爪蟾胚胎脊髓胆碱能神经元上两类非去极化激活的钙通道

用膜片箝方法,在标定的爪蟾胚胎脊髓胆碱能神经细胞膜上记录钙离子单通道电流。结果显示,在静息膜电位时钙通道即有开放活动。根据通道的电导及动力学等特性,我们将其分为两种类型:NS 型钙通道,斜率电导7.5pS,静息膜电位时平均开放时间0.58ms,其活动受牵张膜片的影响;NL 型钙通道,斜率电导16.7pS,静息膜电位时二级平均开放时间分别是2ms 和19.3ms。鉴于它们在膜静息以及负电位相的显著活动,我们推测这两类钙通道可能参与神经元静息膜电位时钙依赖的细胞活动过程。     

非洲爪蟾卵细胞膜去极化后由超极化激活的瞬间内向电流

以双微电极电压钳制技术研究了未成熟非洲爪蟾卵细胞膜的离子流,发现在较长时间的去极化（－３０ｍｖ,５秒）前脉冲后由超极化引出的一个内向电流,其潜伏期约为０．５秒,经过０．４秒左右到达高峰,随近经０．８秒左右完全回复。其幅值随超极化程度增强而增大,其翻转电位接近氯平衡电位,并随胞外ＣＬ－浓度改变而变化。降低胞外ＣＬ－浓度使其幅值增大。降低胞外Ｎａ＾＋浓度对其无明显影响,提示此内几电流可能是氯离子流。以     

爪蟾卵母细胞表达猫脊髓背根神经节初级感觉神经元的速激肽受体

提取猫的背根神经节 (DRG)中的Poly(A) ⁺RNA ,注射到非洲爪蟾卵母细胞中进行表达 ,2d后通过双电极电压箝技术检测爪蟾卵母细胞对速激肽受体激动剂的反应 .NK- 1受体特异激动剂 [Sar⁹,Met(O₂ ) ¹¹]SP (Sar -SP) ,NK -2受体特异激动剂 [β- Ala⁸] neurokininA ( 4～ 1 0 ) (Ala -NKA)和神经激肽A(NKA)产生相似的内向电流 .由一个快速的锋电流和持续数分钟的振荡电流组成 .Sar- SP的反应只被NK- 1受体特异拮抗剂L- 6 6 8,1 6 9( 1 μmol/L)阻断 ,而NKA及Ala- NKA的反应则仅被NK -2受体特异拮抗剂L -6 5 8,877( 1 μmol/L)阻断 .注射猫脊髓背角Poly(A) ⁺RNA在爪蟾卵母细胞中表达的速激肽受体的反应基本相似 .这些速激肽受体的反应都具有强烈的脱敏性 .这是首次在爪蟾卵母细胞受体表达系统证明DRG神经元有速激肽NK- 1和NK -2受体 ,从而提示在伤害性初级传入末梢上可能存在速激肽突触前自身受体     

大鼠脑mRNA在爪蟾卵母细胞内表达的电压门控钙通道的电压箝研究

本工作用电压箝记录方法研究了爪蟾卵母细胞经注射大鼠脑mRNA后表达的电压门控钙通道。钙通道的特性由通过钙通道的钡离子流(I_(Ba))来描述。本研究采用的卵母细胞均取自被鉴定的爪蟾。这些爪蟾卵母细胞内源性I_(Ba)大多为零,或小于15nA。将从出生后10d的大鼠全脑中提取的mRNA微量注入这些卵母细胞。在注射mRNA后的5d内,I_(Ba)逐渐增大。在mRNA注射后第三天,由大鼠脑mRNA表达的电压依赖性I_(Ba)最大值一般超过100nA。作为对比,在注射从胚胎大鼠脑提取的mRNA的卵母细胞,几乎测不到电压依赖性I_(Ba)的表达。我们研究了由大鼠脑mRNA表达的I_(Ba)的电压依赖性激活及失活特性和I_(Ba)的药理。发现镧系金属离子(La~(+3),Nd~(+3),Sm~(+3),Eu~(+3),Gd~(+3),Dy~(+3),Er~(+3)在微摩尔浓度数量级即能有效抑制I_(Ba)。L-型钙通道配体nifedipine和Bay K 8644在浓度100μmmol/L时,抑制I_(Ba),而另一dihydropyridine类配体(±)-nimodipine在相同浓度却增加I_(Ba)。     

TGF_β相关mRNAs在爪蟾早期胚胎中的表达

用TGF_β-1 cDNA片段作为探针,在爪蟾早期胚胎中检测到了能同此探针杂交的转录产物,分子量分别为4.2 kb,3.2 kb和2.3 kb,其中4.2 kb和3.2 kb的mRNA 在囊胚期(分期7—8)中的含量最高,而在卵裂期胚胎,原肠胚以及神经胚中含量极微。但2.3 kb的mRNA以卵裂期胚胎到神经胚中含最比较稳定。我们称这些能同TGF_β-1cDNA 探针杂之的mRNAs为TGF_β相关的mRNAs (TGFB_β-relatedmRNAs),它们在囊胚中主要分布在植物性半球,但背方和腹方的含量无明显差别。根据TGF_β相关mRNAs 在囊胚国的含量最高和主要分布在植物性半球,我们推测它们可能与中胚层诱导有关。     

TGFβ相关蛋白在爪蟾早期胚胎中的免疫组织化学的研究

By the method of immunocytochemistry, using the polyclonal antibodies raised against the 1-29 N-terminal residues of TGF beta-1, we found that the protein could bind to the antibodies was present in the early embryos of Xenopus. The protein was named TGF beta-related protein. It was distributed mainly in the endoderm from blastula (stg. 7) to late neurula. In the blastula (stg. 8), the protein was localized in the vegetal hemisphere near the floor of the blastocoel [Plate I, Fig. 1]. In the early gastrula (stg. 10.5) [Plate I, Fig. 2], it was localized in the central part of the vegetal hemisphere. In late gastrula (stg. 12), it was mainly distributed around the gastrocoel [Plate I, Fig. 3], but the fluorescence in endoderm cells (ventral part beneath the gastrocoel) was stronger than in the mesoderm cells (dorsal part of the gastrocoel). In the early neurula (stg. 14), the whole endoderm displayed strong fluorescence and the part of dorsal mesoderm (presumptive somite & notochord) close to endoderm was also found to be positively stained [Plate I, Fig. 4,5], but the part close to neural plate was negative. In The late neurula (stg. 20) [Plate I, Fig. 6], it was found in the central area of yolk mass (endoderm cells). No positive stain was detected in the unfertilized egg, embryos earlier than stage and later than stage 20/21.2+ protein in early development.     

DIG标记DNA探针检测noggin在爪蟾胚胎中的表达

整体原位杂交（Whole-mount in situ hybridization)用于基因表达定位和表达分布模式的研究,已经成为一种非常重要的手段。PCR方法进行探针标记,可以获得特异性高,片断大小可变的探针。采用DIG标记,检测已知基因noggin在爪蟾胚胎时空分布,所得结果与文献报道一致,表明PCR方法获得的DNA探针在一定的条件下可以用于爪蟾胚胎整体原位杂交。     

爪蟾胚胎内胚层发育中细胞间连接通讯能力的广泛增强(英文)

本研究以羧基荧光素为示踪物,发现在爪蟾胚胎内胚层大多数细胞之间,这种分子量为376 D的染料的转移,只有到原肠胚末期后才能测出,在此之前,这些细胞之间仅有电耦联存在。观察表明,这一差异不可能单由于细胞分裂体积减小所致,它反映了在原肠胚末期细胞间连接通讯能力由低水平到高水平的转变,是细胞的这一通讯能力发育的结果。这个转变与已知的两栖类内胚层细胞分化区域模式在神经胚早中期的建立在时间上相衔接,因此它是否是后者的必要条件值得进一步查明。在早期囊胚及以后割离的植物性半球的内胚层细胞中,或在中囊胚期之后割离再由单细胞重新聚合成的小细胞团中,其细胞间普遍出现羧基荧光素转移的时间与这些细胞在在体条件下的一致,说明其细胞间连接通讯能力的发育在这样的离体标本中能完全自主地进行。这些标本可以代替整体标本用来方便地测定该转变出现的时间。这种测定对研究各种因素对细胞间连接通讯能力发育的影响具有重要意义。     

Single calcium channels in neurons of rat spinal ganglia

Using a refined patch clamp technique, a study was made of single calcium channels of spinal ganglia neurons on a cell-attached membrane site in newborn rats; these convey the basic (high threshold) component of calcium current. Findings show that currents carried by calcium ions at a concentration of 60 mM in the recording pipet changes from 0.58±0.05 to 0.43±0.05 pA with a change in potential of 20 mV. This corresponds to a channel conductance of 7±0.5 pS. The distribution of open time was monoexponential with a time constant of about 0.75 msec, independent of membrane potential. Distribution of closed time approached a biexponential time course. The fast component (0.8 msec) was voltage-dependent, while the slow component decreased from 22 to 4 msec when depolarization increased by 20 mV. Using experimentally obtained time parameters which describe single calcium channel function, and assuming a three-tier model of the channel, the numerical values of the constants of transition rates between individual states were determined.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 17, No. 5, pp. 673–682, September–October, 1985.     

Effect of protein phosphorylation on neurite outgrowth in cultured embryonic Xenopus spinal neurons

Intracellular signaling pathways involved in neurite outgrowth have been extensively studied in a variety of cell systems. While most of these studies utilized continuous neuronal-like cell lines, fewer studies have been conducted in primary neuronal culture. One primary culture system that has recently been used to dissect the signaling pathways involved in axon guidance consists of spinal neurons derived from embryonic Xenopus laevis. In this study, we used Xenopus to study neurite outgrowth by treating neuronal cultures with pharmacological agents that activate or inhibit various protein kinases or that inhibit protein phosphatases. We found that agents which affected signaling via cAMP-dependent protein kinase, calmodulin, cyclin-dependent kinase 5, or protein phosphatases had effects on Xenopus neurite outgrowth that were similar to those reported in other primary neurons or in neuronal-like cell lines. However, agents which affected protein kinase C signaling had effects on Xenopus neurite outgrowth that were distinct from those reported in neuronal-like cell lines. Although continuous cell lines have several advantages for the dissection of signaling pathways involved in neurodevelopment, these observations underscore the importance of also using primary neurons to examine these pathways.     

Zebrafish transgenic constructs label specific neurons in Xenopus laevis spinal cord and identify frog V0v spinal neurons

A correctly functioning spinal cord is crucial for locomotion and communication between body and brain but there are fundamental gaps in our knowledge of how spinal neuronal circuitry is established and functions. To understand the genetic program that regulates specification and functions of this circuitry, we need to connect neuronal molecular phenotypes with physiological analyses. Studies using Xenopus laevis tadpoles have increased our understanding of spinal cord neuronal physiology and function, particularly in locomotor circuitry. However, the X. laevis tetraploid genome and long generation time make it difficult to investigate how neurons are specified. The opacity of X. laevis embryos also makes it hard to connect functional classes of neurons and the genes that they express. We demonstrate here that Tol2 transgenic constructs using zebrafish enhancers that drive expression in specific zebrafish spinal neurons label equivalent neurons in X. laevis and that the incorporation of a Gal4:UAS amplification cassette enables cells to be observed in live X. laevis tadpoles. This technique should enable the molecular phenotypes, morphologies and physiologies of distinct X. laevis spinal neurons to be examined together in vivo . We have used an islet1 enhancer to label Rohon‐Beard sensory neurons and evx enhancers to identify V0v neurons, for the first time, in X. laevis spinal cord. Our work demonstrates the homology of spinal cord circuitry in zebrafish and X. laevis , suggesting that future work could combine their relative strengths to elucidate a more complete picture of how vertebrate spinal cord neurons are specified, and function to generate behavior. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 77: 1007–1020, 2017     

Two peroxiredoxins 6 of Xenopus laevis

Two different genes of peroxiredoxin 6 are encoded in the genome of Xenopus laevis: xen1 (Acc.no. EMBL Data Bank - BCO54278) and xen2 (Acc.no. EMBL Data Bank - BC540309). Both genes were cloned and expressed in Escherichi coil. Proteins were purified and analyzed. The amino acid sequences of the enzymes Xen1 and Xen2 are 95% identical with the same peroxidase activity, pH and temperature optimums, as well as thermostability, being approximately equal. The expression of peroxiredoxin 6 genes significantly differ during ontogenesis of X. laevis. The expression of xen1 starts on a later stage of development 47-48, while the gene xen2 is expressed on all stages of development with the same increase starting from stage 0-5. The level of xen2 expression in embryos increased after incubation in presence of hydrogen peroxide. The comparison of amino acid sequences of proteins Xen1 and Xen2 shows that only the enzyme Xen2 may have phospholipase activity, since it has residues of phospholipase A2 active center: Ser31, His25, Asp139.     

Purification of a calcium dependent ribonuclease from Xenopus laevis.

We have purified a Ca2+ dependent ribonuclease from the oocytes of Xenopus leavis. Two properties of this ribonuclease set it apart from other known nucleases. First, Ca2+ was required for ribonuclease activity, and Mg2+ would not substitute. Second, the enzyme specifically degraded RNA and digestion of double or single stranded DNA was not observed. Ca2+ dependent ribonuclease activity of the purified 36-kDa protein was directly observed after renaturation of the protein following electrophoresis in an SDS-Laemmli gel. In addition, the enzyme was shown to have endoribonuclease activity at numerous sites. The Ca2+ dependence suggests that the ribonuclease activity may be modulated by changes in the level of intracellular Ca2+ and thereby provide a direct link to signal transduction systems.     

Purposeful patterns of spontaneous calcium transients in embryonic spinal neurons

Embryonic cultured Xenopus spinal neurons generate two types of spontaneous elevation of intracellular calcium that encode developmental information in the frequency with which they are produced. Calcium spikes regulate the appearance of GABA and maturation of potassium current. Calcium waves in growth cones regulate neurite extension. Spikes and waves are also observed in neurons differentiating in situ. Because differentiation is dependent on the frequency of calcium transients, neurons that are coactive and fire spikes in concert would be expected to differentiate together. Consistent with this prediction, segmentally arrayed clusters of putative motoneurons on the ventral aspect of the neural tube fire together during development.     

Effect of antikeratin microinjection on the embryonic development of Xenopus laevis

Anti-keratin monoclonal antibody AF5 was introduced into fertilized eggs of Xenopus laevis.,and its effects on embryonic development were studied.Survival rate of the antikeratin-injected embryos was much lower(only 35.67% at gastrula)than that of the control(74.85% at gastrula),in which embryos were injected with mouse IgG.Most of survivors in the experimental series showed aberrant external appearance.On the other hand,in cleavage stage,ie 2-7h after fertilization,immunohistochemical staining of embryos showed that the expermental embryos were mostly keratin negative,while embryos of the control ones were keratin positive.When introducing this antikeratin into one cell of a 2-cell embryo,only the uninjected half of the embryo continued its development while the other half could not develop at all.These results suggested that intact keratin cytoskeleton in early embryos is indispensable to the embryonic development of Xenopus laevis.     

Action of hypoxia on different types of calcium channels in hippocampal neurons

Whole-cell patch clamp and polarographic oxygen partial pressure (pO2) measurements were used to establish the sensitivity of high-voltage-activated (HVA) Ca2+ channel subtypes of CA1 hippocampal neurons of rats to hypoxic conditions. Decrease of pO2 to 15-30 mm Hg induced a potentiation of HVA Ca2+ currents by 94%. Using selective blockers of N- and L-types of calcium channels, we found that inhibition of L-type channels decreased the effect by 54%, whereas N-type blocker attenuated the effect by 30%. Taking into account the ratio of currents mediated by these channel subtypes in CA1 hippocampal neurons, we concluded that both types of HVA Ca2+ channels are sensitive to hypoxia, however, L-type was about 3.5 times more sensitive to oxygen reduction.     

Three types of transmitter release from embryonic neurons

Prior to the contact with their target muscle cells in culture, growth cones of many isolated Xenopus embryonic neurons release acetylcholine (ACh) spontaneously. Using patch clamp techniques, this release can be detected by an outside-out patch of muscle membrane placed near the growth cone. Intracellular recording from innervated muscle cells showed spontaneous miniature endplate potentials (MEPPs) of varying amplitudes. Amplitude histograms showed a skewed distribution with multiple peaks, suggesting the existence of subunits in either the quantal packages of ACh released by the nerve terminal or in the postsynaptic muscle response. In addition to the quantal ACh release reflected by MEPPs, nerve terminal also release a large amount of ACh in a non-quantal fashion. This non-quantal ACh release is revealed by the hyperpolarization of the muscle membrane following extracellular application of curare or alpha-bungarotoxin, as well as by denervation of the muscle cell.