Mohammed Errami

Localization of N‐Methyl‐d‐Aspartate Receptors in the Rat Striatum: Effects of Specific Lesions on the [3H]3‐(2‐Carboxypiperazin‐4‐yl)propyl‐1‐Phosphonic Acid Binding

Abstract: The binding of [3H]3‐(2‐carboxypiperazin‐4‐yl)propyl‐1‐phosphonic acid ([3H]CPP), a rigid analogue of 2‐amino‐7‐phosphonoheptanoic acid (AP7) and reported to be a selective N‐methyld‐aspartate (NMDA) antagonist, was studied in rat striatal membranes using a centrifugation procedure to separate bound and free radioligand. [3H]CPP bound with high affinity (Kd= 272 nM) in a saturable, reversible, and protein concentration‐dependent manner, Specific binding was suggested to involve a single class of noninteracting binding sites. The most potent [[3H]CPP binding inhibitors tested were CPP, l‐glutamate, 2‐amino‐5‐phosphonovalerate, and AP7, NMDA, l‐aspartate, and α‐aminoadipate were also shown to be efficient in inhibiting the binding, whereas quisqualate, d,l‐2‐amino‐4‐phosphonobutyrate, kainate, l‐glutamate diethylester, and α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid were found to be essentially inactive. These data are therefore consistent with the view that [3H]CPP selectively binds to NMDA receptors in the rat striatum. Lesions of intrastriatal neurons using local injections of kainic acid revealed a marked decrease in [3H]CPP binding, suggesting an almost exclusively postsynaptic location of binding sites in the striatum. Conversely, bilateral lesion of corticostriatal glutamatergic fibers resulted in an increased number of [3H]CPP striatal binding sites, providing evidence for a putative supersensitivity response to this striatal deafferentation. Interestingly, lesion of the nigrostriatal dopaminergic neurons using intranigral 6‐hydroxydopamine injections resulted, 2–3 weeks later, in a similar increase in the number of [3H]CPP striatal binding sites. These data suggest the occurrence of functional receptor‐receptor interregulations at the postsynaptic level between dopaminergic and NMDA receptors in complement with the interactions occurring at the presynaptic level between glutamatergic and dopaminergic nerve terminals.

Chronic hyperammonemia alters motor and neurochemical responses to activation of group I metabotropic glutamate receptors in the nucleus accumbens in rats in vivo.

Hyperammonemia leads to altered cerebral function and neurological alterations in patients with hepatic encephalopathy. We studied the effects of hyperammonemia in rats on the modulation by group I metabotropic glutamate receptors (mGluR) of motor and neurochemical functions in vivo. Locomotion induced by injection of the mGluR agonist DHPG into nucleus accumbens was increased in hyperammonemic rats. In control rats DHPG increased extracellular dopamine (ca. 400%) but not glutamate. In contrast, in hyperammonemic rats DHPG increased extracellular glutamate (ca. 600%), while DHPG-induced dopamine increase was reduced. Blocking mGluR1 receptor with CPCCOEt prevented all DHPG effects, indicating that this receptor mediates its locomotor and neurochemical effects. Hyperammonemic rats showed increased (32%) mGluR1alpha, but not mGluR5 content in nucleus accumbens. These results show that modulation of locomotor and neurochemical functions by mGluRs in nucleus accumbens is strongly altered in hyperammonemia. These alterations may contribute to the neurological alterations in hyperammonemia and liver failure.

Development of a micromethod to study the Na+-independentl-[3H]glutamic acid binding to rat striatal membranes. II. Effects of selective striatal lesions and deafferentations

An apparent single class of Na+-independent L-[3H]glutamate (L-[3H]Glu) binding sites was biochemically and pharmacologically identified on rat striatal tissue. The Kd value was 1.75 microM and the Bmax 3.89 nmol/g protein. In order to further elucidate the putative physiological role of these binding sites and to valid our binding assay, experiments were conducted to determine the anatomical location of the L-[3H]Glu binding sites in the striatum. Local injection of the neurotoxin, kainic acid into the striatum caused degeneration of target cells in the structure followed by an important decrease (-37%) in the number of these binding sites, with no significant change in the affinity constant. Lesions of the cortical frontal and parietal areas projecting to the striatum via putative glutamatergic fibres led, on the other hand, after 3 weeks to one month, to a significant increase (+23%) in the number of L-[3H]Glu binding sites. The Kd value does not significantly change after decortication. Finally, suppression either of the nigrostriatal dopaminergic input or of the partially cholinergic thalamostriatal tract do not affect [3H]Glu to striatal tissue. These results suggest that about 40% of our [3H]Glu binding sites are located on striatal target cells sensitive to the neurotoxic effect of kainic acid. Therefore, they seem to be partly postsynaptic. The existence of a relation between these binding sites and the corticostriatal glutamatergic input was shown by the development of a supersensitivity response after suppression of this cortical input. These sites might therefore constitute one of the receptor subclasses for Glu linked to the excitatory action of the corticostriatal afferent.(ABSTRACT TRUNCATED AT 250 WORDS)

α-[3H]Amino-3-Hydroxy-5-Methyl-4-Isoxazolepropionic Acid Binding to Rat Striatal Membranes: Effects of Selective Brain Lesions

Abstract: The binding of α‐[3H]amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid ([3H]AMPA), a structural Glu analog, to rat striatal membranes was studied. In the absence of potassium thiocyanate and Cl‐/Ca2+, saturation‐curve analysis of [3H]AMPA binding suggested that a single class of noninteracting binding sites with a KD value of 340 ± 27 nM was involved, although AMPA inhibition of [3H]AMPA binding set at a concentration of 100 nM suggested, in contrast, the presence of multiple populations of striatal binding sites. Several otner excitatory amino acid receptor agonists and antagonists were tested, and the most potent and selective quisqualic acid (QA) receptor agonists (QA, l‐G1u, and AMPA) were found to represent the most potent inhibitors of [3H]AMPA binding. N‐Methyl‐D‐aspartate receptor agonists and antagonists were ineffective as displacers of the [3H]AMPA binding. Lesions of intra‐striatal neurons (using kainic acid local injections) and of corticostriatal afferent fibers led 2–3 weeks later to large decreases (63 and 30%, respectively) in striatal [3H]AMPA binding, whereas selective lesion of the nigrostriatal dopainergic pathway (using nigral injection of 6‐hydroxydopamine) was without any influence. Taken together, these results suggest that [3H]AMPA binding is primarily associated with postsynaptic intrastriatal neurons. Some [3H]AMPA binding sites may also be located presynaptically on corticostriatal nerve endings. So, in addition to the possibility that [3H]AMPA binding sites may be involved in corticostriatal synaptic transmission, it is interesting that these putative QA‐preferring excitatory amino acid receptor sites may also play some role in autoregulatory processes underlying this excitatory synaptic transmission.

Chronic hyperammonemia induces tonic activation of NMDA receptors in cerebellum

J. Neurochem. (2009) 112, 1005–1014.

Effects of pyroglutamic acid on corticostriatal glutamatergic transmission.

The effects of L-pyroglutamic acid, a molecule structurally derived from L-glutamic acid (Glu), were measured on the high affinity of uptake of glutamic acid from striatal synaptosomes of the rat and on the binding of [L-3H]glutamic acid to striatal membranes. The results showed a competitive inhibition of the high affinity transport of glutamic acid by L-pyroglutamic acid in vitro with no effect on the uptake of gamma-aminobutyric acid (GABA). An inhibition of the binding of [L-3H]glutamic acid to striatal membranes was also detected. Significant high affinity uptake of [L-3H]pyroglutamic acid was evident in synaptosomes from the striatum. A regional distribution study of the uptake processes for [L-3H]glutamic acid and [L-3H]pyroglutamic acid in different areas of the brain showed a similar distribution, suggesting that an uptake of [L-3H]pyroglutamic acid, although weak, occurs in glutamatergic nerve terminals. This proposal was further reinforced by measuring the effects of a large cortical lesion involving frontal and parietal areas on the uptake of [L-3H]glutamic acid and [L-3H]pyroglutamic acid in synaptosomes from the striatum. The results showed a large decrease in the uptake processes of both labelled molecules showing that the uptake of [L-3H]pyroglutamic acid, as for glutamic acid mainly occurred in corticostriatal nerve terminals, although other uptake sites are not excluded.

Chronic Hyperammonemia Alters the Circadian Rhythms of Corticosteroid Hormone Levels and of Motor Activity in Rats

Patients with liver cirrhosis may present hepatic encephalopathy with a wide range of neurological disturbances and alterations in sleep quality and in the sleep‐wake circadian rhythm. Hyperammonemia is a main contributor to the neurological alterations in hepatic encephalopathy. We have assessed, in an animal model of chronic hyperammonemia without liver failure, the effects of hyperammonemia per se on the circadian rhythms of motor activity, temperature, and plasma levels of adrenal corticosteroid hormones. Chronic hyperammonemia alters the circadian rhythms of locomotor activity and of cortisol and corticosterone levels in blood. Different types of motor activity are affected differentially. Hyperammonemia significantly alters the rhythm of spontaneous ambulatory activity, reducing strongly ambulatory counts and slightly average velocity during the night (the active phase) but not during the day, resulting in altered circadian rhythms. In contrast, hyperammonemia did not affect wheel running at all, indicating that it affects spontaneous but not voluntary activity. Vertical activity was affected only very slightly, indicating that hyperammonemia does not induce anxiety. Hyperammonemia abolished completely the circadian rhythm of corticosteroid hormones in plasma, completely eliminating the peaks of cortisol and corticosterone present in control rats at the start of the dark period. The data reported show that chronic hyperammonemia, similar to that present in patients with liver cirrhosis, alters the circadian rhythms of corticosteroid hormones and of motor activity. This suggests that hyperammonemia would be a relevant contributor to the alterations in corticosteroid hormones and in circadian rhythms in patients with liver cirrhosis.

Effects of voltage-sensitive calcium channel blockers on extracellular dopamine levels in rat striatum.

Various subtypes of voltage-sensitive calcium channels (VSCCs) support the release of dopamine (DA) in the central nervous system. Using in vivo microdialysis, we investigate the influence of these subtypes of calcium channels on dopaminergic terminals in the rat striatum. L-type (nifedipine-sensitive), N-type (ω-conotoxin GVIA-sensitive), or N- and P/Q-type (ω-conotoxin MVIIC-sensitive) Ca2+ channels were blocked using selective antagonists injected locally, and K+-evoked DA release was measured in freely moving animals. K+(100 mM) induced a massive increase of basal DA extracellular levels (930%) and was without significant effect on extracellular levels of DA metabolites DOPAC and HVA, and on the serotonin metabolite 5HIAA. ω-Conotoxin GVIA (1 μM) and ω-conotoxin MVIIC (1 μM) significantly reduced the K+-evoked DA release by 55 and 62%, respectively. The simultaneous application of the two conotoxins at the same concentration reduced K+-evoked DA release by 66%. Nifedipine (10 μM) had no significant effect on K+-evoked DA release, while neomycin, a nonspecific VSCC blocker, produced a highly significant decrease when applied at 250 and 500 μM (56 and 75%, respectively). The compounds, however, had no effect on basal DA release and on the levels of extracellular DOPAC, HVA, and 5HIAA. These results suggest that under high and persistent conditions of membrane depolarization (15 min, 100 mM K+), striatal DA release is mainly mediated by N-type VSCCs.

Impaired release of corticosterone from adrenals contributes to impairment of circadian rhythms of activity in hyperammonemic rats.

Patients with liver cirrhosis may present impaired sleep-wake and circadian rhythms, relative adrenal insufficiency and altered hypothalamus-pituitary-adrenal gland (HPA) axis. The underlying mechanisms remain unclear. Circadian rhythms are modulated by corticosteroids which secretion is regulated by HPA axis. Hyperammonemia alters circadian rhythms of activity and corticosterone in rats. The aims were: (1) assessing whether corticosterone alterations are responsible for altered circadian rhythm in hyperammonemia: (2) to shed light on the mechanism by which corticosterone circadian rhythm is altered in hyperammonemia. The effects of daily corticosterone injection at ZT10 on circadian rhythms of activity, plasma corticosterone, adreno-corticotropic hormone (ACTH) and hypothalamic corticotropic releasing hormone (CRH) were assessed in control and hyperammonemic rats. ACTH-induced corticosterone release was analyzed in cultured adrenal cells. Corticosterone injection restores the corticosterone peak in hyperammonemic rats and their activity and circadian rhythm. Plasma ACTH and CRH in hypothalamus are increased in hyperammonemic rats. Corticosterone injection normalizes ACTH. Chronic hyperammonemia impairs adrenal function, reduces corticosterone content and ACTH-induced corticosterone release in adrenals, leading to reduced feedback modulation of HPA axis by corticosterone which contributes to impair circadian rhythms of activity. Impaired circadian rhythms and motor activity may be corrected in hyperammonemia and hepatic encephalopathy by corticosterone treatment.

Altered modulation of motor activity by group I metabotropic glutamate receptors in the nucleus accumbens in hyperammonemic rats.

One of the neurological complications in hepatic encephalopathy is the impairment of motor coordination and function. Clinical signs of basal ganglia, cortico-spinal and cerebellar dysfunction have been commonly detected in these patients. We are studying the molecular bases of the alterations in motor coordination and function in hepatic encephalopathy. Hyperammonemia is considered the main factor responsible for the neurological alterations in patients with hepatic encephalopathy. Activation of metabotropic glutamate receptors (mGluRs) in the nucleus accumbens (NAcc) induces locomotion in rats. Asa first step in our studies on the alterations in motor coordinationand function in hyperammonemia and hepatic encephalopathy we studiedwhether the control of motor function by mGluRs in the NAcc is altered in hyperammonemic rats. The locomotor activity induced by injection into the nucleus accumbens (NAcc) of DHPG, an agonist of group I mGluRs was significantly increased in hyperammonemic rats. Injectionof DHPG increased extracellular dopamine but not glutamate in the NAcc of control rats. In hyperammonemic rats DHPG-induced increase in dopamine was significantly reduced, and extracellular glutamate increased 6-fold. The content of mGluR 1 but not mGluR 5, is increased in the NAcc of hyperammonemic rats. Blockade of mGluR 1 completely prevented motor and neurochemical effects induced by DHPG. These results show that modulation of both motor function and extracellular concentration of neurotransmitters by mGluRs in the NAcc is altered in hyperammonemia. This may contribute to the alterations in motor function in hepatic encephalopathy.