Sonic Hedgehog Promotes Neurite Outgrowth of Primary Cortical Neurons Through Up-Regulating BDNF Expression

Sonic hedgehog (Shh), a secreted glycoprotein factor, can activate the Shh pathway, which has been implicated in neuronal polarization involving neurite outgrowth. However, little evidence is available about the effect of Shh on neurite outgrowth in primary cortical neurons and its potential mechanism. Here, we revealed that Shh increased neurite outgrowth in primary cortical neurons, while the Shh pathway inhibitor (cyclopamine, CPM) partially suppressed Shh-induced neurite outgrowth. Similar results were found for the expressions of Shh and Patched genes in Shh-induced primary cortical neurons. Moreover, Shh increased the levels of brain-derived neurotrophic factor (BDNF) not only in lysates and in culture medium but also in the longest neurites of primary cortical neurons, which was partially blocked by CPM. In addition, blocking of BDNF action suppressed Shh-mediated neurite elongation in primary cortical neurons. In conclusion, these findings suggest that Shh promotes neurite outgrowth in primary cortical neurons at least partially through modulating BDNF expression.     

Amyloid Beta Peptide Impaired Carbachol but not Glutamate-Mediated Phosphoinositide Pathways in Cultured Rat Cortical Neurons

Signal transduction systems, including cholinergic pathways, which are likely to be of pathophysiological significance are altered in Alzheimer's disease (AD). Muscarinic cholinergic receptors are linked to the hydrolysis of phosphoinositide, involving the production of inositol 1,4,5-trisphosphate [Ins (1,4,5)P₃] and the mobilization of cytosolic free calcium concentrations ([Ca²⁺]_i). Effects of amyloid peptide (A) on these signals prior to neuronal degeneration were examined in cultured rat cortical cells. A increased the release of lactate dehydrogenase (LDH) in a concentration-dependent manner, however, it was blocked by B27 supplement. Prolonged exposure to a sublethal dose of A 25–35 or 1–42 disrupted carbachol-mediated release of Ins(1,4,5)P₃ and [Ca²⁺]_i, which was inhibited in media supplemented with B27 or the antioxidant vitamin E. In order to determine the specificity of the effect of A, various agonists glutamate or KCl but not bradykinin which utilize the phosphoinositide cascade were investigated. Our results indicated that A did not affect the stimulation of glutamate or KCl-mediated production of Ins(1,4,5)P₃ or cause elevation in [Ca²⁺]_i. Furthermore, metabotropic agonist trans-1-amino-cyclopentane-1,3,-dicarboxylate (ACPD) elevated calcium level was not inhibited by A pretreatment. Taken together, the results demonstrate that a sublethal dose of A selectively impaired cholinergic receptor-mediated signal transduction pathways, and antioxidant or B27 supplement attenuated this effect of A. Alterations of cholinergic signaling by prolonged exposure to A could be involved in cortical neurodegeneration that occurs in AD. Because functional loss of cholinergic pathways is an important aspect of AD, the differences in susceptibility of these two types of receptors prior to other signs of A action is important and requires further investigation.     

缺氧诱导因子-1结构及功能的研究进展

缺氧诱导因子(hypoxia inducible factor-1,HIF-1)是一种介导机体对缺氧环境产生应答的转录因子。在炎症及实体肿瘤周围的组织大多存在缺氧现象。在缺氧条件下,HIF-1α和HIF-1β两个亚基结合,形成HIF-1并迅速活化,参与机体缺氧环境的适应,并在胚胎发育、多种肿瘤及心肺疾病等发生发展中起到重要作用。因此,更好地认识HIF-1的功能及意义,对进一步地认识与其相关生命过程和疾病本质以及研发新的治疗手段具有重要意义。     

Bilaminar Co-culture of Primary Rat Cortical Neurons and Glia

This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or co-culture model. This system is suitable for a variety of experimental needs requiring either a glass or plastic growth substrate and can also be used for culture of other types of neurons.Rat cortical neurons obtained from the late embryonic stage (E17) are plated on glass coverslips or tissue culture dishes facing a feeder layer of glia grown on dishes or plastic coverslips (known as Thermanox), respectively. The choice between the two configurations depends on the specific experimental technique used, which may require, or not, that neurons are grown on glass (e.g. calcium imaging versus Western blot). The glial feeder layer, an astroglia-enriched secondary culture of mixed glia, is separately prepared from the cortices of newborn rat pups (P2-4) prior to the neuronal dissection.A major advantage of this culture system as compared to a culture of neurons only is the support of neuronal growth, survival, and differentiation provided by trophic factors secreted from the glial feeder layer, which more accurately resembles the brain environment in vivo. Furthermore, the co-culture can be used to study neuronal-glial interactions¹.At the same time, glia contamination in the neuronal layer is prevented by different means (low density culture, addition of mitotic inhibitors, lack of serum and use of optimized culture medium) leading to a virtually pure neuronal layer, comparable to other established methods^1-3. Neurons can be easily separated from the glial layer at any time during culture and used for different experimental applications ranging from electrophysiology⁴, cellular and molecular biology^5-8, biochemistry⁵, imaging and microscopy^4,6,7,9,10. The primary neurons extend axons and dendrites to form functional synapses¹¹, a process which is not observed in neuronal cell lines, although some cell lines do extend processes.A detailed protocol of culturing rat hippocampal neurons using this co-culture system has been described previously^4,12,13. Here we detail a modified protocol suited for cortical neurons. As approximately 20x10⁶ cells are recovered from each rat embryo, this method is particularly useful for experiments requiring large numbers of neurons (but not concerned about a highly homogenous neuronal population). The preparation of neurons and glia needs to be planned in a time-specific manner. We will provide the step-by-step protocol for culturing rat cortical neurons as well as culturing glial cells to support the neurons.Download video file.^{(75M, mov)}     

Neuropilin 1 Directly Interacts with Fer Kinase to Mediate Semaphorin 3A-induced Death of Cortical Neurons

Neuropilins (NRPs) are receptors for the major chemorepulsive axonal guidance cue semaphorins (Sema). The interaction of Sema3A/NRP1 during development leads to the collapse of growth cones. Here we show that Sema3A also induces death of cultured cortical neurons through NRP1. A specific NRP1 inhibitory peptide ameliorated Sema3A-evoked cortical axonal retraction and neuronal death. Moreover, Sema3A was also involved in cerebral ischemia-induced neuronal death. Expression levels of Sema3A and NRP1, but not NRP2, were significantly increased early during brain reperfusion following transient focal cerebral ischemia. NRP1 inhibitory peptide delivered to the ischemic brain was potently neuroprotective and prevented the loss of motor functions in mice. The integrity of the injected NRP1 inhibitory peptide into the brain remained unchanged, and the intact peptide permeated the ischemic hemisphere of the brain as determined using MALDI-MS-based imaging. Mechanistically, NRP1-mediated axonal collapse and neuronal death is through direct and selective interaction with the cytoplasmic tyrosine kinase Fer. Fer RNA interference effectively attenuated Sema3A-induced neurite retraction and neuronal death in cortical neurons. More importantly, down-regulation of Fer expression using Fer-specific RNA interference attenuated cerebral ischemia-induced brain damage. Together, these studies revealed a previously unknown function of NRP1 in signaling Sema3A-evoked neuronal death through Fer in cortical neurons.     

Alteration of Phospholipase C Expression in Rat Visual Cortical Neurons by Chronic Blockade of Orexin Receptor 1

International Journal of Peptide Research and Therapeutics - Orexin-A well-known as hypocretin-1 is a neuropeptide that is produced in the lateral hypothalamus area (LHA). It promotes widespread...     

Sensitivity of Rat Cortical Neurons in Distinguishing Taste Qualities by Individual and Correlative Activities

To examine the possibility that different taste qualities arerepresented by the correlative activity of cortical gustatoryneurons, we made simultaneous recordings of neuron pairs duringapplication of four basic tastes into the oral cavity of anesthetizedrats and the following observations were made: (i) in 30 of67 pairs of taste responsive neurons, peaks (troughs in a fewcases) were produced in the cross-correlograms (CCs) duringstimulation with some tastants; (ii) the correlative dischargesoccupied 6–8% of the total spikes discharged by individualneurons during taste stimulation and occurred, in a considerablenumber of cases, even during stimulation with tastants to whichone or both of the component neurons of a pair were apparentlynon-responding (often sucrose and quinine); (iii) the numberof tastants to which a neuron pair responded with a significantcorrelative activity was often greater than the number of tastantsto which the component neurons of the same pair responded withsignificant changes in discharge rate; (iv) there was no significantdifference between the correlative (formation of peaks or troughsin the CC) and individual (change in discharge rate of individualneurons) ways of coding in the sensitivity to distinguish betweentwo taste qualities ranked to be adjacent on the basis of thenumber of spikes composing the response; and (v) the peaks ortroughs appearing in two CCs during stimulations with two kindsof tastants were compared with regard to overlapping of theirdelay ranges and widths. The spikes in the non-overlapping portionof each peak (suppressed spike number in the case of troughs)are supposed to be able to contribute to two-taste discrimination:the correlated discharges occurring with a delay time that correspondsto the overlapping portion can in no way be judged differently,but the spikes falling in the non-overlapping portion may contributeto the differentiation. The ratio of the non-overlapping portionto the entire peak (or trough) was 0.35 on average. It is concludedthat temporal coding of taste qualities seems to operate effectivelyin the gustatory cortex. Chem. Senses 22: 363–373, 1997.     

Modulation of Synapsin I Gene Expression in Rat Cortical Neurons by Extracellular Matrix

1. Neuronal differentiation depends on crosstalk between genetic program and environmental cues. In this study we tried to dissect this complex interplay by culturing neurons from fetal rat brain cortices in a chemically defined, neuron-specific, medium and on different substrata, either artificial (poly-D-lysine) or natural.2. Among the extracellular matrix compounds used in this study, two (collagen I and fibronectin) allowed only a weak attachment of cortical neurons to the substratum, while the others (collagen IV, laminin, and basal lamina from Engelbreth-Holm-Swarm sarcoma) allowed both firm attachment and moderate to extensive neurite outgrowth from neuronal cell bodies.3. By using synapsin I gene expression as a parameter of neuronal differentiation, we found that neurite outgrowth and neuronal differentiation are not linearly linked. Synapsin I gene expression, in fact, was maximal in neurons cultured on laminin, while the fastest neuritic outgrowth was recorded in cultures on poly-D-lysine.4. The data presented in this paper are consistent with the hypothesis that the extracellular matrix plays an active role in modulating the differentiative program of neurons.     

Phorbol Esters but Not the Cholinergic Agonists Oxotremorine-M and Carbachol Increase Release of the Amyloid Precursor Protein in Cultured Rat Cortical Neurons

Abstract: Conventional secretory processing of the amyloid precursor protein is nonamyloidogenic, releasing carboxyl-terminus-truncated amyloid precursor protein derivatives while cleaving the amyloid β-peptide within its sequence. Alternative processing routes are potentially amyloidogenic, yielding the amyloid β-peptide segment intact. In continuous cell lines, secretory processing of the amyloid precursor protein is regulated by both protein kinase C and muscarinic receptor stimulation. However, the first and second messenger systems that regulate amyloid precursor protein release in central neurons are still under investigation. In the present investigation, we examined whether or not first and second messengers of cholinergic neurotransmission increase production of soluble derivatives of the amyloid precursor protein in primary cultures of rat cortical neurons. Activation of protein kinase C by the phorbol esters phorbol 12,13-dibutyrate and phorbol 12-myristate 13-acetate increased production of the soluble form of the amyloid precursor protein dramatically. In contrast, activation of muscarinic receptors by oxotremorine-M or carbachol did not result in a significant increase in amyloid precursor protein release. Similarly, chemically induced depolarization using 35 m M KCI did not alter production of soluble amyloid precursor protein derivatives. Our data suggest that although protein kinase C stimulation plays an important role in regulating release of the amyloid precursor protein, cholinergic neurotransmission does not regulate its release in cultured rat cortical neurons.     

HDAC6 Inhibitor Blocks Amyloid Beta-Induced Impairment of Mitochondrial Transport in Hippocampal Neurons

Even though the disruption of axonal transport is an important pathophysiological factor in neurodegenerative diseases including Alzheimer's disease (AD), the relationship between disruption of axonal transport and pathogenesis of AD is poorly understood. Considering that α-tubulin acetylation is an important factor in axonal transport and that Aβ impairs mitochondrial axonal transport, we manipulated the level of α-tubulin acetylation in hippocampal neurons with Aβ cultured in a microfluidic system and examined its effect on mitochondrial axonal transport. We found that inhibiting histone deacetylase 6 (HDAC6), which deacetylates α-tubulin, significantly restored the velocity and motility of the mitochondria in both anterograde and retrograde axonal transports, which would be otherwise compromised by Aβ. The inhibition of HDAC6 also recovered the length of the mitochondria that had been shortened by Aβ to a normal level. These results suggest that the inhibition of HDAC6 significantly rescues hippocampal neurons from Aβ-induced impairment of mitochondrial axonal transport as well as mitochondrial length. The results presented in this paper identify HDAC6 as an important regulator of mitochondrial transport as well as elongation and, thus, a potential target whose pharmacological inhibition contributes to improving mitochondrial dynamics in Aβ treated neurons.     

Effects of Estradiol and IGF-1 on the Sodium Calcium Exchanger in Rat Cultured Cortical Neurons

The Na⁺/Ca²⁺ exchanger (NCX) is an important bidirectional transporter of calcium in neurons and has been shown to be involved in neuroprotection. Calcium can activate a number of cascades that can result in apoptosis and cell death, and NCX is a key factor in regulating the cytoplasmic concentration of this ion. 17-β-estradiol and insulin-like growth factor 1 (IGF-1) are known neuroprotective hormones with interacting mechanisms and effects on intracellular calcium; however, their relationship with the NCX has not been explored. In this article, the effects of these two hormones on neuronal NCX were tested using the whole-cell patch clamp technique on rat primary culture neurons. Both 17-β-estradiol and IGF-1 produced an increase in the NCX-mediated inward current and a decrease in the NCX-mediated outward current. However, the IGF-1 effect was lower than that of 17-β-estradiol, and the effect of both agents together was greater than the sum of each agent alone. Neither of the agents affected the pattern of regulation by extracellular or intrapipette calcium. Inhibitors of the IGF-1 and 17-β-estradiol receptors and inhibitors of the main signaling pathways failed to change the observed effects, indicating that these actions were not mediated by the classical receptors of these hormones. These effects on the NCX could be a mechanism explaining the neuroprotective actions of 17-β-estradiol and IGF-1, and these findings could help researchers to understand the role of the NCX in neuroprotection.     

Proteomic Analysis of Primary Cultured Rat Cortical Neurons in Chemical Ischemia

To elucidate the molecular events involved in early ischemic neuronal death, we performed two-dimensional proteome profiling of primary cultures of rat cortical neurons following chemical ischemia induced by the administration of sodium azide under glucose-free conditions. Using a lactic dehydrogenase assay and Western blot analysis of dephosporylation of the voltage-gated potassium channel Kv2.1, we determined duration of chemical ischemia of 2 h to be the relevant time-point for early ischemic neuronal death. Sixty-one proteins were differentially expressed, and 26 different proteins were identified by MALDI-TOF with Mascot database searching. The proteome data indicated that chemical ischemia altered the expression of 20 proteins that are involved in stress response/chaperone, brain development, cytoskeletal/structural proteins, metabolic enzymes, and calcium ion homeostasis. Western blotting and immunocytochemical studies of the 6-most functionally significant proteins showed that, in the ischemia-treated group, the expression of glucose-related protein 78, heat shock protein 90 alpha, and α-enolase was significantly increased, while the expression of inositol triphosphate receptor 1 and ATP synthase beta subunit was decreased. In addition, the expression of dihydropyrimidinase-like 3 showed a truncated pattern in the ischemia group. The changes in the expression of these proteins might be significant indicators of early ischemic neuronal death.     

n-6脂肪酸去饱和酶fat-1基因对神经元细胞凋亡的抑制作用

将C．elegans n-6脂肪酸去饱和酶基因fat-1的cDNA插入到腺病毒的穿梭载体pAd-CMV中,并与骨架载体同源重组,构建腺病毒重组体（Ad．GFP．fat1）,通过包装细胞系（293）产生重组腺病毒,感染原代培养的大鼠皮层细胞．在显微镜下观察、细胞增殖试剂盒（MTT）和凋亡染色试剂盒分析fat-1基因对大鼠皮层细胞凋亡的影响,核糖核酸酶保护性分析,检测fat-1基因在大鼠皮层细胞内的表达,酶联免疫分析花生四烯酸类（Eicosanoids）前列腺素（Prostaglandin E2）的含量．结果表明,通过基因重组技术,得到预期的重组病毒;fat-1基因在原代培养的大鼠皮层细胞中能有效异源表达,2d后,可检测到fat-1 mRNA的条带,与对照Ad．GFP细胞相比,fat-1基因明显抑制了大鼠皮层细胞因诱导产生的凋亡（35％）,受保护细胞的前列腺素含量也明显地减少（30％）．     

大鼠Shh-N融合蛋白的表达、纯化及功能研究

为了获得重组Sonic hedgehog N端蛋白（Shh-N）并研究其功能,应用PCR技术扩增Shh-N cDNA,然后克隆至原核表达质粒中,转化E.coli后获得表达菌株, 经1 mmol/L IPTG诱导高效表达出带有His-tag的融合蛋白,其中大部分为可溶性蛋白,少量为包涵体.用His-tag特异性结合树脂纯化可溶性融合蛋白,经SDS-聚丙烯酰胺凝胶电泳鉴定为单一区带,凝胶自动扫描分析表明,Shh-N的纯度达85%以上.纯化后的Shh-N在成纤维生长因子8(FGF₈)的协同作用下,能诱导神经前体细胞（NPC）向酪氨酸羟化酶阳性神经元（TH⁺）发育.在此基础上可以诱导不同类型的人类干细胞定向分化为多巴胺(DA)神经元,从而为临床治疗帕金森病（PD）提供充足的供体细胞.     

Induction of Glutamine Synthetase in Rat Astrocytes by Co-Cultivation with Embryonic Chick Neurons

Co-cultivation of confluent rat astrocyte cultures with embryonic chick neurons resulted in induction of glutamine synthetase activity in the astrocytes. This induction of glutamine synthetase in astrocytes by neurons was independent of induction by hydrocortisone and forskolin, but was dependent on the length of co-cultivation and the number of neurons present in the co-culture. Cycloheximide and actinomycin D inhibited the induction of glutamine synthetase in astrocytes by neurons, whereas cytosine arabinoside had no apparent effect. Results suggest that this induction of glutamine synthetase in astrocytes is mediated by cell contact with neurons and may represent a specific neuronal and glial interaction.