specifically, the minimal effective doses of SCH 58261 and CPT in aged rats were 1 and 0.6 mg/kg i.p, respectively. The results indicate that both adenosine A1 and A2A receptors play a functional role in the control of motor activity, and, therefore, the blockade of both receptor subtypes is involved in the motor stimulating properties of methylxanthines. Also the evidence indicates, for the first time, that in aged animals the motor inhibitory adenosinergic tone seems to be increased with respect to young animals.

Age-related alteration of the adenosine/dopamine balance in the rat striatum.

An antagonistic interaction between adenosine A2A- and dopamine D2-receptors has been described. Radioligand binding experiments showed a predominant reduction in the number of D2 vs. A2A-receptors in the striatum of aged compared to young rats. The A2A-receptor-mediated antagonistic modulation of D2-receptor binding remained unchanged in aged animals. In striatal homogenates a significant increase in adenosine and no change in dopamine content was found in aged vs. young rats. These results reveal the existence of an age-dependent imbalance of adenosine vs. dopamine in favor of adenosine, which involves both presynaptic and postsynaptic mechanisms.

Adenosine A1 and A2 receptor agonists significantly prevent the electroencephalographic effects induced by MK-801 in rats.

Both N6-cyclopentyladenosine (CPA, adenosine A1 receptor agonist) and 2-[4-(2-carboxyethyl)phenethylamino]-5'-N-ethylcarboxamido-adenosi ne (CGS 21680, adenosine A2 receptor agonist) inhibited the electroencephalographic (EEG) effects induced by the noncompetitive NMDA receptor antagonist (+)-5-methyl-10,11-dihydro-5H-dibenzo-(a,d)cyclohepten-5,10-imine maleate (MK-801) in rats. While the inhibitory effects of CPA were evident at doses (0.1 and 0.5 mg/kg i.p.) devoid of intrinsic behavioral effects, CGS 21680 was effective only when administered at depressant doses (2 mg/kg i.p.). Since the effects induced by NMDA receptor antagonists may be regarded as a model of psychosis, these results suggest a possible role of adenosine receptor agonists as antipsychotics.

Baseline electroencephalographic tracings in rabbits differ significantly according to 'acute' or 'chronic' preparation. A computerized study.

Baseline electroencephalographic (EEG) tracings recorded from 'acute' and 'chronic' rabbits were compared by computerized spectral analysis. The results showed that baseline EEG activity of rabbits differ significantly and markedly according to acute or chronic preparation. It is suggested that this finding should be taken into account when studying the EEG effects of centrally acting drugs.

Adenosine A1 receptor blockade selectively potentiates the motor effects induced by dopamine D1 receptor stimulation in rodents.

An antagonistic interaction between adenosine A1 and dopamine D1 receptors has previously been found in the basal ganglia. However, direct evidence for a selective adenosine A1 antagonist-induced potentiation of dopamine D1-mediated motor activation is lacking. The systemic administration of the adenosine A1 antagonist 8-cyclopentyl-1,3-dimethylxanthine significantly potentiated the motor activating properties of the systemically administered dopamine D1 agonist SKF 38393 in both reserpinized mice and unilaterally 6-hydroxy-dopamine-lesioned rats. However, 8-cyclopentyl-1, 3-dimethylxanthine did not modify the motor effects of the dopamine D2 agonist quinpirole. The present work shows that an antagonistic interaction between adenosine A1 and dopamine D1 receptors may be involved in the motor activating effects of adenosine antagonists, like caffeine.

Stimulation of adenosine A1 receptors prevents the EEG arousal due to dopamine D1 receptor activation in rabbits.

The influence of adenosine A1 (N6-cyclopentyladenosine, CPA) and A2 (2-[4-(2-carboxylethyl)phenethylamino]-5'-N-ethylcarboxamido -adenosine hydrochloride, CGS 21680) receptor agonists on SKF 38393-induced electroencephalographic (EEG) arousal was studied in rabbits. While CPA (0.1 mg/kg i.v.) significantly prevented the EEG effects of SKF 38393, CGS 21680 (0.2 mg/kg i.v.) did not affect them. These results demonstrate that adenosine A1 receptors can modulate dopamine D1 receptor-induced EEG arousal and show, for the first time, that adenosine-dopamine interactions are involved in brain functions other than motor activity.