Receptors In Rat Striatum Research Articles

Glutaminergic tonic action potentiate MPP+-induced hydroxyl radical production in rat striatum

The effect of glycine on 1-methyl-4-phenylpyridinium ion (MPP+)-induced hydroxyl radical (OH) formation in the extracellular fluid of rat striatum were investigated. Rats were anesthetized and sodium salicylate in Ringer's solution (0.5 nmol/μl/min) was infused through a microdialysis probe to detect the generation of OH as reflected by the non-enzymatic formation of 2,3-dihydroxybenzoic acid (2,3-DHBA) in rat striatum. MPP+ (5 mmol/L) produced an increase in OH formation. When glycine (1 mmol/L) was infused into the rat striatum through a microdialysis probe after MPP+ treatment, the marked in the level of 2,3-DHBA was observed in the brain dialysate. However, in the presence of MK-801 (100 μmol/L), a non competitive antagonist of N-methyl-D-aspartate (NMDA), glycine failed to increase the 2,3-DHBA formation by MPP+. When corresponding experiments were performed with nitro-L arginine (L-NNA) (1 mmol/L), a nitric oxide synthase (NOS) inhibitor, same result was obtained. These results suggest that MPP+-induced OH generation may modulated by glycine via NMDA receptor in rat striatum. This increase might be explained because of the presence of a glutaminergic tonic action

The Density of N-Methyl-D-Aspartate Receptor in Rat Striatum Varies with Differing Psychotropic Drug Treatments

Background: A number of studies in rodents suggest that antipsychotic drug treatment can either alter the levels or functionality of the N-methyl-D-aspartate receptor (NMDAR). This raises the possibility that some of the therapeutic actions or side-effects of antipsychotic drugs could be mediated through the NMDAR. Objective: To determine if treatments with combinations of psychotropic drugs change the levels of the NMDAR in rat Central Nervous System (CNS). Methods: In situ radioligand binding and autoradiography were used to quantify [3H]MK-801 binding to NMDAR in frozen CNS sections from rats treated with vehicle, haloperidol (0.1 mg/kg/day), olanzapine (1.0 mg/kg/ day), lithium (25.5 mg/kg/day) or, at the same doses, haloperidol and lithium or olanzapine and lithium for 28 days. Results: There was a significant variation of [3H]MK-801 binding with treatment in the rat striatum due to higher levels of radioligand in rats treated with haloperidol compared to those treated with olanzapine and lithium (p < 0.01; Cohen’s d = 1.47). Levels of radioligand binding did not vary with drug treatment in the cortex. Conclusion: These data argue for some effect of polypharmacy on NMDAR levels, possibly localized to the striatum. Further studies of mood stabilisers with known effects on NMDAR functionality, such as valproate and lamotrigine, with and without antipsychotic drugs could be worthwhile as they may show a greater effect on NMDAR density in the CNS. Keywords: Glutamate, haloperidol, lithium, olanzapine, NMDA, psychotropic drug.

Hints on the Lateralization of Dopamine Binding to D1 Receptors in Rat Striatum.

Dopamine receptors in striatum are important for healthy brain functioning and are the target of levodopa-based therapy in Parkinson's disease. Lateralization of dopaminergic neurotransmission in striata from different hemispheres occurs in patients, but also in healthy individuals. Our data show that the affinity of dopamine binding to dopamine D1 receptors is significantly higher in left than in right striatum. Analysis of data from radioligand binding to striatal samples from naïve, 6-hydroxydopamine lesioned, levodopa-treated and levodopa-induced dyskinetic rats shows differential receptor structure and gives hints on the causes of right/left lateralization of dopamine binding to striatal D1 receptors. Moreover, binding data showed loss of lateralization in levodopa (L-DOPA)-induced dyskinetic rats.

Compensation for cranial spill‐in into the cerebellum improves quantitation of striatal dopamine D2/3 receptors in rats with prolonged [18F]‐DMFP infusions

The condition of steady-state receptor binding in positron emission tomography (PET) studies is best obtained through the use of a bolus plus steady-infusion paradigm. This is a particularly important consideration in the context of in vivo competition studies, where a pharmacological challenge can be administered during the interval of steady-state ligand binding, as in the case of [¹¹C]-raclopride studies with amphetamine challenge. However, the short half-life of ¹¹C imposes limits on the practical duration of constant infusions. Therefore, we chose to test [¹⁸F]-DMFP as a tracer for dopamine D₂/₃ receptors in rat striatum in the paradigm. Using a conventional bolus injection, the [¹⁸F]-DMFP BP(ND) was 3.8 in striatum of anesthetized rats. When followed by a constant infusion, we obtained quasi-stable BP(ND) estimates of 4.5 within an interval of 45 min. During infusions lasting up to 4 h, BP(ND) declined progressively. This seemed due to the progressive spill-in of radioactivity from the cranium to the cerebellum reference region, despite optimized iterative reconstruction of the images. Therefore, we propose a new concept of compensation for this spill-in effect using pharmacokinetic considerations, without requiring high-resolution anatomical images. Challenge with amphetamine (1 and 4 mg/kg) evoked an ∼25% reduction in BP(ND) . There was no clear evidence of dose-dependence in the striatal-binding changes, despite the considerably greater physiological effect, as documented by ECG. Thus, the general applicability of the bolus plus infusion method with [¹⁸F]-DMFP for small animal studies is impeded by the substantial labeling of the cranium. The cranial uptake was linear, indicating first-order kinetics for the enzymatic defluorination of the tracer. Based on this phenomenon, we developed an analytic method compensating for the effects of progressive cranial labeling on the estimation of specific binding in striatum.

CAMP production mediated through P2Y11-like receptors in rat striatum due to severe, but not moderate, carbon monoxide poisoning

We examined the effect of carbon monoxide (CO) poisoning on the production of cAMP, an intracellular second messenger, in rat striatum in terms of extracellular cAMP, which is highly correlated with intracellular cAMP, by using microdialysis. Severe poisoning due to 3000ppm CO, but not moderate poisoning due to 1000ppm CO, caused an increase in cAMP, which was susceptible to a voltage-dependent Na+ channel blocker, tetrodotoxin, and more profound than that under comparable hypoxia caused by 5% O2. These results were similar to our previous findings on the production of hydroxyl radical (•OH), suggesting a close relationship between cAMP and •OH production. The increase in cAMP was suppressed by a non-selective purine P2 receptor antagonist, suramin. However, other non-selective P2 receptor antagonists, pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid and reactive blue 2, exhibited no effect and weak non-significant suppression, respectively. A P2Y11 receptor antagonist, NF157, dose-dependently suppressed the increase in cAMP, although rats lack the P2Y11 receptor. These results suggest that a threshold for cAMP production mediated through P2Y11-like receptors following depolarization triggered by Na+ influx exists in rat striatum during CO poisoning, and that the threshold is reached only in cases of severe CO poisoning. It is also likely that the threshold is related to the generation of •OH, contributing to the toxicity of CO in the brain.

In vitro and in vivo comparison of [3H](+)‐PHNO and [3H]raclopride binding to rat striatum and lobes 9 and 10 of the cerebellum: A method to distinguish dopamine D3 from D2 receptor sites: A method to distinguish dopamine D3 from D2 receptor sites

In vitro binding characteristics of the dopamine D₃/D₂ antagonist [³H]raclopride were compared to the D₃/D₂ agonist [³H](+)-PHNO in membrane preparations from rat striatum, cerebellum Lobules 9 and 10 (CB L9,10), and other cerebellar regions. In striatum, both radioligands labeled a single binding site. [³H](+)-PHNO showed higher affinity, though lower B(max) , compared with [³H]raclopride and was sensitive to inhibition by Gpp(NH)p. [³H](+)-PHNO showed significant specific binding to CB L9,10 membranes with higher affinity compared to striatal membranes. [³H](+)-PHNO binds to a high- and a low-affinity binding site in CB L9,10 membranes; the high-affinity site was not Gpp(NH)p-sensitive. [³H](+)-PHNO did not significantly bind cerebellum left hemisphere membranes. Very low specific binding of [³H]raclopride was found in CB L9,10. The selective dopamine D₃ antagonist SB-277011 did not displace the binding of either ligand to striatal membranes but potently inhibited the binding of [³H](+)-PHNO in CB L9,10 membranes. The highly selective D₂ antagonist SV-156 showed the opposite profile. In vivo experiments were consistent with and supported by in vitro results. In summary, [³H](+)-PHNO and [³H]raclopride mainly label dopamine D₂ receptors in rat striatum, with [³H](+)-PHNO labeling a D₂(High) population. In vitro and in vivo, [³H](+)-PHNO labels CB L9,10 dopamine D₃ receptors that are apparently in a high affinity state whereas [³H]raclopride gave only very low signal in this region. The present approaches appear useful for selectively labeling dopamine D₃ and D₂ receptors in different rat brain regions and offer the possibility to demonstrate D₃ versus D₂ receptor selectivity of compounds using native rat brain tissue.

The effects of stressful stimuli and hypothalamic–pituitary–adrenal axis activation are reversed by the melanin-concentrating hormone 1 receptor antagonist SNAP 94847 in rodents

Melanin-concentrating hormone (MCH) is an orexigenic and dipsogenic neuropeptide that has been reported to mediate acute behavioral and neuroendocrine stress-related responses via MCH 1 receptor activation in rodents. The purpose of the present investigation was to use the MCH 1 receptor antagonist SNAP 94847 ( N-(3-{1-[4-(3,4-difluoro-phenoxy)-benzyl]-piperidin-4-yl}-4-methyl-phenyl)-isobutyramide) to determine the effects of MCH 1 receptor blockade on MCH-evoked adrenocorticotropic hormone (ACTH) release, chronic mild stress-induced anhedonia, stress-induced hyperthermia and forced swim stress-induced immobility. The appropriate dose range for testing SNAP 94847 was determined by measuring MCH-evoked water drinking. The corresponding occupancy of MCH 1 receptors in rat striatum was also measured across a broad dose range. Orally administered (p.o.) SNAP 94847 (1–10 mg/kg) corresponds to 30–60% occupancy at MCH 1 receptors and significantly blocks water drinking induced by the intracerebroventricular (i.c.v.) injection of MCH. MCH (i.c.v.) significantly elevates plasma levels of ACTH in rats, and SNAP 94847 (2.5 mg/kg, p.o.) blocks MCH-evoked ACTH release. Using the chronic mild stress paradigm, we show that repeated daily exposure to environmental stressors for 5 weeks significantly suppresses sucrose intake in rats, and that SNAP 94847 (1 mg/kg, BID) for 1–5 weeks restores baseline sucrose intake. Moreover, a single administration of SNAP 94847 attenuates stress-induced hyperthermia and the behavioral effects of forced swim stress with minimal effective doses of 2.5 and 30 mg/kg (p.o.), respectively. The regulation of ACTH release and reversal of the effects of chronic and acute stress by SNAP 94847 are suggestive of a role for MCH 1 receptor blockade in the treatment of disorders characterized by high allostatic load.

Increased Expression of 5-HT6 Receptors in the Rat Dorsomedial Striatum Impairs Instrumental Learning

A number of studies have shown that systemic 5-HT(6) receptor antagonists can improve learning and memory, but the mechanism for these observations is not known. As striatum normally expresses 5-HT(6) receptors abundantly and is important in consolidating stimulus-response learning, we used targeted gene delivery to further increase the expression of 5-HT(6) receptors in rat striatum and then examined learning. Increased 5-HT(6) expression had no effect on performance in the Morris water maze, a hippocampal-dependent learning paradigm, and did not alter the latency to approach or consume sucrose tablets. However, rats with increased 5-HT(6) expression failed to acquire a reward-based instrumental learning task, a striatum-dependent learning model, during 3 days of successive sessions as compared to sham surgery or GFP-expressing control rats. This behavioral deficit was observed in rats overexpressing 5-HT(6) receptors in the dorsomedial striatum, but not in rats with increased dorsocentral striatal expression. The 5-HT(6) receptor-associated deficit was reversed by administration of a 5-HT(6) antagonist, SB-258585, before each training session. When animals learned the instrumental learning task before gene transfer, increased 5-HT(6) receptor expression had no effect on long-term recall or performance of the task or on extinction of operant responding. Thus, 5-HT(6) receptor activity in rat striatum disrupts acquisition of new instrumental learning but does not impair memory or performance of reward-motivated behavior once established.

Effect of one-week ethanol treatment on monoamine levels and dopaminergic receptors in rat striatum.

We studied the effects of ethanol on the levels of norepinephrine, dopamine, serotonin (5-HT) and their metabolites as well as on D1- and D2-like receptors in the rat striatum. Ethanol (2 or 4 g/kg, po) was administered daily by gavage to male Wistar rats and on the 7th day, 30 min or 48 h after drug administration, the striatum was dissected for biochemical assays. Monoamine and metabolite concentrations were measured by HPLC and D1- and D2-like receptor densities were determined by binding assays. Scatchard analyses showed decreases of 30 and 43%, respectively, in D1- and D2-like receptor densities and no change in dissociation constants (Kd) 48 h after the withdrawal of the dose of 4 g/kg. Ethanol, 2 g/kg, was effective only on the density of D2-like receptors but not on Kd of either receptor. Thirty minutes after the last ethanol injection (4 g/kg), decreases of D2 receptor density (45%) as well as of Kd values (34%) were detected. However, there was no significant effect on D1-like receptor density and a 46% decrease was observed in Kd. An increase in dopamine and 3,4-dihydroxyphenylacetic acid (DOPAC), a decrease in norepinephrine, and no alteration in 5-HT levels were demonstrated after 48-h withdrawal of 4 g/kg ethanol. Similar effects were observed in dopamine and DOPAC levels 30 min after drug administration. No alteration in norepinephrine concentration and a decrease in 5-HT levels were seen 30 min after ethanol (4 g/kg) administration. Our findings indicate the involvement of the monoaminergic system in the responses to ethanol.

Effect of nooglutil on benzodiazepine withdrawal syndrome and binding of 3H-spiperone with D2 receptors in rat striatum.

A new nootropic preparation nooglutil (N-(5-oxynicotinoyl)-L-glutamic acid), a positive modulator of AMPA receptors for glutamate, administered intraperitoneally in a dose of 70 mg/kg reduced anxiety of rats in the Vogel conflict test after 24-h withdrawal from chronic diazepam treatment (4 mg/kg intraperitoneally for 45 days). Nooglutil (5 nM-750 microM) had no effect on in vitro binding of (3)H-spiperone in intact rats. Systemic administration of 50 mg/kg nooglutil in vivo increased the dissociation constant and density of D(2)receptors. Increasing the dose to 100 mg/kg abolished this effect. Our findings suggest that nooglutil produces an indirect effect on the brain dopaminergic system under normal and pathological conditions and this effect is probably mediated via the glutamatergic system.

Morphine acutely and persistently attenuates nonvesicular GABA release in rat nucleus accumbens.

Withdrawal from repeated exposure to morphine causes a long-lasting increase in the reactivity of nucleus accumbens nerve terminals towards excitation. The resulting increase in action potential-induced exocytotic release of neurotransmitters, associated with behavioral sensitization, is thought to contribute to its addictive properties. We recently showed that activation of N-methyl-D-aspartate (NMDA) as well as dopamine (DA) D1 receptors in rat striatum causes tetrodotoxin-insensitive transporter-dependent GABA release. Since sustained changes in extracellular GABA levels may play a role in drug-induced neuronal hyperresponsiveness, we examined the acute and long-lasting effect of morphine on this nonvesicular GABA release in rat nucleus accumbens slices. The present study shows that morphine, through activation of mu-opioid receptors, reduces nonvesicular NMDA-induced [(3)H]GABA release in superfused nucleus accumbens slices. Moreover, prior repeated morphine treatment of rats (10 mg/kg, sc, 14 days) caused a reduction in NMDA-stimulated [(3)H]GABA release in vitro until at least 3 weeks after morphine withdrawal. This persistent neuroadaptive effect was not observed studying dopamine D1 receptor-mediated [(3)H]GABA release in nucleus accumbens slices. Moreover, this phenomenon appeared to be absent in slices of the caudate putamen. Interestingly, even a single exposure of rats to morphine (>2 mg/kg) caused a long-lasting inhibition of NMDA-induced release of GABA in nucleus accumbens slices. These data suggest that a reduction in nonvesicular GABA release within the nucleus accumbens, by enhancing the excitability of input and output neurons of this brain region, may contribute to the acute and persistently enhanced exocytotic release of neurotransmitters from nucleus accumbens neurons in morphine-exposed rats.

Extended treatment with typical and atypical antipsychotic drugs Differential effects on the densities of dopamine D 2-like and GABA A receptors in rat striatum

In situ radioligand binding and quantitative autoradiography have been used to measure the density of striatal D 1-like, D 2-like, and GABA A receptors in rats treated with haloperidol at 0.01 or 0.1 mg/kg/day or chlorpromazine, olanzapine or clozapine at 0.1 or 1.0 mg/kg/day for 1, 3 or 7 months. [ 3H]SCH23390 binding to D 1-like receptors was not changed by any drug treatments. There were significant increases in [ 3H]nemonapride binding to D 2-like receptors at different time points due to treatment with haloperidol, chlorpromazine and olanzapine. By contrast, treatment with clozapine and olanzapine caused a time-dependent decrease in [ 3H]muscimol binding to the GABA A receptor. These data suggest that treatment with atypical antipsychotic drugs, but not typical antipsychotic drugs, affect striatal GABAergic neurons. In addition, it would appear that clozapine might be unique in that it does not increase dopamine-D 2 like receptor density at doses which would be predicted to have antipsychotic effects in humans. The extent to which such changes are involved in the therapeutic effects of drugs such as olanzapine and clozapine remains to be determined.

Autoradiographic analysis of dopamine receptor-stimulated [ 35S]GTPγS binding in rat striatum

Autoradiographic analysis of [ 35S]GTPγS binding was used to investigate functional activation of dopamine receptors in rat striatum. Dopamine-stimulated [ 35S]GTPγS binding was observed with a maximal increase of 38% over basal activity. A similar stimulatory response was obtained with the D 2 agonist quinpirole, but not SKF-238393, a D 1 agonist. The effect of dopamine was blocked by the D 2 antagonist raclopride, but was unaffected by SCH-23990, a D 1 antagonist. There appeared to be a differential distribution of dopamine-stimulated [ 35S]GTPγS binding, with the lowest activity obtained in the medial portion of the caudal striatum. These results demonstrate, using an autoradiographic approach, (i) that dopamine stimulated [ 35S]GTPγS binding in the rat striatum occurs through activation of D 2 receptors, and (ii) that the effects of dopamine activation vary in different areas of the rat striatum.

A caspase-3-like protease is involved in NF-κB activation induced by stimulation of N-methyl- d-aspartate receptors in rat striatum

Glutamate receptor stimulation reportedly activates NF-κB in vitro and in vivo, although underlying mechanisms remain to be elucidated. Here we evaluated the role of proteases in mediating N-methyl- d-aspartate (NMDA) receptor agonist-induced NF-κB activation and apoptosis in rat striatum. The intrastriatal infusion of quinolinic acid (QA, 60 nmol) had no effect on levels of NF-κB family proteins, including p65, p50, p52, c-Rel and Rel B. In contrast, QA decreased IκB-α protein levels by 60% ( P<0.05); other members of the IκB family, including IκB-β, IκB-γ, IκB-ϵ and Bcl-3, were not altered. The QA-stimulated degradation of IκB-α was completely blocked by the NMDA receptor antagonist MK-801. QA-induced IκB-α degradation and NF-κB activation were not affected by the proteasome inhibitor MG-132 (1–4 μg). On the other hand, the caspase-3 inhibitor Ac-DEVD·CHO (2–8 μg) blocked QA-induced IκB-α degradation in a dose-dependent manner ( P<0.05). Ac-DEVD·CHO (4 μg) also substantially reduced QA-induced NF-κB activation ( P<0.05), but had no effect on QA-induced AP-1 activation. Furthermore, Ac-DEVD·CHO, but not MG-132, dose-dependently attenuated QA-induced internucleosomal DNA fragmentation. These findings suggest that NF-κB activation by NMDA receptor stimulation involves IκB-α degradation by a caspase-3-like cysteine protease dependent mechanism. Caspase-3 thus appears to contribute to the excitotoxin-induced apoptosis in rat striatal neurons occurring at least partially as a consequence of NF-κB activation.

Regulation of tyrosine hydroxylase activity and phosphorylation at Ser(19) and Ser(40) via activation of glutamate NMDA receptors in rat striatum.

The activity of tyrosine hydroxylase, the rate-limiting enzyme in the biosynthesis of dopamine, is stimulated by phosphorylation. In this study, we examined the effects of activation of NMDA receptors on the state of phosphorylation and activity of tyrosine hydroxylase in rat striatal slices. NMDA produced a time-and concentration-dependent increase in the levels of phospho-Ser(19)-tyrosine hydroxylase in nigrostriatal nerve terminals. This increase was not associated with any changes in the basal activity of tyrosine hydroxylase, measured as DOPA accumulation. Forskolin, an activator of adenylyl cyclase, stimulated tyrosine hydroxylase phosphorylation at Ser(40) and caused a significant increase in DOPA accumulation. NMDA reduced forskolin-mediated increases in both Ser(40) phosphorylation and DOPA accumulation. In addition, NMDA reduced the increase in phospho-Ser(40)-tyrosine hydroxylase produced by okadaic acid, an inhibitor of protein phosphatase 1 and 2A, but not by a cyclic AMP analogue, 8-bromo-cyclic AMP. These results indicate that, in the striatum, glutamate decreases tyrosine hydroxylase phosphorylation at Ser(40) via activation of NMDA receptors by reducing cyclic AMP production. They also provide a mechanism for the demonstrated ability of NMDA to decrease tyrosine hydroxylase activity and dopamine synthesis.

In vivo imaging of adenovirus-mediated over-expression of dopamine D2 receptors in rat striatum by positron emission tomography.

PET was used to provide in vivo imaging of the over-expression of dopamine D2 receptor (D2R) induced by adenovirus vector-mediated gene transfer in rat striatum. The uptake of three kinds of D2R-specific ligands, [11C]raclopride, [11C]nemonapride and [11C]N-methylspiperone, measured by PET was higher in the striatum injected with the vectors for D2R than the contralateral striatum injected with a control vector 2-3 days after injection. However, the uptake of [11C]SCH 23390, a dopamine D1 receptor specific ligand, or [11C]beta-CIT-FP, a dopamine transporter specific tracer, was not different between bilateral striata. Co-injection of excess unlabeled raclopride inhibited the uptake of [11C]raclopride. At day 16 the increased uptake of [11C]raclopride declined to basal level, consistent with past in vitro assessment of this vector. In vivo imaging of D2R will permit longitudinal assessment of the efficiency of this and similar vectors in rat brain that can be related to functional changes being observed.

Sustained Behavioral Stimulation Following Selective Activation of Group I Metabotropic Glutamate Receptors in Rat Striatum

Group I metabotropic glutamate receptors (mGluRs) are densely expressed in the medium-sized spiny projection neurons of striatum. Activation of this group of the mGluRs modifies neuronal physiology through stimulation of phosphoinositide hydrolysis and intracellular Ca 2+ release. Unlike the ionotropic glutamate receptors that mediate rapid synaptic transmission, activation of the mGluRs produces long-lasting actions brought about by modulation of activities of intracellular effectors. In this study, the role of the group I mGluRs in the modulation of extrapyramidal motor function was examined using a group I selective agonist, 3,5-dihydroxyphenylglycine (DHPG), in chronically cannulated rats. Bilateral injections of DHPG at a series of subtoxic doses (20, 40, 80, and 160 nmol) into the central part of the dorsal striatum produced hyperlocomotion and a unique stereotypical behavior (spontaneous and repetitive twitching movement of the head and forepaws) in a dose-dependent manner. The characteristic twitchy behavior usually commenced 30 min to 1 h, and could last as long as 10 to 12 h, after a single injection of the group I agonist. The behavioral responses to DHPG administration were markedly antagonized by intrastriatal injection of the group I antagonist PHCCC (10 nmol), but not the group II/III antagonist MSOPPE (10 nmol). Intrastriatal administration of 20 nmol dantrolene, an inhibitor of intracellular Ca 2+ mobilization, also prevented DHPG-stimulated motor activities. However, blockade of dopamine D 1 receptors with systemic administration of SCH-23390 (0.1 mg/kg, SC) did not alter the ability of DHPG to provoke behavioral activities. These data indicate that selective activation of the DHPG-sensitive group I mGluRs in the striatum can produce long-lasting stimulation of motor activity. DHPG-induced motor stimulation involves the mobilization of intracellular Ca 2+ stores, but appears to be independent of D 1 dopaminergic transmission.

Co-stimulation of cyclic-AMP-linked metabotropic glutamate receptors in rat striatum attenuates excitotoxin-induced nuclear factor-κB activation and apoptosis

Interactions between glutamatergic mechanisms mediated by receptors of the ionotropic and metabotropic classes in the central nervous system are complex and incompletely understood. To explore the consequences of these interactions on excitotoxicity, we examined the influence of group II and group III selective metabotropic glutamate receptor agonists on the N-methyl- d-aspartate-induced apoptotic destruction of GABAergic neurons in rat striatum. The intrastriatal administration of a group III metabotropic glutamate receptor agonist (amino-4-phosphonobutyric acid, 900–1800 nmol), but not of a group II agonist [(2 S,1′ S,2′ S)-(carboxycyclopropyl)glycine, 100–1800 nmol] produced internucleosomal DNA fragmentation. Similarly, amino-4-phosphonobutyric acid (600 nmol) but not (2 S,1′ S,2′ S)-(carboxycyclopropyl)glycine (100–1800 nmol) destroyed some striatal neurons as indicated by a loss of D 1 dopamine receptors and 67,000 mol. wt glutamate decarboxylase (glutamate decarboxylase-67) messenger RNA. On the other hand, the intensity of internucleosomal DNA fragmentation induced by N-methyl- d-aspartate (150 nmol) was substantially decreased by the intrastriatal co-administration of either (2 S,1′ S,2′ S)-(carboxycyclopropyl)glycine or amino-4-phosphonobutyric acid (100–600 nmol). Both (2 S,1′ S,2′ S)-(carboxycyclopropyl)glycine and amino-4-phosphonobutyric acid also reduced the N-methyl- d-aspartate-induced loss of striatal D 1 dopamine receptors by 67% and 68% (both P<0.001), and glutamate decarboxylase-67 messenger RNA by 68% and 61%, respectively. Furthermore, both (2 S,1′ S,2′ S)-(carboxycyclopropyl)glycine and amino-4-phosphonobutyric acid also attenuated the N-methyl- d-aspartate-induced decline in striatal IκB-α protein levels by 62% and 37%, as well as the increase in nuclear transcription factor nuclear factor-κB binding activity by 135% and 94% (both P<0.001), and the subsequent rise in p53 and c-Myc protein levels. These results suggest that stimulation of cyclic-AMP-linked metabotropic glutamate receptors inhibits ionotropic glutamate receptor-mediated activation of apoptotic cascades involving IκB-α degradation and nuclear factor-κB nuclear translocation, as well as p53 and c-Myc induction. Certain selective metabotropic glutamate receptor agonists might thus find utility as adjuncts to N-methyl- d-aspartate antagonists in the protection against the neurotoxicity initiated by excessive ionotropic glutamate receptor stimulation.

Calcium channels involved in the inhibition of acetylcholine release by presynaptic muscarinic receptors in rat striatum.

1. The mechanism of the inhibitory action of presynaptic muscarinic receptors on the release of acetylcholine from striatal cholinergic neurons is not known. We investigated how the electrically stimulated release of [3H]-acetylcholine from superfused rat striatal slices and its inhibition by carbachol are affected by specific inhibitors of voltage-operated calcium channels of the L-type (nifedipine), N-type (omega-conotoxin GVIA) and P/Q-type (omega-agatoxin IVA). 2. The evoked release of [3H]-acetylcholine was not diminished by nifedipine but was lowered by omega-conotoxin GVIA and by omega-agatoxin IVA, indicating that both the N- and the P/Q-type (but not the L-type) channels are involved in the release. The N-type channels were responsible for approximately two thirds of the release. The release was >97% blocked when both omega-toxins acted together. 3. The inhibition of [3H]-acetylcholine release by carbachol was not substantially affected by the blockade of the L- or P/Q-type channels. It was diminished but not eliminated by the blockade of the N-type channels. 4. In experiments on slices in which cholinesterases had been inhibited by paraoxon, inhibition of [3H]-acetylcholine release by endogenous acetylcholine accumulating in the tissue could be demonstrated by the enhancement of the release after the addition of atropine. The inhibition was higher in slices with functional N-type than with functional P/Q-type channels. 5. We conclude that both the N- and the P/Q-type calcium channels contribute to the stimulation-evoked release of acetylcholine in rat striatum, that the quantitative contribution of the N-type channels is higher, and that the inhibitory muscarinic receptors are more closely coupled with the N-type than with the P/Q-type calcium channels.

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 γ-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 μM), an antagonist of NMDA receptors. In addition, a change in the pattern of local NMDA (500 μM)- 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 Parkinson’s disease, as well as in the treatment of this neurological disorder.