Low-threshold N-methyl- -aspartate receptor function correlates negatively with learning

The intermediate, medial hyperstriatum ventrale (IMHV) is an area of the forebrain of the domestic chick which exhibits great plasticity. Moreover, there is a strong link between plasticity in the IMHV and specific changes in behaviour. The IMHV in vitro is still plastic, and many of its physiological properties are age-dependent, peaking in slices taken from 3- or 4-day-old birds. This 'window' coincides with an important transitional period in a chick's normal behavioural development. It has also been claimed that reversal training is at its most effective in 3- and 4-day-old birds - a proposition which was confirmed by the experiments reported here. A combination of in vivo training followed by in vitro electrophysiology also revealed that the function of low-threshold N-methyl-D-aspartate receptors (one of the age-related variables) is negatively related to the effectiveness of reversal training, when age is held constant.     

Postnatal stress selectively upregulates striatal N-methyl- -aspartate receptors in male rats

Early life events have been thought to contribute towards vulnerability to drug addiction later in life. In the present investigation, the effect of daily neonatal maternal isolation stress on NMDA channel activity was studied. [³H]MK-801 binding was measured in several brain regions from neonatally isolated (ISO) and nonhandled (NH) adult male and female rats. Maximal [³H]MK-801 binding in the caudate–putamen of male ISO rats was 58% higher compared to same sex NH rats. Unlike male rats, maximal [³H]MK-801 binding in the caudate–putamen of female ISO rats was lower than female NH rats. No other brain region showed any significant difference in maximal [³H]MK-801 binding between ISO and NH male and female rats, respectively. There was no effect of pup isolation on the binding affinity (K_d value) in either sex. Repeated maternal isolation is associated with alterations in the NMDA channel activity in the caudate–putamen of adult rats, and may be responsible for the augmentation in the addictive behavior reported.     

Developmental maturation of the N-methyl- -aspartic acid receptor channel complex in postnatal rat brain

The N-methyl- -aspartate (NMDA) receptor plays an important role in developmental plasticity. Previous studies have reported differences between the NMDA receptor-channel complex in the rat pup brain and the adult brain. In the present study, modulation of the NMDA channel complex as a function of age was measured to determine when the temporal switching of the NMDA receptor from the immature form to the adult mature form takes place. [³H]MK-801 binding was measured in the rat forebrain from postnatal day 1 to day 21. Our data suggest the presence of two types of NMDA receptors — an immature type and a mature type. The immature NMDA receptor, seen during the early postnatal period (day 1–day 14) is highly sensitive to spermidine, -glutamate alone potentiates [³H]MK-801 binding, and glycine failed to potentiate an -glutamate-induced increase in [³H]MK-801 binding. During the late postnatal period (after day 14) spermidine alone did not increase [³H]MK-801 binding as potently as it did during the early postnatal period, high-affinity [³H]MK-801 binding was not seen in the presence of -glutamate alone, and -glutamate and glycine or -glutamate and spermidine or -glutamate, glycine and spermidine together, significantly increased [³H]MK-801 binding in a manner similar to that reported in the adult brain. Together, the pharmacology of the NMDA receptor during the early postnatal period differs from the adult-like receptor seen during the late postnatal period, and that in rats the apparent switching of the NMDA receptor from the immature type to the mature type takes place after the second postnatal week.     

Age-Related Changes in the N-Methyl- -aspartate Receptor Binding Sites within the Human Basal Ganglia

The present study examined the regional differences in dopamine transporter binding sites and NMDA receptor complex binding based on autoradiographic images obtained in postmortem sections of human normal brain tissues. In middle-aged control tissues, high and comparable levels of [³H]CFT binding were observed in the caudate nucleus, putamen, and accumbens nucleus without significant alteration along the rostrocaudal axis and ventral and dorsal parts of these nuclei. In aging normal brain tissues, dopamine binding sites for [³H]CFT were significantly reduced in the caudate nucleus, putamen, and accumbens nucleus. -[³H]Glutamate, [³H]MK-801, and [³H]glycine binding to the NMDA receptor complex was lower in aging brain tissues than in middle-aged controls. Significant correlation did occur between age and [³H]CFT binding and between age and -[³H]glutamate, [³H]MK-801, and [³H]glycine binding sites. These results demonstrate that the basal ganglia have age-associated reductions in dopamine transporter uptake and NMDA receptors. These data support hypoactive activity of the NMDA receptor complex system with advancing age. The dopamine transporter uptake and NMDA receptors appear to be vulnerable to the aging process in the basal ganglia.     

Epileptic seizures induced by N-acetyl- -aspartate in rats: in vivo and in vitro studies

Tremor rat (tm/tm), the parent strain of spontaneously epileptic rat (SER: zi/zi, tm/tm), exhibits absence-like seizures characterized by 5–7 Hz spike-wave-like complexes on cortical and hippocampal electroencephalograms (EEG) after 10 weeks of age, prior to development of convulsive seizures. Recently, this animal model has been demonstrated to display a genomic microdeletion within the critical region of tm, where aspartoacylase hydrolyzing N-acetyl- aspartate (NAA) is located, besides showing the ability to accumulate NAA in the brain. Therefore, the present study was performed to determine the involvement of NAA in the induction of epileptic seizures. When NAA (4 μmol) was applied intracerebroventricularly (i.c.v.) to normal Wistar rats, 4–10 Hz polyspikes and/or spike-wave-like complexes followed by absence-like seizure before persistent 1–5 Hz waxing high-voltage after-discharges were observed on cortical and hippocampal EEG. At a higher dose (8 μmol), NAA induced convulsive seizures. The absence-like seizures with polyspikes and/or spike-wave-like complexes on the EEG were also observed with i.c.v. NAA in premature tremor rats without seizures. The NAA-induced seizures in normal rats were antagonized by i.c.v. glutamic acid diethyl ester, a non-selective glutamate receptor antagonist. In addition, NAA applied to the bath rapidly induced a long-lasting depolarization concomitantly with repetitive firings in hippocampal CA3 neurons of normal rat brain slice preparations. These findings suggest that NAA is involved in the induction of absence-like seizures and/or convulsion, probably via glutamate receptors.     

Effects of -arginine on the afferent resting activity in the cephalopod statocyst

The effects of bath application of the nitric oxide (NO) precursor -arginine ( -ARG) on the resting activity (RA) of afferent crista fibers were studied in isolated statocysts of the cuttlefish Sepia officinalis under various experimental conditions. -ARG (threshold 10⁻⁷ M) had three different effects: inhibition, excitation, and excitation followed by an inhibition; only the inhibitory effect of -ARG was dose-dependent. -Arginine ( -ARG) had no effect. When the preparation was pre-treated with NO synthase inhibitors (N^G-Nitric- -arginine methyl ester HCl ( -NAME), N^G-Nitro- -arginine ( -NOARG)), both the inhibitory and the excitatory effects of -ARG significantly decreased at higher concentrations (10^{−5 to −4} M), or were completely blocked at lower concentrations (10^{−7 to −6} M), of -ARG. When the preparation was pre-treated with guanylate cyclase inhibitors (1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ), methylene blue (M-BLU), cystamine (CYS)), -ARG had only excitatory effects, whereas its effects were only inhibitory when the preparation was pre-treated with adenylate cyclase inhibitors 2′,3′-dideoxyadenosine (DDA), MDL-12330A (MDL), nicotinic acid (NIC-A)). -ARG had no effects when the pre-treatment was with a guanylate cyclase inhibitor and an adenylate cyclase inhibitor combined; in that situation, the RA of the afferent fibers remained. These data indicate that in cephalopod statocysts, a cGMP and a cAMP signal transduction pathway (presumably via the generation of NO) are responsible for the effects of -ARG on the RA of crista afferent fibers. They also indicate that the -ARG–cGMP pathway is the dominant pathway and is inhibitory, and that both pathways have only modulatory effects on, but are not essential for, the generation of the RA.     

Intracellular ATP depletion inhibits swelling-induced -[H]aspartate release from primary astrocyte cultures

Volume expansion-sensing outward rectifier (VSOR) anion channel, also referred to as volume-sensitive organic osmolyte-anion channel (VSOAC), appears to be responsible for cell swelling-induced amino acid release in a variety of cells. One prominent feature of the VSOR/VSOAC is that non-hydrolyzed intracellular ATP binding to the channel or an accessory protein is required for its activation. In this study, the effect of intracellular ATP depletion on the swelling-induced release of -[³H]aspartate from rat primary astrocyte cultures due to exposure to either high K⁺ or hypotonic media was studied. When the cells were pretreated for 10 min with a combination of the metabolic inhibitors 2-deoxyglucose and rotenone, 100 mM K⁺ media- or hypotonic media-induced -[³H]aspartate release was completely suppressed. Added separately, each inhibitor showed only partial or no inhibition of -[³H]aspartate release, which correlated with its relative effectiveness in decreasing intracellular ATP levels. These data are consistent with the view that during high [K⁺]_o or hypotonic media-induced swelling of primary astrocyte cultures an ATP-dependent swelling-activated VSOAC channel is responsible for -[³H]aspartate release and close to normal ATP is required for full channel activation.     

Pharmacokinetics and pharmacodynamics of -arginine in rats: a model of stimulated neuronal nitric oxide synthesis

Nitric oxide (NO) is believed to be involved in a variety of central nervous system (CNS) functions, including opioid responsivity. Elucidation of the role of NO in the CNS requires the ability to elevate systematically neuronal NO concentrations in vivo. This study was conducted to assess the pharmacokinetics of -arginine, a NO precursor, and to relate the disposition of this amino acid to the pharmacodynamic endpoint of neuronal NO production. -Arginine (250-, 500-, or 1000-mg/kg/h) or saline was infused intravenously for 6 h to rats. -Arginine was quantified in brain and blood (after in vivo microdialysis) with high-performance liquid chromatography. NO was quantified simultaneously with a sensitive and specific amperometric sensor placed in the hippocampus. The data were fit with a comprehensive pharmacokinetic–pharmacodynamic (PK/PD) model to obtain parameters governing the systemic disposition of -arginine, the uptake of -arginine into the brain, and subsequent NO production. Exogenous administration of -arginine resulted in incremental elevations in hippocampal NO, with a 33, 48, and 50% increase from control for the 250-, 500-, and 1000-mg/kg/h -arginine treated rats, respectively. The PK/PD model, which incorporated known characteristics of the system (saturable uptake of -arginine into brain; NO production governed by circadian changes in enzyme activity) was capable of describing accurately the observed data. The model developed herein will be invaluable in characterizing the numerous roles of NO in the CNS.     

Middle cerebral artery occlusion in the mouse by intraluminal suture coated with poly- -lysine: neurological and histological validation

The present study was conducted to validate a modified method of temporary focal cerebral ischemia in the mouse; neurobehavioral function and histopathological infarction were quantitated following various periods of middle cerebral artery occlusion (MCAo). Male C57BL/6 mice were anesthetized with 3% halothane in a mixture of 30%O₂/70%N₂O delivered by face mask and were subjected to 30- to 180-min of temporary middle cerebral artery occlusion (MCAo) by an intraluminal suture coated with poly- -lysine. Twenty-eight of 40 mice showed an initial high-grade neurological deficit (30-min MCAo, n=7; 60-min, n=8; 120-min, n=8; 180-min, n=5) when examined during MCAo; these were used for subsequent study. One day after MCAo, behavioral function was re-evaluated, and brains were perfusion-fixed and infarct volumes were measured. The initial neurological deficit improved at 24 h in mice with 30- or 60-min of prior MCAo but tended to persist in mice with 120- or 180-min insults. Following each duration of ischemia, mice exhibited ipsilateral infarcts. Small, inconsistent predominantly subcortical infarcts were present after 30-min MCAo, while longer occlusion periods gave rise to consistent foci of subcortical infarction involving striatum, septum, thalamus, and hippocampus, as well as areas of frontoparietal cortical infarction. The major advantages of the improved intraluminal MCAo model reported here, incorporating sutures coated with poly- -lysine, include: a 100% incidence of infarction of predictable location and size in mice having an initial neurological deficit. Periods of 60- to 180-min MCA occlusion in this model yield sufficiently reproducible sequelae to permit the effects of various therapeutic agents on neurological outcome and size of infarction to be readily studied.     

Repeated seizure-associated long-lasting changes of N-methyl- -aspartate receptor properties in the developing rat brain

Glutamate NMDA receptor has been implicated in brain developmental processes as well as in excitotoxicity and seizure mediation. A previous study has shown that an acute episode of seizures for 30 min in rats altered NMDA receptor characteristics, mainly in the very immature animal. In order to assess whether receptor modifications may also account for long-lasting cerebral disabilities, medium- and long-term consequences of repeated seizures in developing rats on brain NMDA receptor properties were investigated. Seizures were induced once a day for 3 consecutive days, either from postnatal day 5 (P5) to P7 or from P15 to P17. NMDA receptors were then analysed at P15, P25 and P60 (adulthood) by measuring specific binding of [³H]MK-801 on brain membrane preparations. In addition, allosteric modulation of NMDA receptors by exogenous glutamate and glycine was investigated. Seizures from P5 to P7 led to a 22% increase in the density of [³H]MK-801 binding sites measured at P15, but did not affect NMDA receptor density or affinity at P25 or P60. P15–P17 seizures led to a 21% decrease in the density of binding sites and to a 33% decrease in receptor dissociation constant at P25, while they were without effect at P60. Moreover, P5–P7 and P15–P17 seizures were both associated with a suppression of the glutamate/glycine-induced receptor activation at P60. These modifications might account for long-term alterations in cerebral excitability or plasticity after early convulsive disorders, with regards to altered cognitive capacities, epileptogenesis and brain susceptibility to recurrent seizures.     

Serotonin innervation of Lurcher mutant mice: basic data and manipulation with a combination of amantadine, thiamine and -tryptophan

The Lurcher (Lc/+) mutant mouse is characterized by a considerable atrophy of the cerebellum due to a massive loss of cerebellar Purkinje and granule cells, as well as of neurons from the inferior olivary nucleus. In this study the effects of a therapeutic combination of amantadine, thiamine and -tryptophan on the serotonin (5-HT) innervation was assessed in Lurcher mice by autoradiography, using [³H]citalopram to label 5-HT transporters. In wild type mice as well as in both saline-treated and drug-treated Lurcher mutants, [³H]citalopram binding remained unchanged in forebrain and brainstem regions. In the cerebellum, labelling of deep cerebellar nuclei (CBnuc) was about twofold higher than in the cortex (CBctx). In saline-treated Lurcher mutants compared to wild type mice, the densities of [³H]citalopram were 98% higher in CBctx, and 180% higher in CBnuc. In CBctx of drug-treated Lurcher mutants, transporter densities were 89% higher than in the wild type, but did not differ from the saline-treated Lurcher. In the CBnuc of the drug-treated Lurcher mutants, [³H]citalopram binding was 50% higher than in the saline-treated Lurcher group, and 320% higher than in wild type mice. The results show that 5-HT transporters, already upregulated in the CBnuc of Lurcher mutant mice, can be further increased by a pharmacological treatment, possibly altering the availability of 5-HT in some of its target areas.     

Acute interactions between -DOPA and the neurotoxic effects of 1-methyl-4-phenylpyridinium or 6-hydroxydopamine in mice

We have compared the effects of an i.p. pretreatment with L-DOPA (200 mg/kg) associated with benserazide (25 mg/kg) on neurotoxic effects of either 6-hydroxydopamine (6-OHDA) (50 microg, 10 microl per mouse) or 1-methyl-4-phenylpyridinium (MPP+) (17.5 microg, 10 microl per mouse). The striatal dopamine (DA) content, the vesicular monoamine transporter (VMAT2) density, as well as the hypothalamic norepinephrine (NE) content were measured 8 days after treatments. The L-DOPA-benserazide pretreatment worsened by 65% the 6-OHDA-induced depletion in striatal DA. On the contrary, it reduced by 42% the MPP+-induced depletion in striatal DA and by 54% the MPP+-induced decrease in VMAT2 density. It was noticed that the L-DOPA-benserazide pretreatment did not modify the marked decrease in hypothalamic NE content induced by 6-OHDA.     

Role of carbon monoxide in -glutamate-induced cardiovascular responses in nucleus tractus solitarius of conscious rats

Heme oxygenase degrades heme to form carbon monoxide. It has been reported that heme oxygenase-derived carbon monoxide may interact with -glutamate ( -Glu) receptors in the nucleus tractus solitarius (NTS). Integrative studies suggest that heme oxygenase inhibitors raise blood pressure, in part, by inhibiting carbon monoxide formation in the NTS. The currents studies were designed to determine if heme oxygenase inhibitors affect the cardiovascular actions of -Glu in the NTS. Accordingly, MAP and HR responses to unilateral microinjections of -Glu (5 nmol/100 nl) into the NTS were measured before and after ipsilateral microinjections of zinc deuteroporphyrin 2,4-bis glycol (ZnDPBG, 4.5 nmol/100 nl) or chromium mesoporphyrin (CrMP, 1.5 nmol/100 nl) in awake rats chronically instrumented with NTS guide cannulaes and arterial catheters. With respect to non-treatment (+36±5 mmHg, −107 bpm, n=10), ZnDPBG pre-treatment attenuated the pressor and bradycardic responses to -Glu (+7±3 mmHg, −10±6 bpm, P<0.05). CrMP similarly attenuated cardiovascular responses to -Glu (+47±3 mmHg, −68±8 bpm vs. +20±5 mmHg, −40±9 bpm; before vs. after, n=10, P<0.05). Matched series yielded no vehicle- or time-related effects. Our findings suggest that a heme oxygenase product, such as carbon monoxide, may affect NTS glutamatergic neurotransmission to participate in cardiovascular control.     

Electromyographic Activity Patterns of Ankle Flexor and Extensor Muscles during Spontaneous and -DOPA-Induced Locomotion in Freely Moving Neonatal Rats

In rats, hindlimb postural and locomotor functions mature during the first 3 postnatal weeks. Previous evidence indicates that maturation of descending monoaminergic pathways is important for the postnatal emergence of locomotion with adequate antigravity postural support. Here we have studied the effect of the monoamine precursor -DOPA on locomotor activity in freely moving postnatal rats (7–9 days old) using electromyographic recordings from ankle extensor (soleus) and flexor (tibialis anterior or extensor digitorum longus) muscles. Before pharmacological treatment, both muscles were usually silent at rest, and during spontaneous movements there was a high degree of coactivation between the two antagonists. This was due to a longer electromyographic (EMG) burst duration in flexors, which partly overlapped with the extensor burst. -DOPA administration (150 mg/kg) resulted in a marked increase in postural tonic EMG activity in extensors which appeared gradually within 10 min after injection and was sufficient for the pups to maintain a standing posture with the pelvis raised above ground. Thereafter, episodes of locomotion characterized by rhythmic reciprocal bursts of EMG activity in flexor and extensor muscles were seen. The -DOPA-induced rhythmic EMG pattern was also seen in postnatal rats subjected to a midthoracic spinal cord transection, indicating that the effect of -DOPA on motor coordination is exerted primarily at the level of the spinal pattern generator. Analysis of EMG burst characteristics showed that the pattern of -DOPA-induced locomotion in both intact and spinalized postnatal rats resembled in some respects that observed in adults during spontaneous locomotion. The appearance of reciprocal activation during - DOPA-induced locomotion in neonates was primarily due to a shortening of the EMG burst duration in flexors, which reduced the degree of antagonist coactivation. These results show that the spinal cord has the potential to produce coordinated overground locomotion several days before such movements are normally expressed in the freely moving animal.     

Nitric Oxide and -Arginine Cause an Accumulation of Utrophin at the Sarcolemma: A Possible Compensation for Dystrophin Loss in Duchenne Muscular Dystrophy

Duchenne muscular dystrophy (DMD), a severe X-linked recessive disorder which results in progressive muscle degeneration, is due to a lack of dystrophin, a membrane cytoskeletal protein. An approach to treatment is to compensate for dystrophin loss with utrophin, another cytoskeletal protein with over 80% homology with dystrophin. Utrophin is expressed, at the neuromuscular junction, in normal and DMD muscles and there is evidence that it may perform the same cellular functions as dystrophin. So, the identification of molecules or drugs that could up-regulate utrophin is a very important goal for therapy. We show that in adult normal and mdx mice (an animal model of Duchenne myopathy) treated with -arginine, the substrate of nitric oxide synthase (NOS), a pool of utrophin localized at the membrane appeared and increased, respectively. In normal and mdx myotubes in culture, -arginine, nitric oxide (NO), or hydroxyurea increased utrophin levels and enhanced its membrane localization. This effect did not occur with -arginine, showing the involvement of NOS in this process. The NO-induced increase in utrophin was prevented by oxadiazolo-quinoxalin-1-one, an inhibitor of a soluble guanylate cyclase implicated in NO effects. These results open the way to a potential treatment for Duchenne and Becker dystrophies.     

Neonatal exposure to monosodium -glutamate induces loss of neurons and cytoarchitectural alterations in hippocampal CA1 pyramidal neurons of adult rats

Glutamatergic post-synaptic receptors are closely related to the known excitotoxic effects of high doses of -glutamate. Several behavioral abnormalities, glial reaction, and an increase of expression of the NMDA receptor sub-units have been observed in the rat hippocampus after early monosodium glutamate exposure. Thus, a quantitative morphological study was carried out to determine the effects of early exposure to monosodium glutamate on post-synaptic structures that mediate glutamate excitatory neurotransmission in the hippocampal CA1 field. Four milligrams per gram body weight of monosodium glutamate was subcutaneously injected into neonatal Wistar rats, at 1, 3, 5, and 7 days. Cell loss and several cytoarchitectonic parameters were evaluated in pyramidal cells from the hippocampal CA1 field in the treated rats at 60 days of age. An untreated group of rats were used as controls. Cell number in the hippocampus of experimental rats was 11.5% less than that in control animals. In addition, both dendritic arborization and dendritic spine density were adversely affected, and thin and mushroom-shaped spines became proportionally more numerous, while the opposite occurred to stubby spines. These results strongly suggest the occurrence of cell death and also show some cytoarchitectural modifications in the surviving neurons. These could lead to functional alterations in the hippocampal integrative activity, due to an early cytoexcitotoxic effect of monosodium glutamate.     

Hyperosmolar -mannitol reverses the increased membrane excitability and the nodal swelling caused by Caribbean ciguatoxin-1 in single frog myelinated axons

The effects of hyperosmolar -mannitol were studied on single frog myelinated nerve fibres previously poisoned with Caribbean ciguatoxin-1 (C-CTX-1), a new toxin isolated from the pelagic fish Caranx latus inhabiting the Caribbean region. In current-clamped myelinated axons, C-CTX-1 (50–120 nM) caused spontaneous and repetitive action potential discharges after a short delay. In addition, the toxin produced a marked swelling of nodes of Ranvier of myelinated axons that reached a steady state within about 90 min, as revealed by using confocal laser scanning microscopy. The increased excitability and the nodal swelling caused by C-CTX-1 were prevented or reversed by an external hyperosmotic solution containing 100 mM -mannitol. Moreover, the C-CTX-1-induced nodal swelling was completely prevented by the blockade of voltage-sensitive sodium channels by tetrodotoxin (TTX). It is suggested that C-CTX-1, by increasing nerve membrane excitability, enhances Na⁺ entry into nodes of Ranvier through TTX-sensitive sodium channels, which directly or indirectly disturb the osmotic equilibrium between intra- and extra-axonal media resulting in an influx of water that was responsible for the long-lasting nodal swelling. The fact, that hyperosmolar -mannitol either reversed or prevented the neurocellular actions of C-CTX-1, is of particular interest since it provides the rational basis for its use to treat the neurological symptoms of ciguatera fish poisoning in the Caribbean area.