APH, an N-methyl-D-aspartate receptor antagonist, blocks the metaphit-induced audiogenic seizures in rats

The effect of the competitive antagonist of the N-methyl-D-aspartate (NMDA) receptor, (±)2-amino-7-phosphonoheptanoic acid (APH) on electrocorticographic (ECoG) activity and behavior was studied in the model of epilepsy induced by systemic application of metaphit (1-(1-(3-isothiocyanatophenyl)-cyclohexyl)-piperidine). Male Wistar rats were injected with metaphit intraperitoneally (10 mg/kg, ip), and exposed to intense audio stimulation (electric bell generating 100 ± 3 dB at animal level for 60 s) 1 h after administration and at 1-h intervals thereafter. ECoG tracings showed appearance of paroxysmal activity in form of spikes, spike-wave complexes and ECoG seizures. Audiogenic seizures consisted of wild running followed by clonic and tonic convulsions. Each behavioral seizure response had a characteristic ECoG correlate. The incidence and severity of seizures increased with time, reaching a peak 8–12 h after metaphit administration, and then gradually decreased until 31 h, when no animal responded to sound stimulation. APH was injected intracerebroventricularly (0.005, 0.01, 0.02, 0.03 and 0.05 μmol icv in 5 μL of sterile saline) after the 8th hour of audiogenic testing (AGS). APH inhibited seizures in a dose-dependent manner. The minimum dose which blocked seizures in all animals was 0.03 μmol. However, ECoG signs of seizure susceptibility were not suppressed by APH. After varying periods of time, behavioral seizures reappeared. It seems that APH blocks epileptiform propagation, but has less influence on the epileptogenic activity caused by metaphit.     

Involvement of synaptosomal neurotransmitter amino acids in audiogenic seizure-susceptibility and-severity of Rb mice

The involvement of synaptosomal neurotransmitter amino-acids in seizure susceptibility and seizure severity was explored. The amino-acid contents of brain synaptosomes were determined in three sublines of Rb mice differing in their response to an acoustic stimulus: Rb1, clonic-tonic seizure-prone, Rb2, clonic seizure-prone, and Rb3, seizure-resistant. Synaptosomes were prepared from 6 brain areas considered to be involved in seizure activity: olfactory bulbs, amygdala, inferior colliculus, hippocampus, cerebellum, pons-medulla. The steady-state levels of GABA and glycine (Gly), inhibitory amino-acids, of taurine (Tau), an inhibitory neurotransmitter of neuromodulator, of aspartate (Asp) and glutamate (Glu), excitatory amino-acids, as well as of serine (Ser) and glutamine (Gln), two precursors of neurotransmitter amino-acids, were determined by HPLC. Low levels of Tau, GABA, and Ser in hippocampus, Gly in amygdala, Glu in hippocampus, inferior colliculus and pons, Gln and Asp in inferior colliculus appeared to correlate with seizure-susceptibility. GABA and Asp in olfactory bulb, Gln in amygdala, hippocampus and pons, ser in olfactory bulb and pons, appeared to be associated either with seizure-severity or-diversity. A strong involvement of hippocampus (Tau, GABA, Ser, Glu, and Gln) and inferior colliculus (Asp, Glu, Gln) in audiogenic seizure-susceptibility, and of olfactory bulb (GABA, Asp) in seizure-severity and/or-diversity is suggested.Special issue dedicated to Dr. Alan N. Davison.     

Long lasting effect of a single audiogenic seizure on GABA turnover rates and steady-state levels

GABA turnover rates (TOR) and steady-state levels (SSL) were determined, 16–18 h after a single acoustic stimulation, in 15 brain areas of 3 mouse sublines. Each subline differs in its response to an acoustic stimulation (Rb1 mice are clonic-tonic seizure-prone, Rb2: clonic seizure-prone, Rb3: seizure-resistant). TOR and SSL were compared to those of unstimulated control mice and to those of repeatedly stimulated mice of the same subline. Following a single acoustic stimulation long-lasting alterations of GABA metabolism, mainly large alterations of GABA TOR, are observed. Most of the effects elicited after repeated stimulations, either on SSL or TOR, are not those of the last stimulation and repeated seizures (and/or stimulations) strengthen the effect of a single one. It appears that, for each of the Rb sublines, a specific and quite simple profile of the alterations of GABA metabolism in response to a single or repeated audiogenic seizures (and/or stimulations) can be given. The global analysis through the correlation of GABA TOR and SSL gives an indication that the alterations of the parameters of the correlation observed are to be allocated to the audiogenic seizures. Furthermore the tonic and clonic components of the audiogenic seizures can be distinguished.     

Long-lasting effects of audiogenic seizures on neurotransmitter amino acids in Rb mice

The existence of long-lasting (15–18 h) alterations of neurotrasmitter amino acid levels following a single or repeated acoustic stimulations in audiogenic seizure-prone Rb1 and Rb2 mice and suizure-resistant Rb3 mice were investigated. The levels of glutamate, aspartate, glycine, taurine, and of some of their precursors: glutamine and serine were determined. Fourteen brain areas were examined. Alterations were found only in 6 brain areas (pons, olfactory bulbs, superior colliculus, inferior colliculus, olfactory tubercles and raphe). Most frequent occuring changes were observed in pons and olfactory tubercles. These changes concerned mainly the excitatory amino acids, glutamate, and aspartate. Alterations of taurine, glycine and serine were also recorded.Abbreviations GABA 4-aminobutyrate - Tau taurine - Gly glycine - Asp aspertate - Glu glutamate - Gln glutamine - Ser serine - OB olfactory bulbs - OT olfactory tubercles - Sr striatum - Se septum - Hy hypothalamus - Th thalamus - Hi hippocampus - A amygdala - SC superior colliculus - IC inferior colliculus - FC frontal cortex - C cerebellum - P pons medulla - Ra raphe - AA neurotransmitter amino acids - I inhibitory - E excitatory - SSL steady-state level Plesant memories of Lawrence Austin's sojourn in my group at Strasbourg gather upon me when I dedicate this article on this occasion for the contribution that Lawrence Austin has made for the cause of neurochemical researchers.     

A model of metformin mitochondrial metabolism in metachromatic leukodystrophy: first description of human Schwann cells transfected with CRISPR-Cas9

Metachromatic leukodystrophy is a neurological lysosomal deposit disease that affects public health despite its low incidence in the population. Currently, few reports are available on pathophysiological events related to enzyme deficiencies and subsequent sulfatide accumulation. This research aims to examine the use of metformin as an alternative treatment to counteract these effects. This was evaluated in human Schwann cells (HSCs) transfected or non-transfected with CRISPR-Cas9, and later treated with sulfatides and metformin. This resulted in transfected HSCs showing a significant increase in cell reactive oxygen species (ROS) production when exposed to 100 µM sulfatides (p = 0.0007), compared to non-transfected HSCs. Sulfatides at concentrations of 10 to 100 µM affected mitochondrial bioenergetics in transfected HSCs. Moreover, these analyses showed that transfected cells showed a decrease in basal and maximal respiration rates after exposure to 100 µM sulfatide. However, maximal and normal mitochondrial respiratory capacity decreased in cells treated with both sulfatide and metformin. This study has provided valuable insights into bioenergetic and mitochondrial effects of sulfatides in HSCs for the first time. Treatment with metformin (500 µM) restored the metabolic activity of these cells and decreased ROS production.     

Deletion of fatty acid amide hydrolase reduces lyso-sulfatide levels but exacerbates metachromatic leukodystrophy in mice

An inherited deficiency of arylsulfatase A (ASA) causes the lysosomal storage disease metachromatic leukodystrophy (MLD) characterized by massive intralysosomal storage of the acidic glycosphingolipid sulfatide and progressive demyelination. Lyso-sulfatide, which differs from sulfatide by the lack of the N-linked fatty acid, also accumulates in MLD and is considered a key driver of pathology although its concentrations are far below sulfatide levels. However, the metabolic origin of lyso-sulfatide is unknown. We show here that ASA-deficient murine macrophages and microglial cells express an endo-N-deacylase that cleaves the N-linked fatty acid from sulfatide. An ASA-deficient astrocytoma cell line devoid of this activity was used to identify the enzyme by overexpressing 13 deacylases with potentially matching substrate specificities. Hydrolysis of sulfatide was detected only in cells overexpressing the enzyme fatty acid amide hydrolase (FAAH). A cell-free assay with recombinant FAAH confirmed the novel role of this enzyme in sulfatide hydrolysis. Consistent with the in vitro data, deletion of FAAH lowered lyso-sulfatide levels in a mouse model of MLD. Regardless of the established cytotoxicity of lyso-sulfatide and the anti-inflammatory effects of FAAH inhibition seen in mouse models of several neurological diseases, genetic inactivation of FAAH did not mitigate, but rather exacerbated the disease phenotype of MLD mice. This unexpected finding was reflected by worsening of rotarod performance, increase of anxiety-related exploratory activity, aggravation of peripheral neuropathy, and reduced life expectancy. Thus, we conclude that FAAH has a protective function in MLD and may represent a novel therapeutic target for treatment of this fatal condition.     

Amino acid neurotransmitter alterations in three sublines of Rb mice differing by their susceptibility to audiogenic seizures

The levels of inhibitory amino acids (Tau, Gly), or excitatory amino acids (Glu, Asp) and Gln, precursor of GABA, have been determined, under resting conditions, in 17 brain areas of 3 sublines of inbred Rb mice displaying different responses to an acoustic stimulus. Rb1 mice were clonictonic seizure-prone, Rb2 mice were clonic seizure-prone and Rb3 mice were seizure resistant. Profile of distribution in the brain of each one of these amino acids differed. Maximum to minimum level ratio was higher for Tau (3.8) than for Glu or Asp or Gln (2). The level of Gly was similar in 13 out of the 17 areas examined. Multiple inter-subline differences were recorded for each amino acid. These differences have been analyzed considering the seizure susceptibility or severity of the three Rb sublines. Common lower levels (approximately –20%: Rb1/Rb3, Rb2/Rb3) of Gln in Temporal Cortex may be implicated in seizure susceptibility. Seirure severity (Rb1/Rb2) seems to correlate, in some areas, with additional lower amounts of GABA already reported and, to a lower extent, of Asp (–19% in striatum, inferior colliculus and cerebellum), of Tau and Gly; a tendency for a rise in Gln content was observed in certain others (10–20% in olfactory bulb, thalamus, hypothalamus, substantia nigra, and frontal, temporal and occipital cortex). The data and correlations recorded provide guidelines for further investigations for synaptosomal and metabolic alterations in the three sublines of the same strain of Rb mice.Abbreviations used GABA 4-aminobutyrate - Tau taurine - Gly glycine - Asp aspartate - Glu glutamate - Gln glutamine - GEPR genetically epilepsy-prone rat - OB olfactory bulbs - OT olfactory tubercles - Sr striatum - Se septum - Hy hypothalamus - Hi hippocampus - Th thalamus - A amygdala - SC superior colliculus - IC interior colliculus - SN substantia nigra - FCx frontal cortex - TCx temporal cortex - OCx occipital cortex - C cerebellum - P pons - Ra raphe     

听源性惊厥致P77PMC大鼠杏仁核内胆囊收缩素mRNA短暂性增加

铃声刺激诱发惊厥 ,原位杂交法检测遗传性听源性癫痫易感大鼠 (P77PMC)一次与多次惊厥发作对杏仁核内胆囊收缩素 (CCK)mRNA含量的影响。结果发现 :( 1)惊厥未发作组大鼠杏仁核单位面积内的CCKmRNA阳性神经元数 (No/ 0 0 1mm2 )较少 ,为 8± 1;( 2 )惊厥发作一次组大鼠杏仁核单位面积内CCKmRNA阳性神经元数显著增加 ,发作后 30min时达到峰值 ,为 5 8± 5 (P <0 0 1) ,但是 2h后迅速降为正常 ,为 9± 2 (P >0 0 5 ) ;( 3)惊厥多次发作组大鼠在惊厥发作后 30min时 ,CCKmRNA阳性神经元数亦显著增加 ,为 2 2± 3 (P <0 0 1) ,但明显低于惊厥发作一次组大鼠 (P <0 0 1) ,1h即恢复正常 ,为 9± 3 (P >0 0 5 )。结果表明 ,惊厥发作后P77PMC大鼠杏仁核内CCKmR NA含量呈现迅速而短暂增加的特点 ,表明CCKmRNA参与惊厥的急性发作过程 ,提示CCKmRNA在惊厥发作早期阶段发挥了重要的抗惊厥作用     

转人APP基因小鼠视网膜视神经细胞超微结构改变

目的观察转人APP基因AD模型小鼠视网膜各层细胞超微结构的改变。方法实验采用C57BL/6J转人APP基因小鼠6只,采用同背景10个月龄的C57 BL/6J正常小鼠6只做为对照。灌注处死小鼠,完整取出右眼球分离视网膜,制作电镜标本观察视网膜各层细胞超微结构改变。结果与对照组相比,模型组视网膜各层细胞异常明显:膜盘排列结构模糊,局部间隙溶解、甚至消失;外核层细胞体干枯变形,染色质聚集浓缩;内核层可见固缩细胞;神经节细胞胞膜不完整,线粒体水肿,并可见染色质聚集。结论转人APP基因AD模型小鼠视网膜各层神经细胞超微结构存在明显病理改变。     

Glutamate receptor GluR1 expression is altered selectively by chronic audiogenic seizures in the Frings mouse brain

The audiogenic seizure-susceptible mouse, Frings, is genetically susceptible to sound-induced seizures and provides a reliable model of reflex epilepsy that lasts throughout the life span of the animal. We used immunohistochemistry to examine if the expression of the non-N-methyl-D -aspartate glutamate receptor (GluR) subunits GluR1, GluR2, or GluR3 were altered subsequent to multiple seizures. Following a regimen of one seizure per day for 3 weeks, GluR1 immunoreactivity, but not GluR2 or GluR3, was substantially elevated in the outer shell of the nucleus accumbens in 21 of 31 chronically seized Frings mice. No other brain regions such as the hippocampus exhibited any qualitative changes in expression of these subunits. In 9 of the 21 Frings mice exhibiting increased GluR1, but in none of the controls, bilateral structural lesions were observed in the lateral hypothalamus. These results support a model where highly localized changes in the expression of GluR1 occur in response to repeated audiogenic seizure. © 1998 John Wiley & Sons, Inc. J Neurobiol 35: 209–216, 1998     

Increased seizure susceptibility induced by guanidinoethane sulfonate in El mice and its relation to glutamatergic neurons

Convulsions and brain levels of amino acids and 5-hydroxytryptamine (5-HT) in El mice were examined after oral administration of a 1% guanidinoethane sulfonate (GES) solution. The incidence of convulsions increased 3 days after starting GES administration, and this effect continued throughout the 6 months of drug administration. Glutamate levels were increased in the cerebrum, and glutamine levels were increased in the cerebellum three days after starting GES administration. Brain 5-HT levels were not changed at that time. These results suggest that increased seizure susceptibility induced by GES in mice is related to glutamatergic neurons.     

利用转基因小鼠及转染色体小鼠产生人抗体的研究进展

自单克隆抗体（mAb)技术问世以来,解决了生命科学的许多重要问题,但其鼠源生导致的HAMA反应在大大限制了它在人体治疗中的应用,因此,制备人抗体成了亟待解决的难题,转入Ig基因组小鼠与转梁色体小鼠 的构建成功为解决这一难题提供了可行途径,本文就这两条小鼠的构建及其在制备人抗体中的应用进展作一综述。     

利用转基因小鼠筛选全人源炭疽致死因子中和抗体

炭疽是由炭疽芽孢杆菌引起的严重威胁人类健康的传染病。炭疽毒素包括3种蛋白质成分:保护性抗原(PA)、致死因子(LF)和水肿因子(EF)。PA与LF形成致死毒素(LT),与EF形成水肿毒素(ET)。由于致死毒素(LT)在感染者损伤及死亡中发挥主要作用,因此在炭疽感染晚期单纯使用抗生素治疗难以发挥疗效,治疗性中和抗体成为目前最有效的炭疽治疗药物。目前国外获得的炭疽毒素抗体多为炭疽PA抗体,美国FDA已批准瑞西巴库(人源PA单抗)用于吸入性炭疽的治疗。一旦炭疽芽孢杆菌被人为改构或PA中和表位发生突变,针对PA单一表位的抗体将可能失效,因此针对LF的抗体将成为炭疽治疗的有效补充。目前国外已有的LF抗体多为鼠源抗体和嵌合抗体,而全人源抗体可以避免鼠源抗体免疫原性高等缺点。本研究首先用LF抗原免疫人抗体转基因小鼠,利用流式细胞仪从小鼠脾淋巴细胞中分选抗原特异的记忆B细胞,通过单细胞PCR方法快速获得两株具有结合活性的抗LF单抗1D7和2B9。瞬时转染Expi 293F细胞制备抗体,通过毒素中和实验(TNA)发现1D7和2B9在细胞模型中均显示较好的中和活性,并且与PA单抗联合使用时,表现出较好的协同作用。总之,本文利用转基因小鼠、流式分选技术和单细胞PCR技术的优势,快速筛选到全人源LF抗体,为快速筛选全人源单克隆抗体开辟了新的思路与方法。     

Analysis of transfer of microinjected zygotes in production of transgenic mice

The data on transfer of mouse eggs microinjected with DNA during production of transgenic mice were analyzed. The transfer of mouse eggs into both oviducts did not lead to a reliably higher birth rate. It did not affect the frequency of recipients’ pregnancy and, although somewhat increased the frequency of multiple birth, led, finally, to unjustified loss of the major part of viable DNA-injected eggs. We recommend transferring no less than 15 microinjected eggs only in one oviduct of each recipient. The transfer into another oviduct is acceptable if the transfer into the first oviduct failed or its outcome is doubtful.     

Changes in skin levels of two neutotrophins （glial cell line derived neurotrohic factor and neurotrophin-3） cause alterations in cutaneous neuron responses to mechanical stimuli

Neurotrophins are important for the development and maintenance of both high and low threshold mechanoreceptors (HTMRs and LTMRs). In this series of studies, the effects of constitutive overexpression of two different neurotrophins, neurotrophin-3 (NT-3) and glial cell line derived neurotrohic factor (GDNF), were examined. Previous studies indicated that both of them may be implicated in the normal development of mouse dorsal root ganglion (DRG) neurons. Neurons from mice transgenically altered to overexpress NT-3 or GDNF (NT-3-OE or GDNF-OE mice) in the skin were examined using several physiological, immunohistochemi-cal and molecular techniques. Ex vivo skin/nerve/DRG/spinal cord and skin/nerve preparations were used to determine the response characteristics of the cutaneous neurons; immunohistochemistry was used to examine the biochemical phenotype of DRG cells and the skin; RT-PCR was used to examine the levels of candidate ion channels in skin and DRG that may correlate with changes in physiologi-cal responses. In GDNF-OE mice, I-isolectin B4 (IB4)-immunopositive C-HTMRs (nociceptors), a large percentage of which are sensitive to GDNF, had significantly lower mechanical thresholds than wildtype (WT) neurons. Heat thresholds for the same cells were not different. Mechanical sensitivity changes in GDNF-OE mice were correlated with significant increases in acid sensing ion channels 2a (ASIC2a) and 2b (ASIC2b) and transient receptor potential channel AI (TRPAI), all of which are putative mechanosensitive ion channels. Overexpression of NT-3 affected the responses of A-LTMRs and A-HTMRs, hut had no effect on C-HTMRs. Slowly adapting type 1 (SA1) LTMRs and A-HTMRs had increased mechanical sensitivity compared to WT. Mechanical sensitivity was correlated with significant increases in acid-sensing ion channels ASIC1 and ASIC3. This data indicates that both neurotrophins play roles in determining mechanical thresholds of cutaneous HTMRs and LTMRs and that sensitivity changes involve the ASIC family of putative mechanoreceptive ion channels.     

Specific changes of sulfatide levels in individuals with pre‐clinical Alzheimer's disease: an early event in disease pathogenesis

To explore the hypothesis that alterations in cellular membrane lipids are present at the stage of pre‐clinical Alzheimer's disease (AD) (i.e., cognitively normal at death, but with AD neuropathology), we performed targeted shotgun lipidomics of lipid extracts from post‐mortem brains of subjects with pre‐clinical AD. We found sulfatide levels were significantly lower in subjects with pre‐clinical AD compared to those without AD neuropathology. We also found that the level of ethanolamine glycerophospholipid was marginally lower at this stage of AD, whereas changes of the ceramide levels were undetectable with the available samples. These results indicate that cellular membrane defects are present at the earliest stages of AD pathogenesis and also suggest that sulfatide loss is among the earliest events of AD development, while alterations in the levels of ethanolamine glycerophospholipid and ceramide occur relatively later in disease.