胍丁胺对NMDA受体药理作用的研究进展

胍丁胺是一种新的神经递质和/或神经调质,是咪唑啉受体的内源性配体。它作为一种阳离子胺类物质,除了咪唑啉受体外,在生物体内还存在多个作用靶点,NMDA受体是其中最重要的作用靶点之一。本文就胍丁胺与NMDA受体在中枢神经系统的分布、胍丁胺在NMDA受体上的作用位点以及胍丁胺通过NMDA受体介导的药理作用等几个方面进行了综述。     

Acquisition deficit and time-dependent retrograde amnesia for contextual fear conditioning in agmatine-treated rats

Cumulative evidence indicates that the hippocampus plays a time-limited role in contextual learning paradigms. Pharmacological studies have indicated that acquisition of background contextual cues during Pavlovian fear conditioning is dependent upon hippocampal function, whereas early inactivation of the hippocampus after training produces retrograde amnesia. When administered prior to contextual fear conditioning, agmatine (5 and 10 mg/kg, i.p.), an endogenous polyamine and N-methyl-D-aspartate (NMDA) receptor ligand found at excitatory synapses in the hippocampus, impaired the acquisition of contextual fear (measured as defensive freezing 26 hours later) without a reduction in baseline motor activity during training. Furthermore, ascending doses of agmatine were found not to exert analgesic effects on response thresholds to peripheral shock. This negated the possibility that the observed learning deficit resulted from a difference in perceived shock intensity. Post-training agmatine treatment produced a time-dependent impairment of consolidation, with subjects approaching a level of fear equivalent to that of a reference group as the delay of treatment increased (up to 6 hours). Since physiologically high levels of agmatine are able to inhibit NMDA receptor activity, these results suggest that polyamine modulation of NMDA receptors, most likely within the hippocampus, is required for the acquisition and consolidation of contextual fear stimuli.     

Design, synthesis, SAR, and biological evaluation of highly potent benzimidazole-spaced phosphono-alpha-amino acid competitive NMDA antagonists of the AP-6 type

A series of 2-amino-(phosphonoalkyl)-1H-benzimidazole-2-alkanoic acids was synthesized and evaluated for NMDA receptor affinity using a [3H]CPP binding assay. Functional antagonism of the NMDA receptor complex was evaluated in vitro using a stimulated [3H]TCP binding assay and in vivo by employing an NMDA-induced seizure model. Several compounds of the AP-6 type demonstrated potent and selective NMDA antagonistic activity both in vitro and in vivo. In particular, [R(-)]-2-amino-3-(5-chloro-1-phosphonomethyl-1H-benzoimidazol-2-yl)-propionic acid (1) displayed an IC(50) value of 7.1 nM in the [3H]CPP binding assay and an ED(50) value of 0.13 mg/kg (ip) in the NMDA lethality model. Compound 1, when administered intravenously as a single bolus dose of 3 mg/kg following permanent occlusion of the middle cerebral artery in the rat, reduced the volume of infarcted brain tissue by 45%. These results support a promising therapeutic potential for compound 1 as a neuroprotective agent.     

Antidepressant-like effect of agmatine and its possible mechanism

In mammalian brain, agmatine is an endogenous neurotransmitter and/or neuromodulator, which is considered as an endogenous ligand for imidazoline receptors. In this study, the antidepressant-like action of agmatine administered p.o. or s.c. was evaluated in three behavioral models in mice or rats. Agmatine at doses 40 and 80 mg/kg (p.o.) reduced immobility time in the tail suspension test and forced swim test in mice or at dose 20 mg/kg (s.c.) in the forced swim test. Agmatine also reduced immobility time at 10 mg/kg (p.o.) or at 1.25, 2.5 and 5 mg/kg (s.c.) in the forced swim test in rats. These results firstly indicated that agmatine possessed an antidepressant-like action. With 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay and lactic dehydrogenase (LDH) assay, 1, 10 and 100 microM agmatine or a classical antidepressant, 2.5 and 10 microM desipramine, protected PC12 cells from the lesion induced by 300 microM N-methyl-D-aspartate (NMDA) treatment for 24 h. Using high-performance liquid chromatography with electrochemical detection (HPLC-ECD), it was found that the levels of monoamines including norepinephrine, epinephrine, dopamine or 5-hydroxytryptamine (5-HT) in PC12 cells decreased after the treatment with 200 microM NMDA for 24 h, while in the presence of 1 and 10 microM agmatine or 1 and 5 microM desipramine, the levels of norepinephrine, epinephrine or dopamine were elevated significantly while 5-HT did not change. Moreover, norepinephrine, 5-HT or dopamine had the same cytoprotective effect as agmatine at doses 0.1, 1 and 10 microM. In the fura-2/AM (acetoxymethyl ester) labeling assay, 1 and 10 microM agmatine, 1 and 5 microM desipramine or monoamines norepinephrine, 5-HT at doses 0.1 and 1 microM attenuated the intracellular Ca(2+) overloading induced by 200 microM NMDA treatment for 24 h in PC12 cells. In summary, we firstly demonstrated that agmatine has an antidepressant-like effect in mice and rats. A classical antidepressant, desipramine, as well as agmatine or monoamines protect the PC12 cells from the lesion induced by NMDA treatment. Agmatine reverses the NMDA-induced intracellular Ca(2+) overloading and the decrease of monoamines (including norepinephrine, epinephrine or dopamine) contents in PC12 cells, indicating that agmatine's antidepressant-like action may be related to its modulation of NMDA receptor activity and/or reversal of the decrease of monoamine contents and Ca(2+) overloading induced by NMDA.     

4-(Phosphonoalkyl)- and 4-(phosphonoalkenyl)-2-piperidinecarboxylic acids: synthesis, activity at N-methyl-D-aspartic acid receptors, and anticonvulsant activity

A series of 4-(phosphonoalkyl)- and 4-(phosphonoalkenyl)-2-piperidinecarboxylic acids were synthesized, and their biological activity was assessed as competitive ligands for the NMDA receptor, both in vitro by using a receptor binding assay ([3H]CGS 19755 binding) and in vivo by using an NMDA seizure model in mice. The analogues were also evaluated in [3H]AMPA and [3H]kainate binding to assess their affinity for non-NMDA excitatory amino acid receptor subtypes. A number of these analogues show potent and selective NMDA antagonistic activity both in vitro and in vivo. Most notable are 4-(phosphonomethyl)-2-piperidinecarboxylic acid (1a) (CGS 19755) and the phosphonopropenyl analogue 1i, both of which show anticonvulsant activity in the 1-2 mg/kg ip range. With the aid of computer-assisted modeling, a putative bioactive conformation for AP-5 is hypothesized from the SAR data presented and a preliminary model for the antagonist-preferring state of the NMDA receptor is presented.     

Dizocilpine (MK-801) arrests status epilepticus and prevents brain damage induced by soman.

The involvement of the NMDA receptor in the neurotoxicity induced by soman, an organophosphorus compound which irreversibly inhibits cholinesterase, was studied in guinea pigs. The drug MK-801 (0.5, 1 or 5 mg/kg, i.p.) was given as a pretreatment before a convulsant dose of soman or as a posttreatment (30, 100 or 300 micrograms/kg, i.m.) 5 min after the development of soman-induced status epilepticus. Pyridostigmine, atropine and pralidoxime chloride were also given to each subject to counteract the lethality of soman. All subjects that were challenged with soman and given the vehicle for MK-801 (saline) exhibited severe convulsions and electrographic seizure activity. Neuronal necrosis was found in the hippocampus, amygdala, thalamus and the pyriform and cerebral cortices of those subjects surviving for 48 hr. Pretreatment with 0.5 or 1 mg/kg doses of MK-801 did not prevent nor delay the onset of seizure activity but did diminish its intensity and led to its early arrest. At the largest dose (5 mg/kg), MK-801 completely prevented the development of seizure activity and brain damage. Posttreatment with MK-801 prevented, arrested or reduced seizure activity, convulsions and neuronal necrosis in a dose-dependent manner. The NMDA receptor may play a more critical role in the spread and maintenance, rather than the initiation of cholinergically-induced seizure activity.     

Effect of CGP39551 administration on the kindling of ethanol-withdrawal seizures

RATIONALE: It has previously been shown that drugs, such as benzodiazepines, that inhibit seizure activity during ethanol withdrawal, fail to alleviate the potentiated withdrawal seen following repeated episodes of withdrawal when administered during each withdrawal episode. Acute administration of the N-methyl- D-aspartate (NMDA) receptor competitive antagonist, CGP39551, has been shown to inhibit seizure activity during ethanol withdrawal, and, when administered during the periods of repeated diazepam withdrawal, it blocked the reduction in pentylenetetrazol (PTZ) seizure threshold seen following a final, untreated withdrawal. OBJECTIVES: The aim of the current study was to determine if CGP39551 could alter final ethanol-withdrawal symptoms when administered during the acute intermittent withdrawal periods. METHODS: Mice were chronically treated with ethanol-containing liquid diet for either 30 days continuously (single withdrawal) or with 8-h withdrawal periods on day 16 and day 23 of treatment (repeated withdrawal). Control animals received a control diet for the same period of time. On the final withdrawal animals were tested for behavioural signs of withdrawal. The effect of CGP39551 administered acutely on withdrawal [up to 5 mg/kg, intraperitoneally (i.p.)] or during the intermittent withdrawal periods (10 mg/kg, i.p.) was examined. RESULTS: Acute administration of CGP39551 failed to inhibit handling-induced convulsions in the single-withdrawal or repeated-withdrawal group and had no effect on either decreased exploration or increased sensitivity to PTZ seen in withdrawal. Surprisingly, when CGP39551 was administered during periods of repeated ethanol withdrawal a potentiation of seizure activity was seen in the final, untreated withdrawal. This potentiation of seizure activity, compared with vehicle-treated animals, was not seen when CGP39551 was given whilst animals had access to ethanol (single-withdrawal group and repeated-withdrawal group where CGP39551 treatment was non-contingent with withdrawal episodes). However, the decrease in exploration seen during withdrawal was potentiated in repeated-withdrawal animals treated with CGP39551 irrespective of the time at which the CGP39551 was administered. CONCLUSIONS: Treatment with an NMDA receptor-competitive antagonist potentiated the ethanol-withdrawal syndrome in animals with previous experience of ethanol withdrawal.     

Protection by imidazol(ine) drugs and agmatine of glutamate-induced neurotoxicity in cultured cerebellar granule cells through blockade of NMDA receptor.

This study was designed to assess the potential neuroprotective effect of several imidazol(ine) drugs and agmatine on glutamate-induced necrosis and on apoptosis induced by low extracellular K+ in cultured cerebellar granule cells. Exposure (30 min) of energy deprived cells to L-glutamate (1-100 microM) caused a concentration-dependent neurotoxicity, as determined 24 h later by a decrease in the ability of the cells to metabolize 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) into a reduced formazan product. L-glutamate-induced neurotoxicity (EC50=5 microM) was blocked by the specific NMDA receptor antagonist MK-801 (dizocilpine). Imidazol(ine) drugs and agmatine fully prevented neurotoxicity induced by 20 microM (EC100) L-glutamate with the rank order (EC50 in microM): antazoline (13)>cirazoline (44)>LSL 61122 [2-styryl-2-imidazoline] (54)>LSL 60101 [2-(2-benzofuranyl) imidazole] (75)>idazoxan (90)>LSL 60129 [2-(1,4-benzodioxan-6-yl)-4,5-dihydroimidazole](101)>RX82 1002 (2-methoxy idazoxan) (106)>agmatine (196). No neuroprotective effect of these drugs was observed in a model of apoptotic neuronal cell death (reduction of extracellular K+) which does not involve stimulation of NMDA receptors. Imidazol(ine) drugs and agmatine fully inhibited [3H]-(+)-MK-801 binding to the phencyclidine site of NMDA receptors in rat brain. The profile of drug potency protecting against L-glutamate neurotoxicity correlated well (r=0.90) with the potency of the same compounds competing against [3H]-(+)-MK-801 binding. In HEK-293 cells transfected to express the NR1-1a and NR2C subunits of the NMDA receptor, antazoline and agmatine produced a voltage- and concentration-dependent block of glutamate-induced currents. Analysis of the voltage dependence of the block was consistent with the presence of a binding site for antazoline located within the NMDA channel pore with an IC50 of 10-12 microM at 0 mV. It is concluded that imidazol(ine) drugs and agmatine are neuroprotective against glutamate-induced necrotic neuronal cell death in vitro and that this effect is mediated through NMDA receptor blockade by interacting with a site located within the NMDA channel pore.     

Agmatine blocks ethanol-induced locomotor hyperactivity in male mice

Ethanol-induced locomotor activity is associated to rewarding effects of ethanol and ethanol dependence. Agmatine is a novel endogenous ligand at α₂-adrenoceptors, imidazoline and N-methyl-d-aspartate (NMDA) receptors, as well as a nitric oxide synthase (NOS) inhibitor. There is no evidence presented for the relationship between the acute locomotor stimulating effect of ethanol and agmatine. Thus, the present study investigated the effects of agmatine on acute ethanol-induced locomotor hyperactivity in mice. Adult male Swiss–Webster mice (26–36 g) were used as subjects. Locomotor activity of the mice was recorded for 30 min immediately following intraperitoneal administration of ethanol (0.5, 1 and 2 g/kg) or saline (n = 8 for each group). Agmatine (5, 10 and 20 mg/kg) or saline was administered intraperitoneally to another four individual groups (n = 8 for each group) of the mice 20 min before the ethanol injection. In these groups, locomotor activity was also recorded immediately following ethanol (0.5 g/kg) injection for 30 min. Ethanol (0.5 g/kg) produced some significant increases in locomotor activity of the mice. Agmatine (5–20 mg/kg) significantly blocked the ethanol (0.5 g/kg)-induced locomotor hyperactivity. These doses of agmatine did not affect the locomotor activity in naive mice when they were administered alone. Our results suggest that agmatine has an important role in ethanol-induced locomotor hyperactivity in mice. There may be a relationship between the addictive psychostimulant effects of the ethanol and central agmatinergic system.     

Convulsions induced by centrally administered NMDA in mice: effects of NMDA antagonists, benzodiazepines, minor tranquilizers and anticonvulsants

1. Convulsions were induced reproducibly by intracerebroventricular injection of N-methyl-D-aspartic acid (NMDA) to conscious mice. 2. Competitive (carboxypiperazine-propylphosphonic acid, CPP; 2-amino-7-phosphonoheptanoic acid, AP7) and non-competitive (MK801; phencyclidine, PCP; thienylcyclohexylpiperidine, TCP; dextrorphan; dextromethorphan) NMDA antagonists prevented NMDA-induced convulsions. 3. Benzodiazepine receptor agonists and partial agonists (triazolam, diazepam, clonazepam, Ro 16-6028), classical anticonvulsants (diphenylhydantoin, phenobarbitone, sodium valproate) and meprobamate were also found to prevent NMDA-induced convulsions. 4. Flumazenil (a benzodiazepine receptor antagonist) and the GABA agonists THIP and muscimol (up to subtoxic doses) were without effect. 5. Flumazenil reversed the anticonvulsant action of diazepam, but not that of MK801. 6. Results obtained in this model differ somewhat from those described in a seizure model with systemic administration of NMDA. An explanation for this discrepancy is offered. 7. This model is a simple test for assessing the in vivo activity of NMDA antagonists and also expands the battery of chemically-induced seizure models for characterizing anticonvulsants not acting at NMDA receptors.     

Learning and memory in agmatine-treated rats

Agmatine, a noncompetitive N-methyl-D-aspartate (NMDA) antagonist, was examined for its role in water maze place learning, contextual and auditory-cued (discrete) fear learning and conditioned taste aversion learning, when administered systemically. Male Wistar rats were given saline or 1, 5, 10 or 50 mg/kg agmatine ip 20 min prior to or 30 min following daily training sessions in a hidden-platform (place learning) water maze task. Agmatine did not affect latencies to find the hidden platform or preference for the training quadrant during probe trials. When administered 20 min prior to contextual or auditory-cued fear-conditioning sessions, these doses of agmatine evoked a linear dose-dependent impairment in the magnitude of learned fear to the contextual stimuli when assessed during extinction trials 24 h later, but had no effect on the magnitude of learned fear to the auditory stimulus. Inferences of baseline motor activity and ability to respond to the presentation of footshock stimuli were not affected by the treatment. Injections of 50 mg/kg agmatine concurrently with a malaise-evoking agent following presentations to a novel sucrose solution abolished learned taste aversions; this agent did not evoke conditioned taste aversions alone. These studies indicate that systemically administered agmatine selectively impairs behavioral inferences of specific types of learning and memory.     

Oral administration of glycine and polyamine receptor antagonists blocks ethanol withdrawal seizures

Cessation of chronic administration of orally administered large amounts of ethanol for 7 days resulted in a markedly increased frequency of audiogenic seizures in Sprague-Dawley rats. Oral administration of the novel glycine receptor antagonist, L-701,324, produced a dose-dependent (2.5 and 5.0 mg/kg; − 30 min) inhibition of ethanol withdrawal signs when measured about 12 h after withdrawal of the ethanol treatment. Similarly, using the same experimental paradigm, oral administration of the specific polyamine receptor antagonist, eliprodil, caused a dose-related (2.0 and 5.0 mg/kg; − 30 min) inhibition of ethanol withdrawal-induced audiogenic seizure activity. The inhibition of ethanol withdrawal seizures produced by L-701,324 and eliprodil, respectively, was obtained at doses which by themselves did not change the locomotor activity in naive Sprague-Dawley rats. The findings that L-701,324 and eliprodil are potent inhibitors of seizure activity induced by cessation of chronic ethanol administration and the fact that they, in contrast to currently available NMDA receptor antagonists, do not produce psychotomimetic and/or sedative effects, suggest that these drugs may represent a new class of therapeutically useful pharmacological agents for the treatment of ethanol withdrawal seizures. Furthermore, since there is evidence that eliprodil produces its pharmacological actions through a specific inhibition of NMDAR1 and/or NMDAR2B subunits, these data may indicate that certain NMDA receptor subunits may be of particular importance for the mediation of seizure activity following the discontinuation of chronic ethanol exposure.     

Oral administration of glycine and polyamine receptor antagonists blocks ethanol withdrawal seizures

Cessation of chronic administration of orally administered large amounts of ethanol for 7 days resulted in a markedly increased frequency of audiogenic seizures in Sprague-Dawley rats. Oral administration of the novel glycine receptor antagonist, L-701,324, produced a dose-dependent (2.5 and 5.0 mg/kg; –30 min) inhibition of ethanol withdrawal signs when measured about 12 h after withdrawal of the ethanol treatment. Similarly, using the same experimental paradigm, oral administration of the specific polyamine receptor antagonist, eliprodil, caused a dose-related (2.0 and 5.0 mg/kg; –30 min) inhibition of ethanol withdrawal-induced audiogenic seizure activity. The inhibition of ethanol withdrawal seizures produced by L-701,324 and eliprodil, respectively, was obtained at doses which by themselves did not change the locomotor activity in naive Sprague-Dawley rats. The findings that L-701,324 and eliprodil are potent inhibitors of seizure activity induced by cessation of chronic ethanol administration and the fact that they, in contrast to currently available NMDA receptor antagonists, do not produce psychotomimetic and/or sedative effects, suggest that these drugs may represent a new class of therapeutically useful pharmacological agents for the treatment of ethanol withdrawal seizures. Furthermore, since there is evidence that eliprodil produces its pharmacological actions through a specific inhibition of NMDAR1 and/or NMDAR2B subunits, these data may indicate that certain NMDA receptor subunits may be of particular importance for the mediation of seizure activity following the discontinuation of chronic ethanol exposure.     

Anticonvulsant effects of eliprodil alone or combined with the glycineB receptor antagonist L-701,324 or the competitive NMDA antagonist CGP 40116 in the amygdala kindling model in rats

The discovery that glutamate's activity at the N-methyl-D-aspartate (NMDA) receptor is positively modulated by glycine and polyamines has led to a new pharmacological strategy that NMDA receptor-mediated events could be antagonized indirectly at the strychnine-insensitive glycine co-agonist site (glycine(B) receptor) and the polyamine modulatory site. Recently we demonstrated that ifenprodil and L-701,324 (7-chloro-4-hydroxy-3(3-phenoxy)phenyl-2(H)quinoline), polyamine and glycine, receptor antagonists, respectively, at subeffective doses markedly increased after-discharge threshold (ADT) when applied together in amygdala-kindled rats. Because ifenprodil and its derivative, eliprodil, exhibit different affinities for NMDA receptors composed of different subunits, our current question was whether a combination of eliprodil and the glycine, receptor antagonist, L-701,324, would produce a super-additive anticonvulsant action. In addition, we examined the combined treatment of eliprodil with a competitive NMDA receptor antagonist CGP 40116 (D-(E)-2-amino-4-methyl-5-phosphono-3-pentenoic acid) in the kindling model. Eliprodil alone (10-40 mg/kg) had no consistent ADT-increasing activity. When eliprodil was combined with an ineffective dose of L-701,324 (2.5 mg/kg), a significant rise in ADT was observed. Likewise, other measures of seizure activity such as severity and duration were modestly but significantly reduced. With respect to behavioral impairments, no signs of synergistic interaction were observed after the drug combinations. On the other hand, no anticonvulsant effects were found when CGP 40116 was administered alone at doses of 1.25-5 mg/kg or CGP 40116 1.25 mg/kg combined with eliprodil 10 mg/kg. These data suggest that combination therapy with antagonists at the polyamine and glycine sites might potentially treat therapy-resistant complex partial seizures.     

The anticholinergic and antiglutamatergic drug caramiphen reduces seizure duration in soman-exposed rats: synergism with the benzodiazepine diazepam

Therapy of seizure activity following exposure to the nerve agent soman (GD) includes treatment with the anticonvulsant diazepam (DZP), an allosteric modulator of γ-aminobutyric acid A (GABA_A) receptors. However, seizure activity itself causes the endocytosis of GABA_A receptors and diminishes the inhibitory effects of GABA, thereby reducing the efficacy of DZP. Treatment with an N-methyl-d-aspartic acid (NMDA) receptor antagonist prevents this reduction in GABAergic inhibition. We examined the efficacy of the NMDA receptor antagonist caramiphen edisylate (CED; 20 mg/kg, im) and DZP (10 mg/kg, sc), administered both separately and in combination, at 10, 20 or 30 min following seizure onset for attenuation of the deleterious effects associated with GD exposure (1.2 LD₅₀; 132 μg/kg, sc) in rats. Outcomes evaluated were seizure duration, neuropathology, acetylcholinesterase (AChE) activity, body weight, and temperature. We also examined the use of the reversible AChE inhibitor physostigmine (PHY; 0.2 mg/kg, im) as a therapy for GD exposure. We found that the combination of CED and DZP yielded a synergistic effect, shortening seizure durations and reducing neuropathology compared to DZP alone, when treatment was delayed 20-30 min after seizure onset. PHY reduced the number of animals that developed seizures, protected a fraction of AChE from GD inhibition, and attenuated post-exposure body weight and temperature loss independent of CED and/or DZP treatment. We conclude that: 1) CED and DZP treatment offers considerable protection against the effects of GD and 2) PHY is a potential therapeutic option following GD exposure, albeit with a limited window of opportunity.     

Is agmatine a novel neurotransmitter in brain?

Recent evidence suggests that agmatine, which is an intermediate in polyamine biosynthesis, might be an important neurotransmitter in mammals. Agmatine is synthesized in the brain, stored in synaptic vesicles in regionally selective neurons, accumulated by uptake, released by depolarization, and inactivated by agmatinase. Agmatine binds to alpha2-adrenoceptors and imidazoline binding sites, and blocks NMDA receptor channels and other ligand-gated cationic channels. Furthermore, agmatine inhibits nitric oxide synthase, and induces the release of some peptide hormones. As a result of its ability to inhibit both hyperalgesia and tolerance to, and withdrawal from, morphine, and its neuroprotective activity, agmatine has potential as a treatment of chronic pain, addictive states and brain injury.     

The relationship between anticonvulsant activity and receptor affinity of N-methyl-D-aspartate antagonists in epileptic fowl

The N-methyl-D-aspartate (NMDA) receptor antagonists [(3-(+/-)2-carboxypiperazin-4-yl)-propyl-l-phosphonic acid (CPP), +/- 2-amino-7-phosphonoheptanoic acid (2AP7), +/- 2-amino-5-phosphonovaleric acid (2AP5), D-alpha-aminoadipic acid (alpha AA), and +/- alpha, epsilon-diaminopimelic acid (DAP)] were tested for anticonvulsant activity in epileptic chickens. There was a high correlation between anticonvulsant potencies (ED50) and the affinity for the NMDA receptor measured by displacement of L-[3H]glutamate from synaptosomal membranes. The high seizure susceptibility is not due to abnormalities in the NMDA receptor as comparison of KD, Bmax and Ki values in synaptosomal preparations from epileptic and non-epileptic chickens indicated no differences in NMDA receptor binding receptor characteristics.     

Agmatine modulates neuroadaptations of glutamate transmission in the nucleus accumbens of repeated morphine-treated rats

It has been proved that agmatine inhibits opioid dependence, yet the neural mechanism remains unclear. In the present study, the effect of agmatine on the neuroadaptation of glutamate neurotransmission induced by morphine dependence, including changes of the extracellular glutamate level and glutamate receptors in the nucleus accumbens was investigated.We found that agmatine (2.5–20 mg/kg, s.c.) inhibited development of morphine dependence, which was consistent with our previous report. In rats repeatedly treated with morphine, the glutamate level in the nucleus accumbens dialysate was markedly increased after naloxone-precipitated withdrawal. When agmatine (20 mg/kg, s.c.) was co-pretreated with morphine or was applied before naloxone-precipitated withdrawal, this elevation of the extracellular glutamate level was inhibited. In the synaptosome model, repeated morphine treatment and naloxone precipitation induced an increase in glutamate release, while agmatine (20 mg/kg, s.c.) co-pretreated with morphine reversed the increase of glutamate release. However, neither morphine or agmatine treatment alone nor morphine and agmatine co-administration had any influence on [3H]-glutamate uptake. It indicated that the elevation of the glutamate level in the nucleus accumbens might be caused by the increase of glutamate release of synaptosome in the withdrawal conditions of morphine-dependent rat. Furthermore, agmatine concomitant treatment with morphine entirely abolished the up-regulation of the NR1 subunit of N-methyl-d-aspartate (NMDA) receptors in the nucleus accumbens in repeated morphine-treated rats.Taken together, the present study demonstrated that agmatine could modulate the neuroadaptations of glutamate transmission in the nucleus accumbens in the case of morphine dependence, including modulating extracellular glutamate concentration and NMDA receptor expression.     

Repeated ethanol withdrawal experience selectively alters sensitivity to different chemoconvulsant drugs in mice

 Repeated ethanol withdrawal experience has been shown to result in exacerbated seizures associated with future withdrawal episodes. This sensitization of the withdrawal response has been postulated to represent a ”kindling” phenomenon. The present study employed an established model of repeated ethanol withdrawals to examine the potential role of GABA_A, and NMDA and non-NMDA glutamate receptor systems in mediating enhanced seizure activity, as assessed by sensitivity to seizures induced by pentylenetetrazol (PTZ), NMDA, and kainic acid (KA) IV infusions, respectively. Adult C3H mice were chronically exposed to ethanol vapor in inhalation chambers. A multiple withdrawal (MW) group received four cycles of 16-h ethanol vapor exposure interrupted by 8-h periods of abstinence; a single withdrawal (SW) group was tested after a single 16-h bout of ethanol intoxication; and the third group was ethanol-naive, serving as controls (C). Results indicated that the MW group evidenced significantly lower PTZ and NMDA seizure thresholds compared to SW and C groups at 8 and 24 h post-withdrawal. In contrast, MW and SW groups exhibited reduced sensitivity (higher seizure threshold) to KA in comparison to controls, and this effect only emerged at 24 h post-withdrawal. Further, MW mice required significantly less additional PTZ or NMDA to induce more severe convulsions once initial signs of seizures were elicited. Conversely, latency and amount of KA required to transition from initial seizure signs to more severe end-stage convulsions was significantly greater for MW and SW groups compared to controls. Taken together, these results suggest that repeated ethanol withdrawal experience does not result in a global non-specific lowering of threshold to convulsive stimuli, but rather, selective changes in CNS mechanisms associated with neural excitability may underlie potentiated withdrawal responses. Thus, reduced GABA_A receptor function and increased NMDA receptor activity may become exaggerated as a consequence of repeated withdrawal experience, while reduced sensitivity to KA induced seizures may represent a compensatory response to withdrawal-related CNS hyperexcitability. Received: 15 December 1997 / Final version: 13 February 1998     

Preclinical evaluation of CHF3381 as a novel antiepileptic agent

CHF3381 [n-(2-indanyl)-glycinamide hydrochloride] has been selected on the basis of a screening program as the compound displaying the highest anticonvulsant activity in the maximal electroshock seizure (MES) test and the best therapeutic index with reference to the rotarod test in mice and rats. In this study, the antiepileptic activity and the behavioural toxicity of CHF3381 were characterised in multiple model systems. CHF3381 effectively prevented MES-induced convulsions when administered i.p. (ED50, 24 mg/kg and 7.5 mg/kg) or p.o. (ED50, 21 mg/kg and 21 mg/kg) in both mice and rats, respectively. The time course of oral anti-MES activity in the rat was related to the brain concentration profile of unchanged CHF3381. Interestingly, the brain drug levels were about 4-5 times higher than in plasma. CHF3381 was very effective in mice against picrotoxin-, and i.c.v. N-methyl-D-aspartate (NMDA)-induced hind limb tonic extension (ED50 Approximately/=10 mg/kg), but was a weaker antagonist of 4-amynopyridine- and bicuculline-induced tonic seizures (ED50 approximately/=100 mg/kg), and ineffective against pentylentetrazole- and picrotoxin-induced clonic seizures. CHF3381 antagonised the behavioural effects and lethality of i.p. administered NMDA (ED50 = 57 mg/kg p.o.), indicating that the compound may act as a functional NMDA antagonist. In keeping with this idea, CHF3381 weakly displaced [(3)H]-TCP from binding to NMDA receptor channels (Ki, 8.8 microM). In the rat amygdala kindling model, CHF3381 was more efficient against kindling development than against kindled seizures (minimally active dose = 80 vs. 120 mg/kg i.p). Furthermore, it significantly increased the seizure threshold in kindled rats at relatively low doses (40 mg/kg i.p.). In contrast with MK-801-induced hyperactivity, CHF3381 moderately reduced the spontaneous locomotor activity in mice at anticonvulsant doses. Toxic effects on motor performance (rotarod test) were found at high doses only (TD50 approximately/= 300 mg/kg p.o., congruent with 100 mg/kg i.p. in both mice and rats). Furthermore, CHF3381 did not impair passive avoidance and Morris water maze responding in the therapeutic range of doses. Finally, the development of tolerance after repeated doses was negligible. These data indicate that CHF3381 exerts anticonvulsant and antiepileptogenic effects in various seizure models and possesses good therapeutic window, with scarce propensity to cause neurological side-effects.