Francesco Beltrame

NEUROINFORMATICS: THE INTEGRATION OF SHARED DATABASES AND TOOLS TOWARDS INTEGRATIVE NEUROSCIENCE

There is significant interest amongst neuroscientists in sharing neuroscience data and analytical tools. The exchange of neuroscience data and tools between groups affords the opportunity to differently re-analyze previously collected data, encourage new neuroscience interpretations and foster otherwise uninitiated collaborations, and provide a framework for the further development of theoretically based models of brain function. Data sharing will ultimately reduce experimental and analytical error. Many small Internet accessible database initiatives have been developed and specialized analytical software and modeling tools are distributed within different fields of neuroscience. However, in addition large-scale international collaborations are required which involve new mechanisms of coordination and funding. Provided sufficient government support is given to such international initiatives, sharing of neuroscience data and tools can play a pivotal role in human brain research and lead to innovations in neuroscience, informatics and treatment of brain disorders. These innovations will enable application of theoretical modeling techniques to enhance our understanding of the integrative aspects of neuroscience. This article, authored by a multinational working group on neuroinformatics established by the Organization for Economic Co-operation and Development (OECD), articulates some of the challenges and lessons learned to date in efforts to achieve international collaborative neuroscience.

Cytofluorometry of electromagnetically controlled cell dedifferentiation.

Cellular morphology changes, which appear related to dedifferentiation (despecialization), have been produced in vitro in the nucleated red blood cell of the frog. This has been achieved by controlled alterations in the electrochemical environment of these living cells, both by a selective modification of the ionic concentrations of an isotonic amphibian Ringer solution, and by the electromagnetic induction of pulsating current having specific waveform parameters. Laser flow microfluorometry shows that the modified Ringer solution is able, per se, to partially trigger the process in the same time interval that certain induced current waveforms can significantly affect the number of cells in the so-called dedifferentiated state. It has also been found that, for a given waveform, the repetition rate appears to have a significant effect on the rate of cell change. Preliminary automated image analysis of cell smears suggests that dedifferentiated and normal cells have the same integrated optical density but different nuclear areas. In conclusion, it appears that, after the initial electrochemical trigger, the early stage of the process, when the cells move from a state of specialized function to one of less specific activity, is the unfolding of their chromatin supercoil, not involving DNA synthesis. Then cytofluorometry allowed us to identify, for the first time, fundamental modifications which occur in the cell nucleus under electromagnetic exposure.

Neuroscience data and tool sharing: a legal and policy framework for neuroinformatics.

The requirements for neuroinformatics to make a significant impact on neuroscience are not simply technical—the hardware, software, and protocols for collaborative research—they also include the legal and policy frameworks within which projects operate. This is not least because the creation of large collaborative scientific databases amplifies the complicated interactions between proprietary, for-profit R&D and public “open science.” In this paper, we draw on experiences from the field of genomics to examine some of the likely consequences of these interactions in neuroscience.Facilitating the widespread sharing of data and tools for neuroscientific research will accelerate the development of neuroinformatics. We propose approaches to overcome the cultural and legal barriers that have slowed these developments to date. We also draw on legal strategies employed by the Free Software community, in suggesting frame-works neuroinformatics might adopt to reinforce the role of public-science databases, and propose a mechanism for identifying and allowing “open science” uses for data whilst still permitting flexible licensing for secondary commercial research.

Computer-based technique for cell aggregation analysis and cell aggregation in in vitro chondrogenesis

No quantitative methods are currently available to measure different aggregation parameters in cell cultures. In this paper we describe a computer-based technique for the automatic and reliable analysis of cellular aggregates, starting from optical microscopy images of living cells grown in suspension. The method allows determination, on the same sample at different time intervals, of quantitative parameters, including aggregation percentage, average number of cells in aggregates, and aggregate size statistical distribution. To determine the number of cells in an aggregate starting from its two-dimensional microscopic profile, a model has been proposed and verified, using sphere packing theory. Algorithms have been tested on chondrocyte suspension cultures, where cell aggregation is a very early and critical event leading to cell differentiation. Using this technique for the analysis of chick embryo chondrocyte cultures, we observed that aggregate size and development kinetics depend on the culture conditions used. The method, with minor adaptations, is of potential use also in other cell systems to evaluate aggregation indexes or to study aggregation kinetics.

Fluorescence microscopy imaging of bone for automated histomorphometry.

We have developed a computer-based method for the automated quantification of bone tissue in histological sections of decalcified specimens. Bone tissue was generated by ectopic implantation of ceramic-based carriers loaded with human bone marrow stromal cells (BMSCs). The method is based on the acquisition of multimodal images, in order to identify and measure the area covered by bone tissue (using fluorescent light) and the total area of tissue (using transmitted light), thereby excluding the regions corresponding to nonresorbed scaffold. The amount of bone as a percentage of the total area of interest (bone/area) and of the newly formed tissue (bone/tissue) is automatically derived. The computer-based results correlated closely with those obtained by manual identification of bone and tissue areas in the same histological fields (R(2) = 0.997; p < 0.0005), with errors dependent on the magnification used but always lower than 9.4%. The method was used to compare the bone/tissue and bone/area percentages in samples of engineered bone based on human BMSCs expanded in the presence of different biochemical factors and loaded onto different scaffolds. The technique thus represents a valuable tool to quantify reproducibly, accurately, and easily bone formation in a variety of tissue-engineering studies.

The Vepsy Updated Project: Virtual Reality in Clinical Psychology

Many of us grew up with the naive assumption that couches are the best used therapeutic tools in psychotherapy. But tools for psychotherapy are evolving in a much more complex environment than a designers chaise lounge. In particular, virtual reality (VR) devices have the potential for appearing soon in many consulting rooms. The use of VR in medicine is not a novelty. Applications of virtual environments for health care have been developed in the following areas: surgical procedures (remote surgery or telepresence, augmented or enhanced surgery, and planning and simulation of procedures before surgery); preventive medicine and patient education; medical education and training; visualization of massive medical databases; and architectural design for health care facilities. However, there is a growing recognition that VR can play an important role in clinical psychology, too. To exploit and understand this potential is the main goal of the Telemedicine and Portable Virtual Environment in Clinical Psychology--VEPSY Updated--a European Community-funded research project (IST-2000-25323, http://www.vepsy.com). The project will provide innovative tools-telemedicine and portable-for the treatment of patients, clinical trials to verify their viability, and action plans for dissemination of its results to an extended audience-potential users and influential groups. The project will also develop different personal computer (PC)-based virtual reality modules to be used in clinical assessment and treatment. In particular, the developed modules will address the following pathologies: anxiety disorders; male impotence and premature ejaculation; and obesity, bulimia, and binge-eating disorders.

Microenvironment complexity and matrix stiffness regulate breast cancer cell activity in a 3D in vitro model

Three-dimensional (3D) cell cultures represent fundamental tools for the comprehension of cellular phenomena both in normal and in pathological conditions. In particular, mechanical and chemical stimuli play a relevant role on cell fate, cancer onset and malignant evolution. Here, we use mechanically-tuned alginate hydrogels to study the role of substrate elasticity on breast adenocarcinoma cell activity. The hydrogel elastic modulus (E) was measured via atomic force microscopy (AFM) and a remarkable range (150–4000 kPa) was obtained. A breast cancer cell line, MCF-7, was seeded within the 3D gels, on standard Petri and alginate-coated dishes (2D controls). Cells showed dramatic morphological differences when cultured in 3D versus 2D, exhibiting a flat shape in both 2D conditions, while maintaining a circular, spheroid-organized (cluster) conformation within the gels, similar to those in vivo. Moreover, we observed a strict correlation between cell viability and substrate elasticity; in particular, the number of MCF-7 cells decreased constantly with increasing hydrogel elasticity. Remarkably, the highest cellular proliferation rate, associated with the formation of cell clusters, occurred at two weeks only in the softest hydrogels (E = 150–200 kPa), highlighting the need to adopt more realistic and a priori defined models for in vitro cancer studies.

Endocytosis of GABAB receptors modulates membrane excitability in the single-celled organism Paramecium.

GABAB receptors modulate swimming behavior in Paramecium by inhibiting dihydropyridine-sensitive Ca2+ channels via G-proteins. Prolonged occupancy of GABAB receptors by baclofen results in a decrease in GABAB receptor functions. Since changes in the number of cell-surface GABAA receptors have been postulated to be of importance in modulating inhibitory synaptic transmission in neurons, we have studied the cell-surface expression and maintenance of GABAB receptors in P. primaurelia. In this study, we use immunostaining in electron and confocal microscopy to demonstrate that constitutive internalization of GABAB receptors in P. primaurelia is mediated by clathrin-dependent and -independent endocytosis. Indeed, GABAB receptors colocalize with the adaptin complex AP2, which is implicated in the selective recruitment of integral membrane proteins to clathrin-coated vesicles, and with caveolin 1, which is associated with uncoated membrane invaginations. Furthermore, when endocytosis is blocked with hypertonic medium, cytosol acidification, filipin or with a peptide that disrupts the association between amphiphysin and dynamin, the effect of baclofen on swimming is increased. These results suggest that GABAB receptor endocytosis into clathrin-coated and -uncoated vesicles represents an important mechanism in the modulation of swimming behavior in Paramecium.

Neuroinformatics as a megascience issue

The greatest increment in acquired knowledge about the brain has occurred in the last two decades, aided by technological advances in molecular biology, molecular genetics, brain imaging, and other new technologies brought about through the computer revolution. The amount of information being generated is increasing in an exponential fashion due to these and ether approaches used by the estimated 50000 neuroscientists working around the globe. These individual investigators working in small groups on highly focused projects publish their results in one of the roughly 200 scientific journals which are published monthly. It is humanly impossible under the current system to integrate all of these data into a meaningful picture of how the brain develops and functions and malfunctions. The complexity and quantity of neuroscience data drives the need to create a global information management system for the neuroscience community. The possibility to do this has already been amply demonstrated by the genome community through bioinformatics. Compared to sequence data, data generated through neuroscience research have many more levels of complexity, including genomic data. This effective demonstration in bioinformatics and the continual advances in the fields of computer and information sciences clearly point to the need for the neuroscience community to embrace and engage the science of informatics, through the global development of the new field of neuroinformatics.

Mapping cholesteryl ester analogue uptake and intracellular flow in Paramecium by confocal fluorescence microscopy

In Paramecium primaurelia the uptake and intracellular flow of cholesteryl ester was studied by fluorescence confocal laser scanning optical microscopy and by the fluorescent analogue cholesteryl‐BODIPY® FL C12 (BODIPY‐CE). The BODIPY FL fluorophore has the characteristic of emitting green fluorescence, which is red‐shifted as the probe concentrates. In cells incubated with 25 µm BODIPY‐CE for 30 s, fluorescence is found in vesicles located around the cytopharynx in the posterior half of the cell. Successively, the lipid is internalized by food vacuoles, the fluorescent vesicles are distributed throughout the cell and the intracellular membranes are labelled. The food vacuole number is maximum after 10–15 min of continuous labelling, then it decreases until no food vacuoles are found in 30‐min fed cells. BODIPY‐CE accumulates in red‐labelled cytoplasmic droplets located in the anterior half of the cell. When food vacuole formation is inhibited by trifluoperazine, fluorescence is found on cellular membranes and in small green‐labelled vesicles at the apical pole. The inhibition of clathrin‐mediated endocytosis does not interfere in P. primaurelia with BODIPY‐CE intracellular flow: intracellular membranes and storage droplets in the cell anterior part are dyed. Conversely, the use of sterol‐binding drugs prevents the lipid accumulation in droplets, stopping the lipid within the cytoplasmic membranes. Furthermore, the cells treated with monensin and cytochalasin B show a labelling of the cellular membranes and lipid droplets, whereas NH4Cl reduces the lipid storage. Low temperature (4 °C) does not prevent the internalization of BODIPY‐CE that, however, is localized at the cytoplasmic membrane level and does not accumulate in storage droplets. In addition, BODIPY‐CE inhibits phagocytosis, as evidenced by comparing the kinetics of food vacuole formation of control cells, only fed with latex particles, with that of cells fed with latex particles and BODIPY‐CE. In conclusion, this study points out that in P. primaurelia the cholesteryl ester enters the cell via food vacuoles and through the plasma membrane and, inside the cell, it alters cell functions.