Low-affinity NMDA Receptor Antagonist Research Articles

Autism spectrum disorder-like behaviors induced by hyper-glutamatergic NMDA receptor signaling through hypo-serotonergic 5-HT1A receptor signaling in the prefrontal cortex in mice exposed to prenatal valproic acid.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by repetitive behaviors, social deficits, and cognitive impairments. Maternal use of valproic acid (VPA) during pregnancy is associated with an increased risk of ASD in offspring. The prevailing pathophysiological hypothesis for ASD involves excitation/inhibition (E/I) imbalances and serotonergic dysfunction. Here, we investigated the association between glutamatergic-serotonergic neuronal interactions and ASD-like behaviors in mice exposed to prenatal VPA. Prenatal VPA exposure induced excessive repetitive self-grooming behavior and impaired social behavior and object recognition memory in young adult period. Prenatal VPA mice showed hyper-glutamatergic function (increase in basal extracellular glutamate levels and CaMKII phosphorylation) and hypo-serotonergic function (decrease in 5-hydroxyindoleacetic acid and stimulation-induced serotonin [5-HT] release, but an increase in 5-HT transporter expression) in the prefrontal cortex. Treatment with a low-affinity NMDA receptor antagonist (memantine), a selective 5-HT reuptake inhibitor (fluoxetine), and a 5-HT1A receptor agonist (tandospirone) attenuated both the increase in CaMKII phosphorylation and ASD-like behavior of prenatal VPA mice. Opto-genetic activation of the serotonergic neuronal system attenuated impairments in social behavior and object recognition memory in prenatal VPA mice. WAY-100635-a 5-HT1A receptor antagonist-antagonized the effect of fluoxetine on impaired social behavior and object recognition memory. These results suggest that E/I imbalance and ASD-like behavior are associated with hypo-serotonergic receptor signaling through 5-HT1A receptors in prenatal VPA mice.

Effect of the Topical External Formulation of the Low-Affinity NMDA Receptor Antagonist Hemantane on Peripheral Sensitization Induced by TRPV1 Receptor Agonist Capsaicin in Mice

Effect of the Topical External Formulation of the Low-Affinity NMDA Receptor Antagonist Hemantane on Peripheral Sensitization Induced by TRPV1 Receptor Agonist Capsaicin in Mice

N-Substituted-3-alkoxy-derivatives of dextromethorphan are functional NMDA receptor antagonists in vivo: Evidence from an NMDA-induced seizure model in rats

Interest in developing NMDA receptor antagonists with reduced side-effects for neurological and psychiatric disorders has been re-energized by the recent introduction of esketamine into clinical practice for treatment-resistant depression. Structural analogs of dextromethorphan bind with low affinity to the NMDA receptor ion channel, have functional effects in vivo, and generally display a lower propensity for side-effects than that of ketamine and other higher affinity antagonists. As such, the aim of the present study was to determine whether a series of N-substituted-3-alkoxy-substituted dextromethorphan analogs produce their anticonvulsant effects through NMDA receptor blockade. Compounds were studied against NMDA-induced seizures in rats. Compounds were administered intracerebroventricularly in order to mitigate confounds of drug metabolism that arise from systemic administration. Comparison of the anticonvulsant potencies to their affinities for NMDA, σ1, and σ2 binding sites were made in order to evaluate the contribution of these receptors to anticonvulsant efficacy. The potencies to block convulsions were positively associated with their affinities to bind to the NMDA receptor ion channel ([3H]-TCP binding) (r = 0.71, p < 0.05) but not to σ1 receptors ([3H]-SKF 10047 binding) (r = −0.31, p = 0.46) or to σ2 receptors ([3H]-DTG binding) (p = −0.38, p = 0.36). This is the first report demonstrating that these dextromethorphan analogs are functional NMDA receptor antagonists in vivo. Given their potential therapeutic utility and favorable side-effect profiles, such low affinity NMDA receptor antagonists could be considered for further development in neurological (e.g., anticonvulsant) and psychiatric (e.g., antidepressant) disorders.

The Role of a Dopaminergic Component in the Analgesic Effect of the Low-Affinity NMDA Receptor Antagonist Hemantane

The role of dopaminergic transmission in the mechanism of analgesic action of the low-affinity NMDAreceptor antagonist hemantane [N-(2-adamantyl)hexamethyleneimine hydrochloride] was demonstrated experimentally. A single i.p. injection of hemantane at a dose of 20 mg/kg has the ability to inhibit dopamine reuptake and exhibits an analgesic effect in the somatic thermal pain model (tail-flick test) and visceral pain model (acetic-acid-induced writhing test) in ICR mice. The dopamine D1 receptor antagonist R(+)-SCH-23390 at a dose of 0.005 mg/kg and dopamine D2 receptor antagonist sulpiride at a dose of 5 mg/kg administered by a single i.p. injection 30 min before hemantane injection prevented manifestation of the antinociceptive activity of the NMDA receptor antagonist in the used methods, which was more pronounced for the spinal reflex (tail-flick test).

Роль дофаминергического компонента в механизме анальгетического действия низкоаффинного блокатора NMDA рецептора гимантана

В проведённом экспериментальном исследовании выявлена роль дофаминергического компонента в механизме анальгетического действия низкоаффинного блокатора NMDA рецептора гимантана (N-(2-адамантил)гексаметиленимина гидрохлорида). Гимантан, обладающий способностью ингибировать обратный захват дофамина, при однократном внутрибрюшинном введении в дозе 20 мг/кг проявляет анальгетический эффект на моделях соматической термической (тест отдёргивания хвоста от теплового излучения) и висцеральной (тест «Уксусные корчи») боли у мышей ICR. Блокатор D1-дофаминергического рецептора R(+)-SCH-23390 в дозе 0,005 мг/кг и блокатор D2-дофаминергического рецептора сульпирид в дозе 5 мг/кг при однократном внутрибрюшинном введении за 30 мин до инъекции гимантана предотвращают проявление антиноцицептивной активности изучаемого блокатора NMDA рецептора в используемых методиках, что более ярко проявляется при оценке его влияния на спинальный флексорный рефлекс.

Memantine effects on auditory discrimination and training in schizophrenia patients.

The uncompetitive low-affinity NMDA receptor antagonist, memantine, acutely increases electrophysiological measures of auditory information processing in both healthy subjects (HS) and patients with schizophrenia. Memantine effects on functional measures of auditory discrimination performance and learning are not known; conceivably, beneficial effects on these measures might suggest a role for memantine in augmenting the cognitive and functional impact of auditory targeted cognitive training (TCT). Here, carefully characterized HS (n = 20) and schizophrenia patients (n = 22) were tested in measures of auditory discrimination performance (words-in-noise (WIN), quick speech-in-noise (QuickSIN), gaps-in-noise) and auditory frequency modulation learning (a component of TCT) on 2 days about a week apart, after ingesting either placebo or 20 mg memantine po, in a double-blind, within-subject cross-over random order design. Memantine modestly enhanced functional measures of auditory discrimination in both schizophrenia patients (WIN) and HS (WIN and QuickSIN), as well as auditory frequency modulation learning in schizophrenia patients. These findings converge with a growing literature showing that memantine can enhance a range of metrics of auditory function. These properties could contribute to the apparent benefits of memantine as an adjunctive treatment in schizophrenia, and suggest that memantine might augment learning and potentially clinical gains from auditory-based TCT.

Effects of Aminoadamantane Derivatives on Morphine-Induced Analgesia in Mice

The effects of low-affinity NMDA-receptor antagonists amantadine (1-aminoadamantane hydrochloride) and hemantane [N-(2-adamantyl)hexamethyleneimine hydrochloride] on morphine-induced analgesia in C57Bl/6 mice were studied. Amantadine (10 and 20 mg/kg, i.p.) per se did not affect the latent period of the response in the hot-plate test while hemantane (10 and 20 mg/kg, i.p.) increased dose-dependently pain thresholds 180 and 240 min after administration. Morphine (20 mg/kg, s.c.) showed a time—effect dependence (30 – 120 min). The aminoadamantanes were administered 90 min after the opioid to assess their effects on morphine-induced antinociception. The responses of the animals were recorded for the next 2.5 h. The aminoadamantanes potentiated and extended the analgesic activity of morphine in the order of efficacy amantadine < hemantane. The results indicated that the aminoadamantanes had different capabilities to cause delayed analgesia and modulated opioid antinociceptive activity at the supraspinal level.

Особенности действия производных аминоадамантана на этанол-индуцированную атаксию, седацию и гиперлокомоторную реакцию у мышей

В опытах in vivo изучено влияние 2 производных аминоадамантана, являющихся низкоаффинными антагонистами NMDA-рецепторов и обладающих выраженной активностью на экспериментальных моделях паркинсонизма и у пациентов с болезнью Паркинсона, на острые эффекты этанола в эксперименте. Гимантан (гидрохлорид N-2-адамантил-гексаметиленимина) в дозах 5 – 20 мг/кг, внутрибрюшинно, дозозависимо предотвращал вызываемую этанолом атаксию у мышей CD-1, седацию у — C57Bl/6 и гиперлокомоторную реакцию у DBA/2 мышей. Амантадин (1-аминоадамантан) в дозах 10 – 20 мг/кг, внутрибрюшинно, не обладал способностью ослаблять острые эффекты этанола (2 г/кг, внутрибрюшинно). Гимантан и амантадин не влияли на продолжительность этанолового наркоза (5,5 г/кг, внутрибрюшинно) у мышей CD-1. Полученные данные указывают на отличие фармакодинамического профиля гимантана как низкоаффинного антагониста NMDA-рецептора при взаимодействии с этанолом в дозе, вызывающей нарушение поведения.

Memantine, a Low-Affinity NMDA Receptor Antagonist, Protects against Methylmercury-Induced Cytotoxicity of Rat Primary Cultured Cortical Neurons, Involvement of Ca2+ Dyshomeostasis Antagonism, and Indirect Antioxidation Effects

Methylmercury (MeHg) is an extremely dangerous environmental pollutant that induces severe toxic effects in the central nervous system. Neuronal damage plays critical roles mediating MeHg-induced loss of brain function and neurotoxicity. The molecular mechanisms of MeHg neurotoxicity are incompletely understood. The objective of the study is to explore mechanisms that contribute to MeHg-induced neurocyte injuries focusing on neuronal Ca2+ dyshomeostasis and alteration of N-methyl-D-aspartate receptors (NMDARs) expression, as well as oxidative stress in primary cultured cortical neurons. In addition, the neuroprotective effects of memantine against MeHg cytotoxicity were also investigated. The cortical neurons were exposed to 0, 0.01, 0.1, 1, or 2μM methylmercury chloride (MeHgCl) for 0.5-12h, or pre-treated with 2.5, 5, 10, or 20μM memantine for 0.5-6h, respectively; cell viability and LDH release were then quantified. For further experiments, 2.5, 5, and 10μM of memantine pre-treatment for 3h followed by 1μM MeHgCl for 6h were performed for evaluation of neuronal injuries, specifically addressing apoptosis; intracellular free Ca2+ concentrations; ATPase activities; calpain activities; expressions of NMDAR subunits (NR1, NR2A, NR2B); NPSH levels; and ROS formation. Exposure of MeHgCl resulted in toxicity of cortical neurons, which were shown as a loss of cell viability, high levels of LDH release, morphological changes, and cell apoptosis. Moreover, intracellular Ca2+ dyshomeostasis, ATPase activities inhibition, calpain activities, and NMDARs expression alteration were observed with 1μM MeHgCl administration. Last but not least, NPSH depletion and reactive oxygen species (ROS) overproduction showed an obvious oxidative stress in neurons. However, memantine pre-treatment dose-dependently antagonized MeHg-induced neuronal toxic effects, apoptosis, Ca2+ dyshomeostasis, NMDARs expression alteration, and oxidative stress. In conclusion, the cytoprotective effects of memantine against MeHg appeared to be mediated not only via its NMDAR binding properties and Ca2+ homeostasis maintenance but also by indirect antioxidation effects.

1MeTIQ Provides Protection Against Aβ-Induced Reduction of Surface Expression of Synaptic Proteins and Inhibits H2O2-Induced Oxidative Stress in Primary Hippocampal Neurons

Alzheimer's disease (AD) is associated with increased brain levels of β-amyloid (Aβ) peptides, which readily self-aggregate into fibrils and oligomers that have particularly deleterious properties toward synapses of excitatory glutamatergic neurons. Here, we examined the neuroprotective effects of 1-methyl-1,2,3,4,-tetrahydroisoquinoline (1MeTIQ) against Aβ-induced loss of synaptic proteins in cultured primary hippocampal neurons. Exposure of mature primary hippocampal neurons to 10 μM synthetic Aβ1-40 over 72 h resulted in ~60 % reduction in the surface expression of NR1 subunit of the NMDA receptor (NMDAR), PSD-95, and synaptophysin, without causing neuronal death. Concomitant treatment with 500 μM of 1MeTIQ, a low-affinity NMDAR antagonist significantly ameliorated the loss of synaptic protein markers. The neuroprotective properties of 1MeTIQ were compared with those of MK-801, which at 0.5 μM concentration also prevented Aβ1-40-induced loss of synaptic proteins in primary neuronal cultures. Furthermore, we provide novel evidence demonstrating effectiveness of 1MeTIQ in reducing the level of reactive oxygen species (ROS) in primary neuronal culture system. As oxidative stress contributes importantly to neurodegeneration in AD, 1MeTIQ may provide a dual neuroproctective effect in AD both as a NMDARs antagonist and ROS formation inhibitor. 1MeTIQ occurs endogenously at low concentrations in the brain and its synthetic form readily penetrates the blood-brain barrier after the systemic administration. Our results highlight a possibility of the application of 1MeTIQ as a neuroprotective agent in AD-related neurodegeneration.

Memantine potentiates hippocampal theta oscillations at a therapeutic dose in anesthetized mice: A mechanistic link to its cognitive-enhancing properties

Memantine is an uncompetitive, low-affinity NMDA receptor antagonist clinically used for the treatment of cognitive deficits in moderate to severe Alzheimer’s disease. Both neurophysiological and behavioral studies in rodents have suggested a beneficial effect of memantine on synaptic plasticity and learning performances. In the present study, we investigated the effect of memantine on pedonculopontine-elicited theta oscillations in the hippocampus of urethane anesthetized mice, a model shown to be sensitive to several pharmacological agents exhibiting cognitive-enhancing properties. We found that a low dose of memantine potentiated elicited theta power while a high dose was disruptive. The low dose of memantine used was shown to yield an unbound brain concentration well within the range of therapeutic concentrations reported in rodent brain extracellular fluid and human cerebrospinal fluid. For further comparison, the effect of another uncompetitive NMDA receptor antagonist with higher affinity, i.e. MK-801, was also investigated. MK-801 was at a low dose devoid of effect on elicited theta power, while a high dose, within the range of doses reported to induce cognitive deficits in a variety of hippocampal-dependent learning paradigms in mice, was found disruptive on elicited theta waves. Taken together, our results suggest that clinically relevant doses of memantine promote neuronal network synchronization in the hippocampus, which may represent an underlying mechanism for the reported cognitive-enhancing properties in both preclinical and clinical studies.

Low doses of dextromethorphan attenuate morphine-induced rewarding via the sigma-1 receptor at ventral tegmental area in rats

Chronic use of morphine causes rewarding and behavioral sensitization, which may lead to the development of psychological craving. In our previous study, we found that a widely used antitussive dextromethorphan (known as a low affinity NMDA receptor antagonist), at doses of 10–20 mg/kg (i.p.), effectively decreased morphine rewarding in rats. In this study, we further investigated the effects and mechanisms of low doses of DM (μg/kg range) on morphine rewarding and behavioral sensitization. A conditioned place preference test was used to determine the rewarding and a locomotor activity test was used to determine the behavioral sensitization induced by the drug(s) in rats. When a low dose of DM (3 or 10 μg/kg, i.p.) was co-administered with morphine (5 mg/kg, s.c.), the rewarding effect, but not behavioral sensitization, induced by morphine was inhibited. The inhibiting effect of DM could be blocked by systemically administering a sigma-1 receptor antagonist, BD1047 (3 mg/kg, i.p.). When BD1047 (5 nmole/site) was locally given at the VTA, it also blocked the effects of a low dose of DM in inhibiting morphine rewarding. Our findings suggest that the activation of the sigma-1 receptor at the VTA may be involved in the mechanism of low doses of DM in inhibiting the morphine rewarding effect and the possibility of using extremely low doses of DM in treatment of opioid addiction in clinics.

Dose-related effects of memantine on a mismatch negativity-like response in anesthetized rats

Memantine is a low-affinity NMDA receptor antagonist that is used in the treatment of Alzheimer's disease to alleviate the cognitive symptoms of the disease. In humans, memantine has been shown to facilitate auditory change detection as reflected in the mismatch negativity (MMN) response recorded in the frontal cortex. In the present study we investigated the effects of memantine on the auditory MMN-like responses recorded in anesthetized rats. Saline, a low (3 mg/kg) or a high (10 mg/kg) dose of memantine was i.p. injected into the animals. Auditory MMN-like responses were recorded during the presentation of a repeated tone of one frequency (standard, P=0.956) that was rarely replaced by a tone of another frequency (deviant, P=0.044). The low dose of memantine did not observably affect the amplitude of the auditory MMN-like response, but it prolonged the duration of the response relative to saline. The high dose of memantine, in contrast, blocked the generation of the auditory MMN-like response. The findings suggest that memantine may, with appropriate doses, facilitate already this early stage of auditory processing.

The rise and fall of Dimebon

Dimebon (latrepirdine) was developed and used in Russia as an over-the-counter oral antihistamine for allergy treatment. In the early 1990s, Dimebon was characterized as a low-affinity NMDA receptor antagonist by Dr. Sergey Bachurin and his colleagues. An initial small-scale, open-label trial of Dimebon in 14 Alzheimer's disease (AD) patients demonstrated potential efficacy. Dimebon was then patented for the treatment of neurodegenerative disorders and licensed by Medivation. Extremely promising results were obtained in a double-blind, placebo-controlled, phase II AD trial in 183 patients; however, a phase II trial of Dimebon in 91 Huntington's disease patients was much less successful. Recently, a phase III AD trial of Dimebon in 598 patients failed to result in any significant improvement in primary or secondary outcomes. The failure of Dimebon may be in large part due to insufficient understanding of its mechanism of action. The NMDA receptor blocking activity of Dimebon is too weak to be physiologically relevant, while the proposed "novel mitochondrial mechanism of action" lacks credible scientific evidence or a molecular target. Independent studies indicate that the clinical effects of Dimebon most likely result from inhibition of histamine H₁ and serotonin 5-HT₆ receptors. Careful preclinical studies of novel potential therapies are needed to minimize chances of making similar costly mistakes in the future.

GluN2B subunit-containing NMDA receptor antagonists prevent Aβ-mediated synaptic plasticity disruption in vivo

Currently, treatment with the relatively low-affinity NMDA receptor antagonist memantine provides limited benefit in Alzheimer's disease (AD). One probable dose-limiting factor in the use of memantine is the inhibition of NMDA receptor-dependent synaptic plasticity mechanisms believed to underlie certain forms of memory. Moreover, amyloid-beta protein (Abeta) oligomers that are implicated in causing the cognitive deficits of AD potently inhibit this form of plasticity. Here we examined if subtype-preferring NMDA receptor antagonists could preferentially protect against the inhibition of NMDA receptor-dependent plasticity of excitatory synaptic transmission by Abeta in the hippocampus in vivo. Using doses that did not affect control plasticity, antagonists selective for NMDA receptors containing GluN2B but not other GluN2 subunits prevented Abeta(1-42) -mediated inhibition of plasticity. Evidence that the proinflammatory cytokine TNFalpha mediates this deleterious action of Ass was provided by the ability of TNFalpha antagonists to prevent Abeta(1-42) inhibition of plasticity and the abrogation of a similar disruptive effect of TNFalpha using a GluN2B-selective antagonist. Moreover, at nearby synapses that were resistant to the inhibitory effect of TNFalpha, Abeta(1-42) did not significantly affect plasticity. These findings suggest that preferentially targeting GluN2B subunit-containing NMDARs may provide an effective means of preventing cognitive deficits in early Alzheimer's disease.

Understanding pseudo

Why do patients with multiple sclerosis (MS) sometimes develop transient worsening of preexisting symptoms? The cause is often attributed to “disruption of compensatory mechanisms.” The observation—and the weak explanation—are not unique to demyelinating disease. Patients who have previously had a stroke or those with progressive dementia can have recurrence of symptoms or clinical worsening with infection. The conventional wisdom is that the same mechanisms underlie this phenomenon wherever it is encountered. While transient worsening with systemic infection is seen after stroke, it is not seen with simple elevation in temperature or routine fever as frequently as it is encountered in MS. It is a mistake of inference to suppose that the same mechanisms operate across the spectrum of neurologic disease. MS specialists are well aware that many of their patients have greater variability of symptoms than stroke patients or those with other CNS injury. In this issue of Neurology ®, Villoslada et al.1 have serendipitously discovered a pharmacologic method to recreate this clinical observation. They conducted a double-blind placebo-controlled trial of the low affinity NMDA receptor antagonist memantine in …

Effects of high- and low-affinity NMDA-receptor antagonists on an auditory MMN-like response recorded in anesthetized rats

Effects of high- and low-affinity NMDA-receptor antagonists on an auditory MMN-like response recorded in anesthetized rats

The effect of 12-week open-label memantine treatment on cognitive function improvement in patients with alcohol-related dementia

There is compelling evidence that alcohol-induced neurotoxicity is related to glutamate excitotoxicity. It was hypothesized that the low-affinity NMDA receptor antagonist memantine would improve the cognitive function of patients with alcoholic dementia. The aim of this study was to test this hypothesis and to evaluate the effect of memantine on the cognitive improvement of patients with alcohol-related dementia (ARD). The study was designed as a 12-wk open-label study investigating the efficacy of 20 mg memantine, a low-affinity NMDA receptor antagonist, as a treatment for cognitive and behavioural problems in 19 patients with probable ARD according to the criteria for ARD proposed by Oslin and colleagues. The CERAD-K (Consortium to Establish a Registry for Alzheimer's Disease - Korean version) and several clinical assessment scales were completed before and after the 12-wk memantine treatment period. Significant improvements in the mean scores from baseline to final assessment were observed in the Global Deterioration Scale (p<0.05), Brief Psychiatric Rating Scale (p<0.01), Geriatric Quality of Life - Dementia scale (p<0.01) and Neuropsychiatric Inventory (p<0.01) at the end of week 12. The CERAD-K subscales of word list recall (p<0.05), word list recognition (p<0.05), time orientation (p<0.01), drawing an interlocking pentagon (p<0.05), and the total MMSE-K (Mini Mental State Examination - Korean version) scores (p<0.01) of the patients all showed significant improvement following the memantine trial. In this open-label study, patients with ARD treated with 20 mg/d memantine for 12 wk showed improvement on global cognition, quality of life and behavioural symptoms. The result of this study suggests the possible usefulness of memantine for the treatment of ARD. As this was an open-label study, the possibility that participants improved cognitively on their own due to protracted abstinence from alcohol cannot be discounted.

Memantine reduces neuronal dysfunctions triggered by in vitro ischemia and 3-nitropropionic acid

Memantine, a low-affinity uncompetitive NMDA receptor antagonist, has been widely utilized for the treatment of Alzheimer's disease. A possible neuroprotective role of this drug in pathophysiological conditions involving an altered energetic metabolism of the basal ganglia has never been addressed. Thus, we have characterized the electrophysiological effect of memantine on striatal spiny neurons recorded under control conditions and after in vitro ischemia (oxygen and glucose deprivation). Memantine reduced in a dose-dependent manner (EC 50 = 5 μM) the irreversible loss of field potential amplitude induced by in vitro ischemia. The neuroprotective effect of memantine against in vitro ischemia was even more potent (EC 50 = 3.2 μM) in the absence of external magnesium, a condition enhancing NMDA-mediated glutamatergic transmission. Memantine was also able to block long-term potentiation recorded from spiny neurons following a brief ischemic episode. Moreover, memantine showed protection against irreversible field potential loss induced by 3-nitropropionic acid (3-NP), an inhibitor of the mitochondrial complex II, without influencing toxicity induced by rotenone, a complex I inhibitor. Memantine could represent a potential neuroprotective agent in pathophysiological conditions involving an altered energy metabolism of basal ganglia.

3α-OL-5-β-pregnan-20-one hemisuccinate, a steroidal low-affinity NMDA receptor antagonist improves clinical rating scores in a rabbit multiple infarct ischemia model: Synergism with tissue plasminogen activator

We previously showed that the neuroactive steroid 3α-ol-5-β-pregnan-20-one hemisuccinate (ABHS), a low-affinity NMDA receptor antagonist, improves behavior in rabbits following spinal cord ischemia. The present study assessed whether this also occurs following cerebral ischemia. We used the rabbit small clot embolic stroke model (RSCEM), a model of multiple infarct ischemia, which has a well-defined behavioral endpoint. Quantal analysis for each treatment determines microclots (mg) that produce neurologic dysfunction in 50% of a group of animals ( P 50), with treatment considered beneficial if it increases the P 50 compared to controls. ABHS (25 mg/kg) injected 5 min post-embolization significantly ( P = 0.046) increased the P 50 to 2.60 ± 0.69 mg compared to 1.15 ± 0.19 mg in controls but was ineffective at longer intervals. For combination studies with tissue plasminogen activator (tPA), a cumulative control curve ( P 50 = 1.18 ± 0.25 mg) was used for statistical comparisons. Low-dose tPA (0.9 mg/kg) and ABHS, given 60 min post-embolization, improved behavior synergistically ( P 50 = 2.44 ± 0.44 mg), compared to tPA ( P 50 = 1.25 ± 0.25 mg) or ABHS ( P 50 = 1.05 ± 0.24 mg) alone. A standard tPA dose (3.3 mg/kg) significantly increased the P 50 at 60 (2.69 ± 0.19 mg) but not 180 min (1.28 ± 0.31 mg) post-embolization. However, when combined with ABHS, an effect was evident even 180 min post-embolization ( P 50 = 2.31 ± 0.48 mg). Although the therapeutic window for ABHS is limited, it significantly enhanced neuroprotection by low-dose tPA and increased the therapeutic window for tPA, suggesting that it may be most useful as a co-therapy with thrombolytics for stroke.