Anti-NMDAR encephalitis induced in mice by active immunization with a peptide from the amino-terminal domain of the GluN1 subunit.

BACKGROUND: Anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis is a recently discovered autoimmune syndrome associated with psychosis, dyskinesia, and seizures. However, the underlying mechanisms of this disease remain unclear, in part because of a lack of suitable animal models. METHODS: This study describes a novel female C57BL/6 mouse model of anti-NMDAR encephalitis that was induced by active immunization against NMDARs using an amino terminal domain (ATD) peptide from the GluN1 subunit (GluN1356-385). RESULTS: Twelve weeks after immunization, the immunized mice showed significant memory loss. Furthermore, antibodies from the cerebrospinal fluid of immunized mice decreased the surface NMDAR cluster density in hippocampal neurons which was similar to the effect induced by the anti-NMDAR encephalitis patients' antibodies. Immunization also impaired long-term potentiation at Schaffer collateral-CA1 synapses and reduced NMDAR-induced calcium influx. CONCLUSION: We established a novel anti-NMDAR encephalitis model using active immunization with peptide GluN1356-385 targeting the ATD of GluN1. This novel model may allow further research into the pathogenesis of anti-NMDAR encephalitis and aid in the development of new therapies for this disease.

Roles of N-methyl-d-aspartate receptors during the sensory stimulation-evoked field potential responses in mouse cerebellar cortical molecular layer.

The functions of N-methyl-d-aspartate receptors (NMDARs) in cerebellar cortex have been widely studied under in vitro condition, but their roles during the sensory stimulation-evoked responses in the cerebellar cortical molecular layer in living animals are currently unclear. We here investigated the roles of NMDARs during the air-puff stimulation on ipsilateral whisker pad-evoked field potential responses in cerebellar cortical molecular layer in urethane-anesthetized mice by electrophysiological recording and pharmacological methods. Our results showed that cerebellar surface administration of NMDA induced a dose-dependent decrease in amplitude of the facial stimulation-evoked inhibitory responses (P1) in the molecular layer, accompanied with decreases in decay time, half-width and area under curve (AUC) of P1. The IC50 of NMDA induced inhibition in amplitude of P1 was 46.5µM. In addition, application of NMDA induced significant increases in the decay time, half-width and AUC values of the facial stimulation-evoked excitatory responses (N1) in the molecular layer. Application of an NMDAR blocker, D-APV (250µM) abolished the facial stimulation-evoked P1 in the molecular layer. These results suggested that NMDARs play a critical role during the sensory information processing in cerebellar cortical molecular layer in vivo in mice.

Panel de auto-anticuerpos para encefalitis autoinmune / Autoantibody panel for autoimmune encephalitis

CONTEXTO CLÍNICO: La encefalitis autoinmune (EA) es un desorden neurológico debilitante que se desarrolla como una encefalopatía rápidamente progresiva y es causada por inflamación cerebral de etiología autoinmune. Se estima que la incidencia anual de encefalitis de cualquier causa en Europa es de 2 a 3 por 100.000 habitantes. De todas las encefalitis, 20% son inmunomediadas y la más común es la encefalitis anti-NMDA (4%). Entre las EA más frecuentes se encuentra la encefalitis anti-NMDA, encefalitis límbica, encefalitis de ganglios basales, síndrome de Morvan, entre otros. Todas estas se han asociado a una seria de auto-anticuerpos ya sean detectados en sangre o en LCR. La EA involucra una serie de enfermedades que son provocadas por auto-anticuerpos dirigidos a epítopes extracelulares de canales de iones, receptores celulares y otras proteínas. La asociación con cáncer es variable en estas patologías y el pronóstico es bueno pudiendo tener efectos reversibles en la función sináptica neuronal. Después de haber excluido las causas más frecuentes de encefalitis como causas infecciosas, se sospecha de una EA cuando el paciente presenta: disminución progresiva de niveles de la conciencia (en días a semanas) con fluctuaciones y alteración de la cognición; alteración de la memoria, especialmente la retención de nueva información; síntomas psiquiátricos; entre otros. Debido a que la sintomatología no es específica, el diagnóstico se realiza con al menos uno de los siguientes: evidencia de nueva lesión focal del sistema nervioso central, convulsiones no explicadas por un desorden convulsivo previo, pleocitosis en el líquido cefalorraquídeo (LCR) o hallazgos sugestivos de encefalitis por resonancia magnética nuclear. En la mayoría de los casos el tratamiento de la EA (ya sea ante la sospecha de EA o con un diagnóstico establecido) se basa en el manejo con corticoesteroides, plasmaféresis y/o inmunoglobulina G endovenosa. Se postula que la detección de un panel de auto-anticuerpos es una herramienta fundamental para el diagnóstico y evaluación de respuesta al tratamiento para pacientes con encefalitis autoinmune. TECNOLOGÍA: Los auto-anticuerpos involucrados en la fisiopatología de la EA se pueden clasificar según su objetivo en: a) anticuerpos contra antígenos intracelulares como anti-GAD; b) anticuerpos contra receptores de sinapsis como: anti-NMDA, anti-AMPA, anti-GABA, entre otros; c) anticuerpos contra canales de iones como: anti-LGI1, anti CASPR-2; y, d) anticuerpos contra otras proteinas de superficie como: antififisina y anti receptor de la glicina. OBJETIVO: Evaluar la evidencia disponible acerca de la eficacia, seguridad y aspectos relacionados a las políticas de cobertura del panel de auto-anticuerpos para encefalitis autoinmune. MÉTODOS: Se realizó una búsqueda en las principales bases de datos bibliográficas (incluyendo Medline, Cochrane y CRD), en buscadores genéricos de Internet, agencias de evaluación de tecnologías sanitarias y financiadores de salud utilizando la siguiente estrategia: (Anti-N-Methyl-D-Aspartate Receptor Encephalitis[MeSH] OR Limbic Encephalitis[MeSH] OR Autoimmune Encephalitis [tiab]) AND (Autoantibodies[Mesh] OR Autoantibod*[tiab] OR autoantibody panel[tiab]) AND (Sensitivity and Specificity[Mesh] OR Sensitivity[tiab] OR Specificity[tiab]). Se priorizó la inclusión de revisiones sistemáticas (RS), estudios clínicos aleatorizados controlados, evaluaciones de tecnologías sanitarias y económicas, guías de práctica clínica y políticas de cobertura de otros sistemas de salud cuando estaban disponibles. RESULTADOS: Para el siguiente informe se incluyeron tres estudios observacionales, dos guías de práctica clínica y tres políticas de cobertura. No se encontraron revisiones sistemáticas, estudios clínicos controlados aleatorizados ni evaluaciones de tecnologías sanitarias que evalúen la detección de algún auto-anticuerpo para EA. De los anticuerpos listados en el Anexo 1, sólo se encontró evidencia con suficiente calidad respecto a la utilización de auto-anticuerpo anti-NMDA, anti-LGI1, anti-CASPR2, anti-GABA y anti-AMPA. CONCLUSIONES: La evidencia con respecto a la utilización de auto-anticuerpos para el diagnóstico de encefalitis autoinmune es de baja calidad. No se encontraron estudios clínicos aleatorizados controlados que evaluaran desenlaces clínicos tras un cambio de estrategia terapéutica guiada por auto-anticuerpos. Para la mayoría de los auto-anticuerpos no se encontraron estudios de precisión diagnóstica. Los estudios observacionales seleccionados sólo evidencian asociación entre la frecuencia de detección de diferentes auto-anticuerpos y encefalitis autoinmune. No se encontraron evaluaciones de tecnologías sanitarias que evalúen algún auto-anticuerpo para encefalitis autoinmune. Las guías de práctica clínica recomiendan anti-NMDA para el diagnóstico definitivo de encefalitis anti-NMDA y; auto-anticuerpos anti-LGI1, anti-GABA y anti-AMPA cuando hay un diagnóstico probable de encefalitis límbica.

Investigating dynamic structural and mechanical changes of neuroblastoma cells associated with glutamate-mediated neurodegeneration.

Glutamate-mediated neurodegeneration resulting from excessive activation of glutamate receptors is recognized as one of the major causes of various neurological disorders such as Alzheimer's and Huntington's diseases. However, the underlying mechanisms in the neurodegenerative process remain unidentified. Here, we investigate the real-time dynamic structural and mechanical changes associated with the neurodegeneration induced by the activation of N-methyl-D-aspartate (NMDA) receptors (a subtype of glutamate receptors) at the nanoscale. Atomic force microscopy (AFM) is employed to measure the three-dimensional (3-D) topography and mechanical properties of live SH-SY5Y cells under stimulus of NMDA receptors. A significant increase in surface roughness and stiffness of the cell is observed after NMDA treatment, which indicates the time-dependent neuronal cell behavior under NMDA-mediated neurodegeneration. The present AFM based study further advance our understanding of the neurodegenerative process to elucidate the pathways and mechanisms that govern NMDA induced neurodegeneration, so as to facilitate the development of novel therapeutic strategies for neurodegenerative diseases.

Intracisternal administration of NR2 antagonists attenuates facial formalin-induced nociceptive behavior in rats.

AIMS: To examine the antinociceptive effects of N-Methyl-D-aspartate (NMDA) receptor NR2 subunit antagonists in a rat model of the facial formalin test. METHODS: Experiments were carried out on adult male Sprague-Dawley rats weighing 220 to 280 g. Anesthetized rats were individually mounted on a stereotaxic frame and a polyethylene tube was implanted for intracisternal injection and, 72 hours later, formalin tests were performed. NMDA receptor antagonists were administered intracisternally 10 minutes prior to subcutaneous injection of 5% formalin (50 MicroL) into the vibrissal pad. RESULTS: The intracisternal administration of 25, 50, or 100 Microg of memantine, an antagonist that acts at the NMDA ion channel site, significantly suppressed the number of scratches in the second phase of the behavioral responses to formalin. Intracisternal administration of a range of doses of 5,7-dichlorokynurenic acid, a glycine site antagonist, or DL-2-amino-5-phosphonopentanoate (AP-5), a nonselective NMDA site antagonist, produced significant antinociceptive effects in the second phase. Intracisternal administration of 1, 2.5, or 5 Microg of (2R,4S)-4-(3 Phosphonopropyl)-2-piperidine_carboxylic acid (PPPA), a competitive NR2A antagonist, significantly suppressed the number of scratches in the second phase, while only the highest dose of PPPA (5 Microg) significantly suppressed the number of scratches in the first phase. The antinociceptive effects of intracisternal injection of (alphaR, betaS)-alpha-(4Hydroxyphenyl)-_ methyl-4-(phenylmethyl)-1-Piperidinepropanol maleate(Ro 25-6981), a selective NR2B antagonist, were similar to those of PPPA. Injection of memantine, AP-5, Ro 25-6981, or vehicle did not result in any motor dysfunction. A low dose of PPPA (1 microg) or 5,7-dichlorokynurenic acid (2.5 microg) did not affect motor function. However, higher doses of PPPA and 5,7-dichlorokynurenic acid produced motor dysfunction. CONCLUSION: The present results suggest that central NR2 subunits play an important role in orofacial nociceptive transmission. Moreover, this data also indicate that targeted inhibition of the NMDA receptor NR2 subunit is a potentially important new treatment approach for inflammatory pain originating in the orofacial area.

Development of a three-dimensional model for the N-methyl-D-aspartate NR2A subunit.

NR2 subunits of N-methyl-d-aspartic acid (NMDA) receptors are known to bind the neurotransmitter glutamate, competitive agonists, and antagonists. Since crystallographic data of these proteins are not available, we built a homology model of the ligand binding domain of the NR2A subunit. A consensus binding mode of selected AP5-like NMDA antagonists has been ascertained using molecular docking. The present 3D model gives insights for the design of new NMDA subtype selective compounds.

The activation of spinal N-methyl-D-aspartate receptors may contribute to degeneration of spinal motor neurons induced by neuraxial morphine after a noninjurious interval of spinal cord ischemia.

We investigated the relationship between the degeneration of spinal motor neurons and activation of N-methyl-d-aspartate (NMDA) receptors after neuraxial morphine following a noninjurious interval of aortic occlusion in rats. Spinal cord ischemia was induced by aortic occlusion for 6 min with a balloon catheter. In a microdialysis study, 10 muL of saline (group C; n = 8) or 30 mug of morphine (group M; n = 8) was injected intrathecally (IT) 0.5 h after reflow, and 30 mug of morphine (group SM; n = 8) or 10 muL of saline (group SC; n = 8) was injected IT 0.5 h after sham operation. Microdialysis samples were collected preischemia, before IT injection, and at 2, 4, 8, 24, and 48 h of reperfusion (after IT injection). Second, we investigated the effect of IT MK-801 (30 mug) on the histopathologic changes in the spinal cord after morphine-induced spastic paraparesis. After IT morphine, the cerebrospinal fluid (CSF) glutamate concentration was increased in group M relative to both baseline and group C (P < 0.05). This increase persisted for 8 hrs. IT MK-801 significantly reduced the number of dark-stained alpha-motoneurons after morphine-induced spastic paraparesis compared with the saline group. These data indicate that IT morphine induces spastic paraparesis with a concomitant increase in CSF glutamate, which is involved in NMDA receptor activation. We suggest that opioids may be neurotoxic in the setting of spinal cord ischemia via NMDA receptor activation.

The N-Methyl-D-aspartate receptor antagonist dizocilpine inhibits associative antinociceptive tolerance to morphine in mice: relation with memory.

I and coauthor previously reported the memory facilitation effect of morphine. The main purpose of this study was to evaluate the involvement of the N-methyl-D-aspartate (NMDA) receptor in associative tolerance to morphine by a contextual procedure. Antinociceptive response latency was measured by the tail-pinch method during repeated morphine (5 mg/kg, s.c.) injection for four consecutive days with pretreatment by dizocilpine (0.01, 0.05, 0.1 mg/kg, i.p.) at 30 min prior to morphine injection in the training phase and before and after morphine injection in the test phase. The nociceptive response latency was shortened by the single administration of dizocilpine (0.05 to 0.25 mg/kg, i.p.). Pretreatment by dizocilpine at 0.05 or 0.1 mg/kg weakened the antinociception to morphine on Day 1, but decreased the tolerance throughout the training phase. In the test phase, the animals were allocated into the same and different contexts. In the test phase, hyperalgesia before morphine injection in the same context and antinociception after morphine injection in the different context were evident in the saline-pretreated group in the training phase, but they were not observed in those contexts in the dizocilpine-pretreated groups. These results suggest that memory dysfunction with dizocilpine inhibits the recovery of associative tolerance to morphine by contextual change.

Atypical antipsychotic profile of flunarizine in animal models.

RATIONALE: Flunarizine is known as a calcium channel blocker commonly used in many countries to treat migraine and vertigo. Parkinsonism has been described as one of its side-effects in the elderly, which is in agreement with its recently characterized moderate D2 receptor antagonism. OBJECTIVES: To perform a pre-clinical evaluation of flunarizine as a potential antipsychotic. METHODS: We evaluated the action of orally administered flunarizine in mice against hyperlocomotion induced by amphetamine and dizocilpine (MK-801) as pharmacological models of schizophrenia, induction of catalepsy as a measure for extrapyramidal symptoms and impairment induced by dizocilpine on the delayed alternation task for working memory. RESULTS: Flunarizine robustly inhibited hyperlocomotion induced by both amphetamine and dizocilpine at doses that do not reduce spontaneous locomotion (3-30 mg/kg). Mild catalepsy was observed at 30 mg/kg, being more pronounced at 50 mg/kg and 100 mg/kg. Flunarizine (30 mg/kg) improved dizocilpine-induced impairment on the delayed alternation test. CONCLUSIONS: These results suggest a profile comparable to atypical antipsychotics. The low cost, good tolerability and long half-life (over 2 weeks) of flunarizine are possible advantages for its use as an atypical antipsychotic. These results warrant clinical trials with flunarizine for the treatment of schizophrenia.

Bimodal effects of MK-801 on locomotion and stereotypy in C57BL/6 mice.

RATIONALE: Systemic injection of the non-competitive NMDA (N-methyl-D-aspartate) receptor antagonist MK-801 (dizocilpine maleate) causes both increased locomotion in rodents and various stereotypic behaviors that are proposed to model certain aspects of schizophrenic symptoms in humans. OBJECTIVES: This study presents a comprehensive characterization of the bimodal effects of MK-801 on locomotion and stereotypy in the C57BL/6 mouse strain, a strain commonly used for genetically modified mice. RESULTS: We found that it is important to analyze both locomotion and stereotypy in parallel, as MK-801-induced stereotypy results in abnormal movements that are recorded as locomotion by automated beam detection systems. Furthermore, it is important to analyze the bimodal effects of MK-801 over an extended time span, rather than the commonly used narrower time window, as at higher doses (e.g., above 0.3 mg/kg) the hyperlocomotion phase develops only after the stereotypic phase subsides. We also observed that the apparent dose-response curve is very sensitive to the particular time window chosen for analysis because MK-801 affects both the time course and maximum value of stimulated locomotion. We show that analyzing the absolute peak value of locomotion induced for each animal, rather than group-averaged time courses, provides a measure that is sensitive over a wider range of MK-801 doses. Interestingly, MK-801 even at a very low dose of 0.02 mg/kg suppressed rather than enhanced rearing behavior, differing in this regard from amphetamine. CONCLUSIONS: The non-competitive NMDA receptor antagonist MK-801 induces a complex pattern of behavioral modification in mice with respect to both the time course and the dose-response relationship of behavioral changes. The results of this study provide a foundation and frame of reference for the growing interest in studying MK-801-induced behavior in mice.

Acute and late effects on induction of allodynia by acromelic acid, a mushroom poison related structurally to kainic acid.

1. Ingestion of a poisonous mushroom Clitocybe acromelalga is known to cause severe tactile pain (allodynia) in the extremities for a month and acromelic acid (ACRO), a kainate analogue isolated from the mushroom, produces selective damage of interneurons of the rat lower spinal cord when injected either systemically or intrathecally. Since ACRO has two isomers, ACRO-A and ACRO-B, here we examined their acute and late effects on induction of allodynia. 2. Intrathecal administration of ACRO-A and ACRO-B provoked marked allodynia by the first stimulus 5 min after injection, which lasted over the 50-min experimental period. Dose-dependency of the acute effect of ACRO-A on induction of allodynia showed a bell-shaped pattern from 50 ag x kg(-1) to 0.5 pg x kg(-1) and the maximum effect was observed at 50 fg x kg(-1). On the other hand, ACRO-B induced allodynia in a dose-dependent manner from 50 pg x kg(-1) to 50 ng x kg(-1). 3. N-methyl-d-aspartate (NMDA) receptor antagonists and Joro spider toxin, a Ca(2+)-permeable AMPA receptor antagonist, inhibited the allodynia induced by ACRO-A, but not by ACRO-B. However, other AMPA/kainate antagonists did not affect the allodynia induced by ACRO. 4. Whereas no neuronal damage was observed in the spinal cord in ACRO-A-treated mice, induction of allodynia by ACRO-A (50 fg x kg(-1)) and ACRO-B (50 ng x kg(-1)) was selectively lost 1 week after i.t. injection of a sublethal dose of ACRO-A (50 ng x kg(-1)) or ACRO-B (250 ng x kg(-1)). Higher doses of ACRO-A, however, could evoke allodynia dose-dependently from 50 pg x kg(-1) to 500 ng x kg(-1) in the ACRO-A-treated mice. The allodynia induced by ACRO-A (500 ng x kg(-1)) was not inhibited by Joro spider toxin or NMDA receptor antagonists. These properties of the late allodynia induced by ACRO-A were quite similar to those of the acute allodynia induced by ACRO-B. 5. ACRO-A could increase [Ca(2+)](i) in the deeper laminae, rather than in the superficial laminae, of the spinal cord. This increase was not blocked by the AMPA-preferring antagonist GYKI52466 and Joro spider toxin. 6. Taken together, these results demonstrate the stereospecificity of ACRO for the induction of allodynia and suggest the presence of a receptor specific to ACRO.

Development of behavioral sensitization to the cocaine-like fungicide triadimefon is prevented by AMPA, NMDa, DA D1 but not DA D2 receptor antagonists.

Triadimefon (TDF) is a triazole fungicide that blocks the reuptake of dopamine (DA) and leads to increased locomotor activity levels in mice and rats, effects similar to those of indirect DA agonists such as cocaine. We recently found in mice that intermittent TDF administration led to robust locomotor sensitization, a phenomenon reflecting neuronal plasticity, following challenge with the same TDF dose after a 2-week withdrawal period. The current study sought to determine whether antagonists to DA D1-like receptors (SCH 23390; SCH), DA D2-like receptors (remoxipride; Rem), ionotropic glutamate n-methyl-d-aspartate (NMDA) receptors (CPP), or ionotropic glutamate alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors (NBQX) could prevent the development of TDF behavioral sensitization, therefore indicating their mechanistic involvement in TDF sensitization. Mice were treated with either vehicle, SCH (0.015 mg/kg), remoxipride (Rem, 0.3 mg/kg), CPP (2.5 mg/kg) or NBQX (10.0 mg/kg), followed 30 min later by vehicle or 75 mg/kg TDF (TDF), twice a week for 7 weeks, with locomotor activity measured post-dosing once a week. After a 2-week withdrawal period, mice were challenged with 75 mg/kg TDF or vehicle, to test for the presence of behavioral sensitization. Pretreatment with SCH, CPP, or NBQX, but not Rem, blocked the development of behavioral sensitization to TDF specifically for vertical activity. Antagonists that blocked TDF vertical sensitization also attenuated the increase in extracellular DA turnover (homovanillic acid [HVA]/DA) normally associated with this behavioral response. Therefore, DA D1, NMDA and AMPA receptors appear to be necessary for the development of behavioral sensitization to TDF. As such, TDF may be considered an environmental risk factor for behavioral dysfunctions linked to glutamatergic and dopaminergic systems.

Animal models of spinal cord injury pain and their implications for pharmacological treatments.

Patients with spinal cord injury often develop chronic pain syndrome, which is difficult to treat. Several animal models of spinal cord injury have been developed in recent years which have significantly advanced our understandings of pathophysiology of this condition. This paper reviews some recent data in the pharmacological treatment of spinal cord injury pain using animal models, and discusses possible clinical applications.

Effects of short-acting NMDA receptor antagonist MRZ 2/576 on morphine tolerance development in mice.

The present study sought to evaluate the ability of a short-acting glycineB site NMDA receptor antagonist, MRZ 2/576, to affect morphine tolerance development in mice. It was found that MRZ 2/576 (10 mg/kg, i.p.) significantly retarded development of morphine analgesic tolerance (20 mg/kg, s.c., 8 days, once a day; tail-flick test) when administered 120 min or 150 min after each daily morphine injection. MRZ 2/576 did not affect the development of morphine tolerance when administered immediately, 15, 30, 60, 90, 180, 240, 300 or 360 min after the daily morphine injections. Thus, short-acting NMDA receptor antagonists may be useful in exploring the temporal characteristics of opioid tolerance (i.e., periods after morphine injection that are critical for tolerance induction) and the present study suggests that after morphine administration there is a period of NMDA receptors activation crucial for the development of tolerance.