NT4-GFP-Ant重组腺相关病毒载体的构建及病毒包装、滴度测定

目的为使基因治疗时,较大分子的功能蛋白能通过血脑屏障,构建NT4-GFP-Ant重组腺相关病毒载体,包装重组病毒,并测定滴度。为进一步动物实验研究该重组病毒液感染鼻粘膜细胞,在鼻粘膜内表达的大分子融合蛋白沿“鼻-脑”通路人脑的可行性奠定基础。方法将已构建成功的NT4-GFP-Ant融合基因插入病毒载体质粒PSSHG中,构建重组腺相关病毒载体质粒,酶切电泳鉴定。用腺病毒辅助质粒PFG140、包装质粒pAAV/Ad及已构建的重组腺伴随病毒载体质粒,三质粒磷酸钙共沉淀法转染80％融合的293细胞系,包装重组腺病毒（rAAV）。回收病毒、斑点杂交方法测定重组病毒滴度。结果成功构建了重组腺相关病毒质粒PSSHG/NT4-GFP- Ant。包装、回收病毒后斑点杂交方法测定重组病毒滴度为3．36×10^9 PFU/ml。结论成功构建了PSSHG/NT4- GFP-Ant重组腺相关病毒载体,并成功包装了较高浓度的重组病毒。     

重组腺相关病毒介导的Dystrophin小基因载体构建及在mdx鼠中表达

目的 :研究重组腺相关病毒载体 (rAAV)介导的人Dystrophin小基因 (SMCKA3 999)载体构建及在DMD模型鼠 (mdx鼠 )的表达。方法 :将SMCKA3 999质粒 ,包装质粒pXX2、腺病毒成分辅助质粒pXX6共转染 2 93细胞 ,包装重组腺相关病毒载体介导的SMCKA3 999基因 (rAAVSMCKA3 999) ,以斑点杂交法测定病毒滴度 ,将rAAVSMCKA3 999单点注射到mdx鼠腓肠肌 ,于注射后 7个月取肌肉提取蛋白质行Westernblot检测。结果 :经三质粒共转染法构建的rAAVSMCKA3 999病毒滴度为 5 0× 10 10 ,在mdx鼠骨骼肌表达持续 7个月以上。结论 :构建的rAAVSMCKA3 999载体为进一步DMD基因治疗研究奠定了基础。     

表达胰高血糖素样肽1重组腺伴随病毒载体的构建

背景：胰高血糖素样肽1(glucagon-like peptide-1, GLP-1)在人体内半衰期过短限制了其应用。 目的：构建可表达GLP-1的重组腺伴随病毒。 方法：将NT4-GLP-1融合基因插入腺伴随病毒包装质粒pSSHG-CMV中,构建pSSHG/NT4-GLP-1重组腺伴随病毒包装质粒。采用磷酸钙共沉淀法将辅助质粒pAAV/Ad、腺病毒质粒pFG140及pSSHG/NT4-GLP-1转染至293细胞系,用其感染Hela细胞。 结果与结论：重组质粒pSSHG/NT4-GLP-1经限制性内切酶EcoRⅠ酶切鉴定可见342 bp的目的片段,说明NT4-GLP-1融合基因已经成功重组于腺伴随病毒包装质粒pSSHG-CMV内。免疫细胞化学结果显示,转染pSSHG/NT4-GLP-1重组腺伴随病毒包装质粒的Hela细胞内有大量棕黄色颗粒,阳性率达到70%以上,说明NT4-GLP-1重组腺伴随病毒在细胞中可以表达GLP-1。     

重组腺病毒载体Ad5-hBDNF-EGFP的构建与鉴定*☆

背景：基因治疗是目前脊髓损伤治疗的方向，目的基因和载体是基因治疗的关键。 目的：构建携带增强型绿色荧光蛋白基因(Enhanced green fluorescence protein, EGFP)标志人脑源性神经营养因子(human brain-derived neurotrophic factor，hBDNF)基因重组腺病毒载体。 设计、时间及地点：单一样本观察，实验于2007-09/2008-06在福建医科大学附属第一医院完成。 材料：感受态大肠杆菌DH-5α购自美国Stratagene公司；pDC316-hBDNF、载体质粒pDC316-mCMV-EGFP、腺病毒骨架质粒pBHGlox_E1,3Cre、腺病毒包装系统AdMax和包装细胞株293购自加拿大Mixcrobix-Biosystems公司。 方法：以pDC316-hBDNF为模板，聚合酶链反应扩增酶切获得hBDNF基因片段，连接到带有EGFP标记基因的载体质粒pDC316-mCMV-EGFP上，构建穿梭质粒pDC316-hBDNF-mCMV-EGFP。利用AdMax包装系统，穿梭质粒与骨架质粒pBHGlox_E1，3Cre共转染293包装细胞, 同源重组产生复制缺陷型重组腺病毒载体Ad5-hBDNF-EGFP，反复感染293细胞扩增病毒后，离子交换法纯化病毒，并测定病毒颗粒数及滴度。 主要观察指标：①hBDNF基因原始质粒聚合酶链反应鉴定。②穿梭质粒pDC316-hBDNF-mCMV-EGFP的构建及鉴定。③重组腺病毒Ad5-hBDNF-EGFP的包装、扩增及纯化。④毒种目的基因的聚合酶链反应鉴定。⑤纯化病毒的滴度测定结果。 结果：经聚合酶链反应鉴定、限制性酶切分析及序列测定，证明已正确构建重组穿梭质粒pDC316-hBDNF-mCMV-EGFP和重组腺病毒载体Ad5-hBDNF-EGFP；扩增纯化后，测得重组腺病毒颗粒数为2.4×1011VP / mL，A260/A280值约为2.0，滴度为0.8×1010 CCID50/ mL。 结论：已成功构建重组腺病毒载体Ad5-hBDNF-EGFP，为hBDNF基因功能及基因治疗的进一步研究奠定实验基础。     

利用Helper-Free系统构建cdk5-siRNA腺相关病毒载体并鉴定

目的构建能表达CDK5特异性小干扰RNA(siRNA)(cdk5-siRNA)的重组腺相关病毒(AAV)载体,体外观察其对CDK5基因的沉默作用。方法采用基因克隆技术将合成的特异性cdk5 RNA干扰寡核苷酸序列克隆至AAV Helper-Free System中的表达质粒pAAV-MCS,构建出质粒pAAV-MCS-cdk5-siRNA,通过酶切测序鉴定重组质粒;将该质粒与系统中的控制质粒pAAV-RC、辅助质粒pHelper用磷酸钙法共转染HEK293细胞,包装得到表达cdk5-siRNA的重组腺相关病毒载体(rAAV-cdk5-siRNA);用斑点杂交法测定重组病毒的滴度,重组病毒感染体外培养的PC12细胞,W estern b lot检测其抑制cdk5表达的效果。结果成功构建并包装出重组腺相关病毒载体rAAV-cdk5-siRNA,病毒滴度达4×1013/m l,重组病毒感染后的PC12细胞cdk5表达明显下调。结论构建的重组腺相关病毒载体rAAV-cdk5-siRNA能明显干扰cdk5的表达,为将其进一步应用于神经变性疾病的治疗研究奠定了基础。     

5/35嵌合型腺病毒载体对脑胶质瘤的抑制作用

目的通过构建一种新型腺病毒载体,验证肿瘤坏死因子相关凋亡诱导配体(TRAIL基因)治疗胶质瘤的疗效,并证实新型载体的优势。方法经美国华盛顿大学实验室授权获得Ad/35△E1-△E3,即E1和E3缺失的5型腺病毒纤维fiber被35型新型载体。利用基因重组,构建出重组腺病毒质粒Ad5/35△E1-△E3-TRAIL。氯化铯密度梯度离心纯化Ad5/35△E1-△E3-TRAIL,并并测定病毒滴度。采用逆转录酶-多聚酶链反应(RT-PCR)、免疫荧光技术、蛋白质印迹法(Western blot)分别检测目的基因的表达。结果纯化后Ad5/35△E1-△E3-TRAIL的病毒滴度为1.22×1015pfu/l。重组质粒经RT-PCR都扩增出104 bp的基因片段。通过免疫荧光和Western blot等方法检测到目的基因的高效表达。结论新型腺病毒载体质粒Ad5/35△E1-△E3-TRAIL能够高效的在脑胶质瘤细胞中表达。     

表达大鼠脑源性神经营养因子(BDNF)腺相关病毒的构建及体外表达分析

目的构建携带大鼠脑源性神经营养因子(BDNF)基因的重组腺相关病毒(AAV)载体,并检测体外表达目的基因的能力。方法应用基因克隆技术将大鼠BDNF的cDNA基因序列克隆入腺病毒质粒pAAV-MCS,PCR酶切测序鉴定序列。与AAV病毒包装质粒pAAV-RC、pHelper用磷酸钙法共转染HEK293细胞,包装得到含转基因过表达BDNF的病毒载体(rAAV-BDNF)。重组病毒感染体外培养的Hela细胞后,用RT-PCR和Western Blot方法检测细胞BDNF基因及蛋白表达情况,用ELISA法测定培养液中表达产物浓度。结果证实BDNFcDNA片段插入到病毒基因组内,并整合到宿主基因组后稳定表达,病毒滴度可以达到1.29×108PFU/mL。重组病毒感染Hela细胞后能高水平表达BDNF,ELISA结果表明,Hela细胞从病毒感染后第2天到第10天表达BDNF水平呈上升趋势,第10天达到2253pg/mL。结论成功地构建了表达BDNF基因的腺相关病毒载体,并可在体外高水平表达其所携带的目的基因,为将其进一步应用于神经系统损伤性疾病治疗的研究奠定了方法学基础。     

重组人骨形态发生蛋白2腺病毒载体的构建与鉴定*

背景：目前报道的重组腺病毒构建方法较复杂,构建周期长,导致重组腺病毒构建的成功率较低。GatewayTM技术是基于λ噬菌体的位点特异性高效重组系统,其构建重组病毒载体具有快捷高效的特点。 目的：构建含人骨形态发生蛋白2 基因的重组腺病毒载体,为应用骨形态发生蛋白2 基因治疗的研究奠定基础。 设计、时间及地点：单一样本观察,实验于2008-02/06在深圳市第二人民医院中心实验室完成。 材料：pOTB7-BMP-2 质粒、HEK293为ATTC产品;BLOCK-iTTM Adenovirus Expression System为Invitrogen产品;大肠杆菌DH5α为Biontex产品。 方法：用聚合酶链反应方法扩增骨形态发生蛋白2 目的基因,并插入载体pMD19-T Simple Vector中进行测序分析。将骨形态发生蛋白2 基因亚克隆到穿梭载体pEC3.1(+)中,构建pEC3.1-BMP-2 穿梭质粒,再将其中的表达盒通过重组克隆到pAd/BLOCK-iT™-DEST腺病毒载体中,线性化后转293 细胞进行包装获得重组腺病毒rAd-BMP2。 主要观察指标：①目的基因骨形态发生蛋白2 的聚合酶链反应扩增。②重组质粒TS-BMP2 的构建。③重组pEC3.1-BMP2 的构建。④重组pAd-BMP2 的构建。⑤重组腺病毒的制备与鉴定。 结果：正确扩增长约1.2 kb的骨形态发生蛋白2 cDNA,并成功地构建其腺病毒载体,经线性化的pAd-BMP2 DNA转染HEK293细胞,包装、扩增后得到人骨形态发生蛋白2 重组腺病毒(rAd-BMP2),其滴度约为1×1010 PFU/mL,该滴度可满足进一步的骨形态发生蛋白2 成骨作用的研究。 结论：成功地构建重组人骨形态发生蛋白2 腺病毒载体。     

大鼠黏结蛋白聚糖1基因重组腺病毒载体的构建及感染效率☆

背景：重组腺病毒质粒的构建方法主要有体外连接法和同源重组法。同源重组法有操作复杂、耗时长、效率低、纯化难的缺点。体外连接法又不可避免非特异性的基因重组及基因突变。 目的：应用改良体外连接法构建携带黏结蛋白聚糖1基因的重组腺病毒载体，测定其在心肌成纤维细胞中的感染效率。 设计、时间及地点：单一样本实验，于2007-08/2008-02在中山大学附属第二医院林百欣实验中心完成。 材料：穿梭载体pShuttle-CMV（含绿色荧光报告基因）和腺病毒骨架质粒pAdxsi购自诺赛基因组研究中心有限公司。 方法：核苷酸序列鉴定pCMV-Sport6.1-Sdc1质粒；用KpnⅠ + XhoⅠ从质粒pCMV-Sport6.1-Sdc1切出黏结蛋白聚糖1基因片段，亚克隆至pShuttle-CMV中，形成重组穿梭质粒。用I-CeuI + I-SceI双酶切出重组穿梭质粒中CMV-Sdc1片段，亚克隆至腺病毒基因组质粒中，得到重组腺病毒质粒。将重组腺病毒质粒转染293细胞包装获得重组腺病毒AdCMVSdc1，转化体外培养的心肌成纤维细胞。 主要观察指标：用DNA测序、酶切及聚合酶链反应法鉴定重组质粒和病毒，并测定重组腺病毒的滴度和感染效率。 结果：①核苷酸序列分析表明，pCMV-Sport6.1-Sdc1质粒正确携带大鼠黏结蛋白聚糖1 cDNA；黏结蛋白聚糖1基因被克隆于载体pShuttle-CMV上，以KpnⅠ + XhoⅠ双酶切可回收3 kb的克隆片段和5.1 kb的载体片段；重组腺病毒质粒用XhoⅠ酶切得到7个片段而空载体仅得到6个片段。②重组腺病毒质粒在293细胞中包装后产生的重组腺病毒对293细胞有致病作用；提取病毒DNA行聚合酶链反应鉴定可扩增出1.13 kb的特异性片段；用病毒上清多次重复感染293细胞扩增重组腺病毒后，病毒滴度检测达2.0×1011 PFU/mL。③用纯化浓缩后的重组腺病毒以感染复数为100感染心肌成纤维细胞，24 h后所有细胞均表达绿色荧光。 结论：成功构建了携带大鼠黏结蛋白聚糖1基因的重组腺病毒载体，经纯化浓缩后具有较高的滴度，能有效转染心肌成纤维细胞。     

携带人血红素加氧酶-1基因重组腺病毒的构建及其在大脑皮层神经元中的表达

目的构建人血红素加氧合酶-1（hHO-1）基因的重组腺病毒,并研究其感染大脑皮层神经元的效率。方法采用基因克隆技术,将hHO-1基因克隆到腺病毒穿梭质粒pShuttle—IRES—hrGFP中,构建pShuttle-hHO-1-IRES—hrGFP质粒,利用细菌BJ5183将穿梭质粒与pAdEasy-1进行重组,获得重组的腺病毒质粒。线性化的腺病毒质粒经脂质体转染AD293细胞,进行重组腺病毒的包装和扩增。采用CsCl梯度离心进行病毒纯化。获得的腺病毒感染大脑皮层神经元,观察其感染效率和hHO-1表达水平。结果通过酶切鉴定证明腺病毒载体构建成功,包装出携带hHO-1基因的腺病毒,病毒滴度为2．0×10^10pfu／ml,获得的腺病毒对大脑皮层神经元的感染效率高于95％以上。结论成功地利用细菌内同源重组方法构建了携带hHO-1基因的腺病毒,并能够在大脑皮层神经元中高效地表达,为利用HO—1进行基因治疗提供了材料。     

Diagnostic Difficulties and Treatment Implications

Summary: Differentiation between types of epileptic seizures has been aided in recent years by the introduction of intensive neurodiagnostic techniques and the development of increasingly detailed classification systems. Paradoxically, these developments have not simplified the task of matching the appropriate antiepileptic drug to a particular seizure type. It is reasonable to assume that anticonvulsant drugs will have different effects on different types of seizures, but faulty, circular reasoning can enter the picture if one also assumes that responses of seizures to different drugs signify different seizure types. There are several examples of differential diagnoses that can fall prey to this problem, including the diagnosis between partial seizures with secondary generalization and generalized tonic-clonic seizures, and the diagnosis between complex partial seizures and absence seizures with automatisms, among others. Considerations of etiology in future classification systems can further complicate the problem: should one then choose an anticonvulsant drug on the basis of individual seizure type or on the basis of the type of epilepsy? Ramifications of this issue extend even to the drug approval process. Official sanction is not given for use of a drug for a seizure type not included in the original efficacy studies, even if later scientific evidence shows that seizure type to be related to a type that is included. New trials must be undertaken. These problems arise from how we choose to classify seizures.     

Cognitive Dysfunction Associated with Antiepileptic Drug Therapy

Summary: Epilepsy is frequently associated with cognitive dysfunction. However, the reasons for this correlation are unclear. Possible influential factors include patient age; duration, frequency, etiology, and type of seizures; hereditary factors; psychosocial issues; and antiepileptic drug (AED) therapy. Whereas many of these factors are beyond the physician's control, AED therapy is one element that can be addressed in treatment decisions by recognizing the potential cognitive effects of particular AEDs. For example, phenobarbital impairs memory and concentration; phenytoin affects attention, problem solving ability, and performance of visuomotor tasks. In contrast, carbamazepine may affect concentration, while valproate would appear to have minimal effects on cognition. Moreover, cognitive effects of AEDs are amplified with coadministration of multiple anticonvulsants (polytherapy). A review of studies on the cognitive effects of monotherapy with AEDs, as opposed to those of polytherapy, provides evidence that drug-related cognitive dysfunction can be reversed if patients are switched to a simpler therapeutic regimen. Future research should be directed toward developing reliable measures for assessing and monitoring cognition, and understanding the particular cognitive side effects of each AED. Physicians also need to revise their opinions about which side effects are "tolerable" for epileptic patients.     

Carbamazepine Efficacy in Adults With Partial and Generalized Tonic-Clonic Seizures

Summary: Carbamazepine and phenytoin are drugs of choice in initial monotherapy for adult partial and secondarily generalized tonic-clonic seizures. These designations reflect the results of the Veterans Administration Epilepsy Cooperative Study Group of 1985. An earlier comparative study of carbamazepine and phenytoin by Ramsay and associates found both drugs equally effective in controlling new-onset seizures. Among the advantages of carbamazepine is that it causes relatively few cognitive and dysmorphic side effects. Its disadvantages are its unavailability in parenteral formulation and its metabolic autoinduction. The latter must be compensated for by planned dosage increases to maintain therapeutic plasma steady-state levels during the first 2 or 3 months of treatment. Carbamazepine is judged a drug of choice in the treatment of these secondarily generalized tonic-clonic seizures, and the drug of choice in children, adolescents, and women susceptible to the dysmorphic side effects associated with other anticonvulsant agents.     

Etiologic and Preventive Aspects of Epilepsy in the Child–Bridging the Gap Between Laboratory and Clinic

Summary: Four broad categories of basic phenomena are pertinent to developing ways to prevent epilepsy. These include mechanisms of epileptogenesis, ictal initiation and temporary entrainment by the seizure discharge of normally functioning brain, seizure propagation, and control mechanisms that function both to restrain the cascade of epileptic events culminating in a seizure and to arrest the epileptic event and restore the interictal state. In newborns and children, hypoxia-ischemia is a major factor leading to epileptogenesis, and several schemes are proposed to classify, quantify, and prevent hypoxic-ischemic encephalopathy. Control mechanisms must be better understood in order to develop prophylactic recommendations for epilepsy, and an experimental model of "kindling antagonism" may increase our understanding of these. Programs of prevention of seizures in children will evolve only if basic researchers and clinicians work productively together to develop an adequate understanding of factors important in epileptogenesis and antiepileptogenic control mechanisms.     

Epilepsy and anomalies of neuronal migration: MRI and clinical aspects

Neuronal migration disorders are the result of disturbed brain development. In such disorders, neurons are abnormally located. In diagnosing these conditions, magnetic resonance imaging is superior to any other imaging technique. This enables us to improve our knowledge of the clinical correlates of neuronal migration. With reference to migrational disorder, a retrospective study of all 303 patients with epileptic seizures referred for magnetic resonance imaging during a 3-year period was performed, 13 patients (aged 12-41, mean age 27) were identified. They represent 4.3% of the entire study group. Of the patients with known epilepsy, 6.7% and of the mentally retarded, 13.7% had migrational disorders. Four patients had schizencephaly as the dominant finding, one was classified as hemimegalencephaly, 2 had isolated heterotopias, and 6 had localized pachy- and/or poly-microgyria. The clinical pictures are complex. Ectopias of grey matter are recognised foci of epilepsy, but from an epileptological and a clinical viewpoint little attention has been given to these disorders. The present study shows that malmigration is not rare in epilepsy patients, especially not in the mentally retarded.     

Predisposing and Causative Factors in Childhood Epilepsy

Summary: We review information from large studies of defined populations, examining the role of known factors and especially of prenatal and perinatal factors in contributing to nonfebrile seizure disorders of early childhood. We depend especially, but not exclusively, on the recently completed analyses from the Collaborative Perinatal Project of the National Institute of Neurological and Communicative Disorders and Stroke, the NCPP. About 4% of children in the NCPP who had at least one non-febrile nonsymptomatic seizure by the age of 7 years had a previous seizure during acute neurologic illness, such as meningitis or during the acute illness after trauma. Many such seizures should potentially be preventable. Of children with seizures, 10% had had a neonatal seizure and 13% had had a febrile seizure. Among the hundreds of prenatal and perinatal factors explored as predictors of childhood seizure disorders, the principal predictors identified were congenital malformations of the fetus, cerebral and noncerebral; family history of certain neurologic disorders; and neonatal seizures. In agreement with the British National Child Development Study, labor and delivery factors in the NCPP appeared to contribute very little to childhood seizure disorders. Maldevelopment, rather than damage at birth to an initially intact nervous system, appeared to be the more common mechanism. Most seizure disorders of early childhood remained unexplained by the large set of prenatal and perinatal characteristics examined.     

Anticonvulsant Drugs and Cognitive Function: A Review of the Literature

Summary: Alterations of cognitive function are separate from disturbances of behavior seen in association with epilepsy. The nature of the cognitive disability may to a certain extent depend on the seizure type. Partial seizures, mainly derived from a temporal lobe focus, impair memory tasks, while generalized seizures seem to have more effect on attentional abilities. A number of studies, reviewed in this paper, suggest that anticonvulsant drugs further impair cognitive function. Maximal impairments are seen in patients receiving polytherapy: rationalization of polytherapy improves cognitive abilities. Studies in children and adults have allowed differentiation of the effects of various commonly used antiepileptic agents. Maximal cognitive deficits are seen with. phenytoin, while phenobarbital and sodium valproate induce moderate disturbances, and carbamazepine seems relatively free from such toxicity. Further research is needed on the interrelationship between types of seizure disorders, types of anticonvulsant medications, and cognitive function.     

Classification of methods in transcranial Electrical Stimulation (tES) and evolving strategy from historical approaches to contemporary innovations

Transcranial Electrical Stimulation (tES) encompasses all methods of non-invasive current application to the brain used in research and clinical practice. We present the first comprehensive and technical review, explaining the evolution of tES in both terminology and dosage over the past 100 years of research to present day. Current transcranial Pulsed Current Stimulation (tPCS) approaches such as Cranial Electrotherapy Stimulation (CES) descended from Electrosleep (ES) through Cranial Electro-stimulation Therapy (CET), Transcerebral Electrotherapy (TCET), and NeuroElectric Therapy (NET) while others like Transcutaneous Cranial Electrical Stimulation (TCES) descended from Electroanesthesia (EA) through Limoge, and Interferential Stimulation. Prior to a contemporary resurgence in interest, variations of transcranial Direct Current Stimulation were explored intermittently, including Polarizing current, Galvanic Vestibular Stimulation (GVS), and Transcranial Micropolarization. The development of these approaches alongside Electroconvulsive Therapy (ECT) and pharmacological developments are considered. Both the roots and unique features of contemporary approaches such as transcranial Alternating Current Stimulation (tACS) and transcranial Random Noise Stimulation (tRNS) are discussed. Trends and incremental developments in electrode montage and waveform spanning decades are presented leading to the present day. Commercial devices, seminal conferences, and regulatory decisions are noted. We conclude with six rules on how increasing medical and technological sophistication may now be leveraged for broader success and adoption of tES.     

Treatment Considerations in Anticonvulsant Monotherapy

Summary: The long-standing practice of polypharmacy in treating epilepsy is giving way to use of monotherapy. Monotherapy can improve seizure control as well as reduce the risk of serious idiosyncratic reactions, dose-related side effects, and complex drug interactions. Monotherapy also offers improved compliance and cost-effectiveness. The basis of monotherapy is accurate diagnosis and assessment of the patient's seizure type(s), followed by selection of a single appropriate anticonvulsant drug. Many patients currently treated with multiple anticonvulsants can be successfully converted to monotherapy with a carefully monitored program in which troublesome and redundant drugs are gradually withdrawn from the therapeutic regimen.     

Effects of NMD A- and AMPA-Receptor Antagonists on Different Forms of Epileptiform Activity in Rat Temporal Cortex Slices

Summary: Lowering extracellular magnesium induces different patterns of epileptiform activity in rat hippocampus and entorhinal cortex. Short recurrent epileptiform discharges in the hippocampus are stable over time, whereas seizurelike events (SLEs) in the entorhinal cortex, the subiculum, and the neighboring neocortex develop into late recurrent discharges which are not blocked by clinically employed antiepileptic drugs. We tested the sensitivity of the different epileptiform discharge patterns to. /V-methyl-D-aspartate (NMDA)- and non-NMDA-receptor antagonists. As NMDA-receptor antagonist we used dextrorphan, ket-amine, and 2-aminophosphonovalerate (2APV); as α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA)-receptor antagonist we employed the quinoxaline derivative glutamate 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). The findings show that the different patterns of epileptiform activity, including the late recurrent discharges, are sensitive to all NMDA-receptor antagonists. However, when dextrorphan was employed to suppress seizure-like events, later recurrent discharges did not develop during the remaining time course of the experiment. CNQX reversibly suppressed recurrent discharges in the hippocampus and SLEs in the entorhinal cortex. However, late recurrent discharges become insensitive to CNQX, even at a high concentration of 60 μM m. This finding suggests a prominent role for NMDA receptors in the generation of late recurrent discharges.