Fluorescent-Protein Stabilization and High-Resolution Imaging of Cleared,Intact Mouse Brains

In order to observe and quantify long-range neuronal connections in intact mouse brain by light microscopy, it is first necessary to clear the brain, thus suppressing refractive-index variations. Here we describe a method that clears the brain and preserves the signal from proteinaceous fluorophores using a pH-adjusted non-aqueous index-matching medium. Successful clearing is enabled through the use of either 1-propanol or tert-butanol during dehydration whilst maintaining a basic pH. We show that high-resolution fluorescence imaging of entire, structurally intact juvenile and adult mouse brains is possible at subcellular resolution, even following many months in clearing solution. We also show that axonal long-range projections that are EGFP-labelled by modified Rabies virus can be imaged throughout the brain using a purpose-built light-sheet fluorescence microscope. To demonstrate the viability of the technique, we determined a detailed map of the monosynaptic projections onto a target cell population in the lateral entorhinal cortex. This example demonstrates that our method permits the quantification of whole-brain connectivity patterns at the subcellular level in the uncut brain.     

Long Term Culture of Cells Derived from Mouse Blastocysts

The development of mouse blastocysts in primary culture has been followed for up to two months. The trophectoderm layer of the blastocyst gives rise to a monolayer of trophoblast cells; cells resembling both ectoplacental cone cells and primary giant cells are observed. The former can transform to giant cells, presumably secondary trophoblast, after several days in culture. Giant trophoblast cells are evident in the culture for much longer than the normal gestation period. Under the culture conditions described, the proportion of blastocysts showing substantial inner cell mass (ICM) proliferation in vitro is higher than that noted in previous studies. The ICM clumps develop into either egg cylinder-like structures, or, more commonly, into spherical, fluid-filled vesicles. The vesicles, which resemble yolk sac morphologically and biochemically [10, 11], continue to enlarge in size during several weeks of culture. The vesicles are attached to the underlying trophoblast monolayers by a stalk. Cells appear to migrate from this stalk out along the culture dish. The result after two to four weeks of culture is the appearance of a mixed monolayer containing a variety of different cell types. Secondary cultures of blastocyst cells have been continuously maintained in vitro for more than one year. Four lines of cells, all developing from the same pool of blastocysts, have been monitored for morphological, growth and biochemical properties, as well as chromosome number. Each line contained two or more morphologically distinct cell types, clearly indicated by cloning studies after eight months of culture. Doubling times and saturation densities among the four lines differed, as did biochemical properties. Although none of the cell lines resembled trophoblast biochemically after 7.5 months in culture, one line, MB4, possessed a number of biochemical properties in common with midgestation yolk sac. After a further five months of culture, some enzymes in the four lines were relatively unchanged; in other cases, notably with alkaline phosphatase, a sharp drop in enzyme activity was observed. One cell line, MB2, and specifically one of the cell types in this line, produced a yellow-orange pigment with a spectrum resembling that of a heme protein. After 7.5 months of culture, two of the four lines, MB21 and MB31, contained large numbers of cells with a diploid number of chromosomes. However, by 12.5 months in culture, the large majority of metaphases in all four cell lines possessed a hypotetraploid chromosome number. In a number of studies carried out to date, none of the cell lines generated tumors when injected into syngeneic hosts.     

Multiphoton Microscopy of Cleared Mouse Brain Expressing YFP

Multiphoton microscopy of intrinsic fluorescence and second harmonic generation (SHG) of whole mouse organs is made possible by optically clearing the organ before imaging.^1,2 However, for organs that contain fluorescent proteins such as GFP and YFP, optical clearing protocols that use methanol dehydration and clear using benzyl alcohol:benzyl benzoate (BABB) while unprotected from light³ do not preserve the fluorescent signal. The protocol presented here is a novel way in which to perform whole organ optical clearing on mouse brain while preserving the fluorescence signal of YFP expressed in neurons. Altering the optical clearing protocol such that the organ is dehydrated using an ethanol graded series has been found to reduce the damage to the fluorescent proteins and preserve their fluorescent signal for multiphoton imaging.⁴ Using an optimized method of optical clearing with ethanol-based dehydration and clearing by BABB while shielded from light, we show high-resolution multiphoton images of yellow fluorescent protein (YFP) expression in the neurons of a mouse brain more than 2 mm beneath the tissue surface.     

Function of Mouse Embryonic Stem Cell-Derived Supporting Cells in Neural Progenitor Cell Maturation and Long Term Cxpansion

Background

In the differentiation of mouse embryonic stem (ES) cells into neurons using the 5-stage method, cells in stage 4 are in general used as neural progenitors (NPs) because of their ability to give rise to neurons. The choice of stage 4 raises several questions about neural progenitors such as the type of cell types that are specifically considered to be neural progenitors, the exact time when these progenitors become capable of neurogenesis and whether neurogenesis is an independent and autonomous process or the result of an interaction between NP cells and the surrounding cells.

Methodology/Principal Findings

In this study, we found that the confluent monolayer cells and neural sphere like cell clusters both appeared in the culture of the first 14 days and the subsequent 6 weeks. However, only the sphere cells are neural progenitors that give rise to neurons and astrocytes. The NP cells require 14 days to mature into neural lineages fully capable of differentiation. We also found that although the confluent monolayer cells do not undergo neurogenesis, they play a crucial role in the growth, differentiation, and apoptosis of the sphere cells, during the first 14 days and long term culture, by secreted factors and direct cell to cell contact.

Conclusions/Significance

The sphere cells in stage 4 are more committed to developing into neural progenitors than monolayer cells. Interaction between the monolayer cells and sphere cells is important in the development of stage 4 cell characteristics.     

Feeding-Related Circadian Variation in tele-Methylhistamine Levels of Mouse and Rat Brains

Circadian changes in the brain histamine (HA) and tele-methylhistamine (t-MH) levels were studied in mice and rats after adaptation to an alternating 12-h light/dark cycle (lights on at 0600). Although there was no significant circadian fluctuation of the brain HA levels, the levels of t-MH, a major metabolite of brain HA, showed a marked circadian variation. In mice, the t-MH levels were about 80 ng/g from 1200 to 1800 but about two times higher values were obtained from 2400 to 0600 of the next morning. In rats, the t-MH levels ranged from 24 to 28 ng/g at 0600 and 1200, slightly increased at 1800, and reached at 2400 a peak twice as high as the levels seen during the light period. The t-MH levels again rapidly decreased during the subsequent 3 h. In mice fasted from 1200, the t-MH levels did not increase during the period of darkness. When mice were fed at 1200 after a 24-h fast, a significant increase in the t-MH levels was observed at 1800. There was no significant circadian variation of the HA and t-MH levels in the plasma of mice and rats. These results suggest that circadian variation in brain t-MH levels is related to feeding and possible subsequent changes in elimination of t-MH from the brain and/or turnover of HA in the brain. This phenomenon should be given due attention when HA dynamics in the brain are being assessed.     

饥饿对小鼠脑中tau蛋白磷酸化和O-GlcNAc糖基化的影响

为了探讨大脑中葡萄糖摄取和代谢障碍在阿尔茨海默病(Alzheimer$sdisease,AD)神经退行性病变中的作用,将昆明种小鼠进行饥饿和再喂食处理,并使用多种磷酸化tau蛋白特异性的抗体和蛋白O-GlcNAc糖基化特异性抗体进行检测,观察饥饿及恢复喂养后不同时间点大脑皮质中tau蛋白糖基化及多个位点磷酸化的变化.结果显示:饥饿处理引起小鼠大脑皮质中总蛋白和tau蛋白的O-GlcNAc糖基化水平降低,同时tau蛋白磷酸化水平升高,饥饿引起的tauO-GlcNAc糖基化和磷酸化改变均在恢复进食后逆转成正常水平.该研究结果提示:大脑中tau蛋白的磷酸化和O-GlcNAc糖基化之间存在相互调节,脑中葡萄糖代谢障碍可能通过下调tau蛋白O-GlcNAc糖基化水平使tau蛋白产生异常过度磷酸化,进而促发AD的病理进程.这一结果为在早期阶段通过逆转tau蛋白异常过度磷酸化治疗AD成为可能提供了实验基础.     

TNF-Overexpression in Borna Disease Virus-Infected Mouse Brains Triggers Inflammatory Reaction and Epileptic Seizures

Proinflammatory state of the brain increases the risk for seizure development. Neonatal Borna disease virus (BDV)-infection of mice with neuronal overexpression of tumor necrosis factor-α (TNF) was used to investigate the complex relationship between enhanced cytokine levels, neurotropic virus infection and reaction pattern of brain cells focusing on its role for seizure induction. Viral antigen and glial markers were visualized by immunohistochemistry. Different levels of TNF in the CNS were provided by the use of heterozygous and homozygous TNF overexpressing mice. Transgenic TNF, total TNF (native and transgenic), TNF-receptor (TNFR1, TNFR2), IL-1 and N-methyl-D-aspartate (NMDA)-receptor subunit 2B (NR2B) mRNA values were measured by real time RT-PCR. BDV-infection of TNF-transgenic mice resulted in non-purulent meningoencephalitis accompanied by epileptic seizures with a higher frequency in homozygous animals. This correlated with lower weight gain, stronger degree and progression of encephalitis and early, strong microglia activation in the TNF-transgenic mice, most obviously in homozygous animals. Activation of astroglia could be more intense and associated with an unusual hypertrophy in the transgenic mice. BDV-antigen distribution and infectivity in the CNS was comparable in TNF-transgenic and wild-type animals. Transgenic TNF mRNA-expression was restricted to forebrain regions as the transgene construct comprised the promoter of NMDA-receptor subunit2B and induced up-regulation of native TNF mRNA. Total TNF mRNA levels did not increase significantly after BDV-infection in the brain of transgenic mice but TNFR1, TNFR2 and IL-1 mRNA values, mainly in the TNF overexpressing brain areas. NR2B mRNA levels were not influenced by transgene expression or BDV-infection. Neuronal TNF-overexpression combined with BDV-infection leads to cytokine up-regulation, CNS inflammation and glial cell activation and confirmed the presensitizing effect of elevated cytokine levels for the development of spontaneous epileptic seizures when exposed to additional infectious noxi.     

小鼠和大鼠的胚胎培养基及若干相关问题

哺乳动物早期胚胎体外培养所用各种化学成份明确的培养基是研究生命科学中一项重要技术。它已被常规用于研究胚胎早期发育,多种动物及人类辅助生殖技术,转基因动物,动物克隆等领域。介绍了用于移植前胚胎培养基的研究和发展历史,当前所用化学成份明确胚胎培养基的主要组成,特别是针对小鼠和大鼠移植前胚胎所用各种培养基及其成份,讨论了这类培养基发展前景和研究方向。     

CREB——长时记忆的介导因子

综述了CREB的研究进展和该领域中需深入研究和注意的问题.CREB作为一种转录因子参与短时记忆向长时记忆的转化,它具有激活型和抑制型两种形式,籍此可以更加精细地调节记忆的转化,这在不同种属动物中已经得到证实,且其基因序列存在着高度的保守性.     

Short and Long Term Investor Synchronization Caused by Decoupling

The dynamics of collective decision making is not yet well understood. Its practical relevance however can be of utmost importance, as experienced by people who lost their fortunes in turbulent moments of financial markets. In this paper we show how spontaneous collective “moods” or “biases” emerge dynamically among human participants playing a trading game in a simple model of the stock market. Applying theory and computer simulations to the experimental data generated by humans, we are able to predict the onset of such moments before they actually happen.     

Micron-scale Resolution Optical Tomography of Entire Mouse Brains with Confocal Light Sheet Microscopy

Understanding the architecture of mammalian brain at single-cell resolution is one of the key issues of neuroscience. However, mapping neuronal soma and projections throughout the whole brain is still challenging for imaging and data management technologies. Indeed, macroscopic volumes need to be reconstructed with high resolution and contrast in a reasonable time, producing datasets in the TeraByte range. We recently demonstrated an optical method (confocal light sheet microscopy, CLSM) capable of obtaining micron-scale reconstruction of entire mouse brains labeled with enhanced green fluorescent protein (EGFP). Combining light sheet illumination and confocal detection, CLSM allows deep imaging inside macroscopic cleared specimens with high contrast and speed. Here we describe the complete experimental pipeline to obtain comprehensive and human-readable images of entire mouse brains labeled with fluorescent proteins. The clearing and the mounting procedures are described, together with the steps to perform an optical tomography on its whole volume by acquiring many parallel adjacent stacks. We showed the usage of open-source custom-made software tools enabling stitching of the multiple stacks and multi-resolution data navigation. Finally, we illustrated some example of brain maps: the cerebellum from an L7-GFP transgenic mouse, in which all Purkinje cells are selectively labeled, and the whole brain from a thy1-GFP-M mouse, characterized by a random sparse neuronal labeling.     

Muscarinic Receptor-Dependent Long Term Depression in the Perirhinal Cortex and Recognition Memory are Impaired in the rTg4510 Mouse Model of Tauopathy

Neurochemical Research - Neurodegenerative diseases affecting cognitive dysfunction, such as Alzheimer’s disease and fronto-temporal dementia, are often associated impairments in the visual...     

The Molecular and Metabolic Influence of Long Term Agmatine Consumption

Agmatine (AGM), a product of arginine decarboxylation, influences multiple physiologic and metabolic functions. However, the mechanism(s) of action, the impact on whole body gene expression and metabolic pathways, and the potential benefits and risks of long term AGM consumption are still a mystery. Here, we scrutinized the impact of AGM on whole body metabolic profiling and gene expression and assessed a plausible mechanism(s) of AGM action. Studies were performed in rats fed a high fat diet or standard chow. AGM was added to drinking water for 4 or 8 weeks. We used ¹³C or ¹⁵N tracers to assess metabolic reactions and fluxes and real time quantitative PCR to determine gene expression. The results demonstrate that AGM elevated the synthesis and tissue level of cAMP. Subsequently, AGM had a widespread impact on gene expression and metabolic profiling including (a) activation of peroxisomal proliferator-activated receptor-α and its coactivator, PGC1α, and (b) increased expression of peroxisomal proliferator-activated receptor-γ and genes regulating thermogenesis, gluconeogenesis, and carnitine biosynthesis and transport. The changes in gene expression were coupled with improved tissue and systemic levels of carnitine and short chain acylcarnitine, increased β-oxidation but diminished incomplete fatty acid oxidation, decreased fat but increased protein mass, and increased hepatic ureagenesis and gluconeogenesis but decreased glycolysis. These metabolic changes were coupled with reduced weight gain and a curtailment of the hormonal and metabolic derangements associated with high fat diet-induced obesity. The findings suggest that AGM elevated the synthesis and levels of cAMP, thereby mimicking the effects of caloric restriction with respect to metabolic reprogramming.     

Short and Long Term Measures of Anxiety Exhibit Opposite Results

Animal models of human diseases of the central nervous system, generalized anxiety disorder included, are essential for the study of the brain-behavior interface and obligatory for drug development; yet, these models fail to yield new insights and efficacious drugs. By increasing testing duration hundredfold and arena size tenfold, and comparing the behavior of the common animal model to that of wild mice, we raise concerns that chronic anxiety might have been measured at the wrong time, for the wrong duration, and in the wrong animal. Furthermore, the mice start the experimental session with a short period of transient adaptation to the novel environment (habituation period) and a long period reflecting the respective trait of the mice. Using common measures of anxiety reveals that mice exhibit opposite results during these periods suggesting that chronic anxiety should be measured during the post-habituation period. We recommend tools for measuring the transient period, and provide suggestions for characterizing the post habituation period.     

Long Term Cryopreservation of the Amastigote Stages of Hemoflagellates

ABSTRACT. Tissue homogenates containing amastigotes of either Leishmania donovani, L. tropica , or Trypanosoma cruzi were rapidly frozen with 10% glycerol as cryoprotectant. Viability and pathogenicity were maintained for at least 23 years with the Khartoum strain of L. donovani , 22 years with the Malakal strain of L. donovani , and 7 years for L. tropica and T. cruzi . Similar results over a shorter period of time were obtained with a slow-freezing technique.     

A Portable Chemotaxis Platform for Short and Long Term Analysis

Flow-based microfluidic systems have been widely utilized for cell migration studies given their ability to generate versatile and precisely defined chemical gradients and to permit direct visualization of migrating cells. Nonetheless, the general need for bulky peripherals such as mechanical pumps and tubing and the complicated setup procedures significantly limit the widespread use of these microfluidic systems for cell migration studies. Here we present a simple method to power microfluidic devices for chemotaxis assays using the commercially available ALZET® osmotic pumps. Specifically, we developed a standalone chemotaxis platform that has the same footprint as a multiwell plate and can generate well-defined, stable chemical gradients continuously for up to 7 days. Using this platform, we validated the short-term (24 hours) and long-term (72 hours) concentration dependent PDGF-BB chemotaxis response of human bone marrow derived mesenchymal stem cells.