The Pig Lhx8 Gene： cDNA Cloning, Bioinformatic Analysis and Expression Level in Tissues and Preimplantation Embryos

Evidence has shown in mouse that Lhx8 is a critical factor for maintenance and differentiation of the oocyte during early oogenesis. In the current paper, attempts were made to clone and characterize a gene encoding Lhx8 from pig. Rapid amplification of cDNA ends （RACE） gave rise to a full-length of Lhx8 which contained 1 681 bp nucleotides, with a complete open reading frame of 885 bp, encoding a 295 amino acid polypeptide. Homology search and sequence multialignment demonstrated that the deduced pig Lhx8 protein sequence shared a high identity with Lhx8 from other mammals, including several highly conservative motifs and amino acids. The phylogenetic tree of the LIM superfamily proteins has been constructed to reveal the evolutionary relationship of various species. RT-PCR analysis showed that the Lhx8 gene was expressed in gonad and immunity tissues. In preimplantation embryos, Lhx8 mRNA expression profiling using realtime PCR revealed that its mRNA levels were highest in 4-cell stage embryos and gradually decreased until the blastocyst stage.     

Cerebellum shapes hippocampal spatial code

Spatial representation is an active process that requires complex multimodal integration from a large interacting network of cortical and subcortical structures. We sought to determine the role of cerebellar protein kinase C (PKC)-dependent plasticity in spatial navigation by recording the activity of hippocampal place cells in transgenic L7PKCI mice with selective disruption of PKC-dependent plasticity at parallel fiber-Purkinje cell synapses. Place cell properties were exclusively impaired when L7PKCI mice had to rely on self-motion cues. The behavioral consequence of such a deficit is evidenced here by selectively impaired navigation capabilities during a path integration task. Together, these results suggest that cerebellar PKC-dependent mechanisms are involved in processing self-motion signals essential to the shaping of hippocampal spatial representation.     

The ground state of the ventral appendage in Drosophila

In Drosophila melanogaster, the antennae, legs, genitalia, and analia make up a serially homologous set of ventral appendages that depend on different selector genes for their unique identities. The diversity among these structures implies that there is a common ground state that selector genes modify to generate these different appendage morphologies. Here we show that the ventral appendage that forms in the absence of selector gene activity is leglike but consists of only two segments along its proximo-distal axis: a proximal segment and a distal tarsus. These results raise the possibility that, during evolution, leglike appendages could have developed without selector gene activity.     

陕北白绒山羊Lhx2基因cDNA序列的克隆与原核表达

根据GenBank中已发表的Lhx2基因序列设计引物,采用RT-PCR方法,从陕北白绒山羊皮肤组织克隆Lhx2基因编码区cDNA,对其进行生物信息学分析,并构建原核表达载体pET-Lhx2,在大肠杆菌BL21(DE3)中表达。最终获得陕北白绒山羊Lhx2基因,长1 221bp;酶切和测序结果显示,原核表达载体pET-Lhx2构建成功;SDS-PAGE和Western blot分析表明,重组原核表达载体在大肠杆菌中成功表达出目的融合蛋白。     

陕北白绒山羊Lhx2毛囊表达载体构建及转染成纤维细胞的研究

以陕北白绒山羊毛囊生长期皮肤组织cDNA为模板,扩增出Lhx2基因的CDS区,成功构建了以KAP6.1为启动子表达Lhx2基因的绿色荧光表达载体pIRES2-KAP-LHX-EGFP.并利用脂质体介导转染绒山羊胎儿成纤维细胞,通过G418筛选获得了稳定转染的细胞系,经PCR检测显示外源基因已整合到细胞基因中.研究结果为进一步开展Lhx2基因对绒山羊毛囊发育的影响和转Lhx2基因绒山羊的生产提供了前期研究基础.     

Correlated bursts of activity in the neonatal hippocampus in vivo

The behavior of immature cortical networks in vivo remains largely unknown. Using multisite extracellular and patch-clamp recordings, we observed recurrent bursts of synchronized neuronal activity lasting 0.5 to 3 seconds that occurred spontaneously in the hippocampus of freely moving and anesthetized rat pups. The influence of slow rhythms (0.33 and 0.1 hertz) and the contribution of both gamma-aminobutyric acid A-mediated and glutamate receptor-mediated synaptic signals in the generation of hippocampal bursts was reminiscent of giant depolarizing potentials observed in vitro. This earliest pattern, which diversifies during the second postnatal week, could provide correlated activity for immature neurons and may underlie activity-dependent maturation of the hippocampal network.     

SV2 is the protein receptor for botulinum neurotoxin A

How the widely used botulinum neurotoxin A (BoNT/A) recognizes and enters neurons is poorly understood. We found that BoNT/A enters neurons by binding to the synaptic vesicle protein SV2 (isoforms A, B, and C). Fragments of SV2 that harbor the toxin interaction domain inhibited BoNT/A from binding to neurons. BoNT/A binding to SV2A and SV2B knockout hippocampal neurons was abolished and was restored by expressing SV2A, SV2B, or SV2C. Reduction of SV2 expression in PC12 and Neuro-2a cells also inhibited entry of BoNT/A, which could be restored by expressing SV2 isoforms. Finally, mice that lacked an SV2 isoform (SV2B) displayed reduced sensitivity to BoNT/A. Thus, SV2 acts as the protein receptor for BoNT/A.     

Regulation of area identity in the mammalian neocortex by Emx2 and Pax6

The contribution of extrinsic and genetic mechanisms in determining areas of the mammalian neocortex has been a contested issue. This study analyzes the roles of the regulatory genes Emx2 and Pax6, which are expressed in opposing gradients in the neocortical ventricular zone, in specifying areas. Changes in the patterning of molecular markers and area-specific connections between the cortex and thalamus suggest that arealization of the neocortex is disproportionately altered in Emx2 and Pax6 mutant mice in opposing manners predicted from their countergradients of expression: rostral areas expand and caudal areas contract in Emx2 mutants, whereas the opposite effect is seen in Pax6 mutants. These findings suggest that Emx2 and Pax6 cooperate to regulate arealization of the neocortex and to confer area identity to cortical cells.     

Spatial selectivity of rat hippocampal neurons: dependence on preparedness for movement

The mammalian hippocampal formation appears to play a major role in the generation of internal representations of spatial relationships. In rats, this role is reflected in the spatially selective discharge of hippocampal pyramidal cells. The principal metric for coding spatial relationships might be the organism's own movements in space, that is, the spatial relationship between two locations is coded in terms of the movements executed in getting from one to the other. Thus, information from the motor programming systems (or "motor set") may contribute to coding of spatial location by hippocampal neurons. Spatially selective discharge of hippocampal neurons was abolished under conditions of restraint in which the animal had learned that locomotion was impossible. Therefore, hippocampal neuronal activity may reflect the association of movements with their spatial consequences.     

"Who" is saying "what"? Brain-based decoding of human voice and speech

Can we decipher speech content ("what" is being said) and speaker identity ("who" is saying it) from observations of brain activity of a listener? Here, we combine functional magnetic resonance imaging with a data-mining algorithm and retrieve what and whom a person is listening to from the neural fingerprints that speech and voice signals elicit in the listener's auditory cortex. These cortical fingerprints are spatially distributed and insensitive to acoustic variations of the input so as to permit the brain-based recognition of learned speech from unknown speakers and of learned voices from previously unheard utterances. Our findings unravel the detailed cortical layout and computational properties of the neural populations at the basis of human speech recognition and speaker identification.     

Neuronal diversity and temporal dynamics: the unity of hippocampal circuit operations

In the cerebral cortex, diverse types of neurons form intricate circuits and cooperate in time for the processing and storage of information. Recent advances reveal a spatiotemporal division of labor in cortical circuits, as exemplified in the CA1 hippocampal area. In particular, distinct GABAergic (gamma-aminobutyric acid-releasing) cell types subdivide the surface of pyramidal cells and act in discrete time windows, either on the same or on different subcellular compartments. They also interact with glutamatergic pyramidal cell inputs in a domain-specific manner and support synaptic temporal dynamics, network oscillations, selection of cell assemblies, and the implementation of brain states. The spatiotemporal specializations in cortical circuits reveal that cellular diversity and temporal dynamics coemerged during evolution, providing a basis for cognitive behavior.     

纳米银对原代培养海马神经细胞凋亡的研究

[目的]探讨纳米银对原代培养海马神经细胞的影响。[方法]用吖啶橙染色观察细胞形态及染色质的变化,用Fluo3荧光染料检测细胞内游离Ca2+浓度变化,并利用ELLSA试剂盒检测Caspase-3凋亡酶活性。[结果]与正常对照组相比,经50μg/ml纳米银处理36h后,海马神经细胞的凋亡率达到最高((48.3±0.33)%),凋亡酶活性增强,并且细胞内游离的Ca2+荧光强度达到41.70±18.78。结论50μg/ml纳米银作用36 h可导致海马神经元凋亡。     

Hemoxygenase-2 is an oxygen sensor for a calcium-sensitive potassium channel

Modulation of calcium-sensitive potassium (BK) channels by oxygen is important in several mammalian tissues, and in the carotid body it is crucial to respiratory control. However, the identity of the oxygen sensor remains unknown. We demonstrate that hemoxygenase-2 (HO-2) is part of the BK channel complex and enhances channel activity in normoxia. Knockdown of HO-2 expression reduced channel activity, and carbon monoxide, a product of HO-2 activity, rescued this loss of function. Inhibition of BK channels by hypoxia was dependent on HO-2 expression and was augmented by HO-2 stimulation. Furthermore, carotid body cells demonstrated HO-2-dependent hypoxic BK channel inhibition, which indicates that HO-2 is an oxygen sensor that controls channel activity during oxygen deprivation.     

Neural systems underlying the suppression of unwanted memories

Over a century ago, Freud proposed that unwanted memories can be excluded from awareness, a process called repression. It is unknown, however, how repression occurs in the brain. We used functional magnetic resonance imaging to identify the neural systems involved in keeping unwanted memories out of awareness. Controlling unwanted memories was associated with increased dorsolateral prefrontal activation, reduced hippocampal activation, and impaired retention of those memories. Both prefrontal cortical and right hippocampal activations predicted the magnitude of forgetting. These results confirm the existence of an active forgetting process and establish a neurobiological model for guiding inquiry into motivated forgetting.     

Model of the origin of rhythmic population oscillations in the hippocampal slice

One goal of mammalian neurobiology is to understand the generation of neuronal activity in large networks. Conceptual schemes have been based on either the properties of single cells or of individual synapses. For instance, the intrinsic oscillatory properties of individual thalamic neurons are thought to underlie thalamic spindle rhythms. This issue has been pursued with a computer model of the CA3 region of the hippocampus that is based on known cellular and synaptic properties. Over a wide range of parameters, this model generates a rhythmic activity at a frequency faster than the firing of individual cells. During each rhythmic event, a few cells fire while most other cells receive synchronous synaptic inputs. This activity resembles the hippocampal theta rhythm as well as synchronized synaptic events observed in vitro. The amplitude and frequency of this emergent rhythmic activity depend on intrinsic cellular properties and the connectivity and strength of both excitatory and inhibitory synapses.     

捻转血矛线虫Hc-hrg-2拯救血红素缺陷型酵母生长表型

【目的】已有研究表明捻转血矛线虫（Haemonchus contortus）Hc-hrg-2属于血红素应答基因,高浓度血红素的刺激可导致该基因的转录水平上调,但其在血红素调控中的功能尚缺乏研究。研究通过同源重组技术构建血红素合成缺陷型酿酒酵母（Saccharomyces cerevisiae）hem1敲除株,通过异源表达Hc-hrg-2对该敲除株进行表型拯救试验,以期验证Hc-hrg-2参与细胞内血红素转运的功能。 【方法】以S. cerevisiae BY4741基因组为模板,设计引物,扩增获得hem1（SGD:S000002640）基因序列5' 和3' 侧翼区同源臂;同时以pYES2-CT质粒为模板设计引物,扩增获得筛选标记URA3序列;利用两次重叠PCR技术依次将测序正确的上游同源臂、URA3、下游同源臂序列串联成基因敲除组件,并通过醇沉法对敲除组件进行纯化。采用醋酸锂转化法将纯化的敲除组件转化至BY4741感受态细胞中, 经SD/- URA（含250 μmol·L^-1 5-氨基乙酰丙酸（ALA））培养基筛选,并利用多对引物进行PCR鉴定,以验证Δhem1敲除株的正确性。同时以含有H. contortus 浙江株Hc-hrg-2 序列（GenBank：MK371241）及其功能域缺失序列Hc-hrg-2(Δgst-n) 和Hc-hrg-2(Δgst-c) 的质粒为模板,利用特异性引物分别进行PCR扩增,通过无缝克隆将扩增得到的目的片段插入酵母pESC-LEU表达载体,经PCR鉴定以及测序验证后,采用醋酸锂转化法将正确的表达载体转化至Δhem1感受态细胞中,通过SD/- URA/- LEU（含250 μmol·L^-1 ALA）培养基筛选以及PCR鉴定验证阳性异源表达株的正确性。通过比较Δhem1敲除株及其各异源表达株在含有或不含有250 μmol·L^-1 ALA的SD/- URA/- LEU液体培养基中的生长情况,进一步验证敲除株的表型同时排除表达载体对表型的影响。用2%半乳糖对含有阳性表达质粒的敲除株进行诱导,取部分菌液进行超裂破碎,收集蛋白,通过Western Blot鉴定目的蛋白的表达;剩余菌体用去离子水重悬至OD₆₀₀ = 0.2,用去离子水进行5倍比稀释,取4 μL稀释后的菌液点至含有250 μmol·L^-1 ALA或者不同浓度血红素的诱导平板上,28℃培养2—3 d后比较敲除株的生长情况。【结果】成功获得hem1基因敲除株Δhem1,与野生株相比,Δhem1不能合成血红素,需外源添加ALA（250 μmol·L^-1）或血红素（≥ 10 μmol·L^-1）才能生长,且异源表达株和敲除株的表型一致。Western Blot结果表明2%半乳糖能诱导Hc-hrg-2及其功能域缺失基因在敲除株中成功表达,且表达Hc-hrg-2能够在低血红素浓度下（≤ 1 μmol·L^-1）促进酵母对血红素的摄取,拯救敲除株的生长缺陷,其硫氧还蛋白样结构域（GST-N）和谷胱甘肽S-转移酶C末端结构域（GST-C）的缺失会降低拯救的效果。【结论】捻转血矛线虫血红素应答基因Hc-hrg-2可促进细胞对血红素的摄取,其GST-N和GST-C功能域在该过程中发挥着重要作用,研究成果为后续深入研究捻转血矛线虫的血红素转运机制奠定基础。     

The role of chloride transport in postsynaptic inhibition of hippocampal neurons

Hippocampal inhibitory postsynaptic potentials are depolarizing in granule cells but hyperpolarizing in CA3 neurons because the reversal potentials and membrane potentials of these cells differ. Here the hippocampal slice preparation was used to investigate the role of chloride transport in these inhibitory responses. In both cell types, increasing the intracellular chloride concentration by injection shifted the reversal potential of these responses in a positive direction, and blocking the outward transport of chloride with furosemide slowed their recovery from the injection. In addition, hyperpolarizing and depolarizing inhibitory responses and the hyperpolarizing and depolarizing responses to the inhibitory neurotransmitter gamma-aminobutyric acid decreased in the presence of furosemide. These effects of furosemide suggest that the internal chloride activity of an individual hippocampal neuron is regulated by two transport processes, one that accumulates chloride and one that extrudes chloride.