“A Fascinating Adventure in Biomedical Research”

Alzheimer disease insidiously attacks the brain and deprives people of their most human qualities, leading to memory loss, behavior changes, and ultimately, death. An essay in this month’s issue of Cold Spring Harbor Perspectives in Medicine provides an excellent overview of modern Alzheimer research, its origins and development, scope, driving forces, and key questions, as well as competing ideas and findings within the field. It was written by Dennis Selkoe, Eckhard Mandelkow, and David Holtzman, editors of our recent book The Biology of Alzheimer Disease.

In the essay, Selkoe and colleagues outline key developments that followed the first description of the disease by Alois Alzheimer in 1907.  They explain how the discoveries of tau and amyloid β-protein precursor in the late 1980s and early 1990s brought Alzheimer research into sync with basic research in molecular genetics and protein chemistry.  Furthermore, the recognition of Alzheimer disease as a common disorder – currently estimated to affect 20-25 million people worldwide – has helped define it as an urgent problem in biomedical research.

But beyond that, Selkoe and colleagues describe how tackling a complicated disease – and one that affects the most human qualities of memory, reasoning, language, and emotion – can be intellectually rewarding. “The complexity of the problem and the diverse ways in which one might think about approaching it make for a fascinating adventure in biomedical research,” they write.

For more on Alzheimer research – including discussion of competing ideas between “BAPtists” and “TAUists” – read the complete essay here.

 

Tracking Worm Behavior

The colorful scribbles on this month’s cover of Cold Spring Harbor Protocols are tracks from one wild-type worm as it crawls through food on an agar dish.  Such images can reveal movement and behavioral patterns in C. elegans. (If you look closely, you can see evidence of pirouettes and foraging behavior.)

Initial methods to analyze behavioral phenotypes in C. elegans relied on human observation, and were therefore subjective and imprecise. Terms like “sluggish” or “loopy” were used to describe the uncoordinated activity of some mutants. And the procedures were often time-consuming, as the observer was required to monitor worm behavior in real time.

But, as described by Bill Schafer and colleagues in the current issue of Cold Spring Harbor Protocols, automated microscopy and image analysis systems for recording and analyzing worm behavior are much more robust.  They allow for precise quantitative definitions of behavioral phenotypes, and permit the analysis of behaviors that occur over long time periods or are difficult to detect by eye.

In the issue, Schafer and colleagues provide protocols for preparing media and worms for automated tracking and image analysis, describe high-throughput worm behavior analysis using Multiworm Tracker, and offer strategies for obtaining uniform illumination during worm tracking.  They also compare and contrast single- and multi-worm tracking approaches, and describe how comparisons of wild-type and genetically modified worms can be used to functionally dissect the molecular mechanisms behind specific behaviors.

 

New Book on Alzheimer Disease Biology

Understanding the complex changes that occur during Alzheimer disease—including the accumulation of amyloid plaques and neurofibrillary tangles in the brain—is critical for the development of successful therapeutic approaches.  Our newest book, The Biology of Alzheimer Disease, provides a current and comprehensive review of the biological basis of Alzheimer disease (AD).

The editors, Dennis Selkoe, Eckhard Mandelkow, and David Holtzman, chose leading researchers in Alzheimer biology to contribute chapters on topics in which they have deep expertise. The 25 chapters include contributions covering all aspects of Alzheimer disease, from our current molecular understanding of it to therapeutic agents that could be used to treat and prevent it.

Additionally, the first and last chapters provide the editors’ perspectives on the disease, its challenges and prospects for developing effective treatments. “[We] have tried to step back from the wealth of details and convey a sense of what has motivated the global quest to understand the biology of AD, how sometimes competing concepts and lines of inquiry have proceeded, and, most importantly, where we believe this scientifically rich and therapeutically promising field is headed,” they write.

The book is a vital reference for neurobiologists, cell biologists, pathologists, and other scientists pursuing the biological basis of Alzheimer disease, as well as investigators, clinicians, and students interested in its pathogenesis, treatment, and prevention.  For more details, click here.

 

Visualizing Synaptic Connections

The cortex of the mouse brain contains ~4,000,000 neurons, so investigating the complex connectivity of these neurons can be difficult.  Recently, this challenge has been overcome by creating transgenic mice that express fluorescent proteins of different colors in individual neurons in the brain.  In this “Brainbow” approach, Cre/lox recombination is used to randomly express two to four different fluorescent proteins in each neuron. The various combinations of fluorescent proteins can produce neurons of about 100 different colors. As a result, adjacent cells are usually different colors, allowing one to clearly visualize individual cells and their contacts with other cells.

The July issue of Cold Spring Harbor Protocols features an article by Jeff Lichtman, Joshua Sanes, and colleagues, who developed the Brainbow technique.  The article describes currently available Brainbow cassettes and transgenic mice, as well as the elements necessary for creating Brainbow transgenes from scratch.  An accompanying protocol provides step-by-step details for introducing Brainbow transgenes into mice, fixing samples from Brainbow animals, and acquiring and analyzing multichannel images from Brainbow samples.

The cover of the July issue shows motor neurons in the spinal cord of a young adult transgenic Brainbow mouse.  For more on the July issue, click here.

 

New Lab Manual on Neuroscience Imaging Techniques

“Directly seeing the nervous system in action—be it a vesicle releasing transmitter, a neuron integrating synaptic input in its dendrites, or a neuronal population generating patterns of activity—is always a fascinating experience and provides us with a sense of immediate and credible understanding.” –Fritjof Helmchen and Arthur Konnerth (Imaging in Neuroscience: A Laboratory Manual)

The latest addition to our fleet of imaging manuals provides neuroscience researchers with a comprehensive set of methods for imaging cells, synapses, neuromolecules, circuits, and brain function in health and disease.  Imaging in Neuroscience covers basic techniques, such as maintaining live cells and tissue slices during imaging, as well as cutting-edge techniques, such as optogenetics, uncaging, calcium imaging, and imaging neuronal activity.

Edited by Fritjof Helmchen (University of Zurich) and Arthur Konnerth (Technical University, Munich), Imaging in Neuroscience includes 92 chapters with step-by-step protocols and background information for visualizing neural dynamics.  The book also features a set of appendices with a glossary of imaging terms and other useful information on spectra, lenses, filters, and safe handling of imaging equipment.

 

Neuroscience Tools with Extraordinary Precision: Quantum Dots and Microbial Opsins

As a preview to the forthcoming laboratory manual Imaging in Neuroscience, due in May, the current issue of Cold Spring Harbor Protocols highlights two articles on neuroscience imaging techniques.  The articles are freely accessible here and here.

Monitoring Individual Molecules with Quantum Dots

The first article details the use of nanometer-sized quantum dots (QDs) to track the motion of individual membrane molecules over time.  QDs possess strong fluorescence and photostability, permitting extended recording times compared to other methods.  In the article, authors Sabine Lévi, Maxime Dahan, and Antoine Triller (Ecole Normale Supérieure, Paris) provide step-by-step methods to stain neurons with QDs and to track QD-labeled molecules using single-fluorophore epifluorescence, as well as guidance for interpreting the data and reconstructing the trajectory of individual QD-labeled molecules.  These methods have been successfully used to follow the diffusion of individual glycine receptors, GABA receptors, NMDA receptors, lipid raft markers, glycophosphatidylinositol-anchored green fluorescent protein (GPI-GFP), and other molecules of interest.

Studying Specific Neural Activities with Microbial Opsins

The second article describes characteristics of various microbial opsins that are used in optogenetics.  Optogenetics is a revolutionary technology that combines optics and genetics to study very specific events, such as action potentials, in their natural context—even in freely moving mammals.  Microbial opsins are light-sensing proteins that regulate ion fluxes to control biological activities, and their corresponding genes can be expressed in mammalian neurons to enable millisecond-precision optical control of neural activity.  The authors of the article, Karl Diesseroth and colleagues (Stanford) and Peter Hegemann (Humboldt-Universität, Berlin), describe the diversity of microbial opsin genes, including those for bacteriorhodopsins, proteorhodopsins, halorhodopsins, and channelrhodopsins, and the structure-function properties of their corresponding proteins.  This overview will be useful to those looking to employ optogenetics as a research tool.

 

Brainbow Fish

The “Brainbow” strategy was originally developed in mice, as a system for labeling neurons in a variety of different colors, allowing one to follow multiple cells regardless of their proximity. Brainbow uses a construct that carries sequences for red, blue and green fluorescent proteins in tandem array, with two pairs of lox sites flanking the first two fluorescent proteins. Recombination occurs in the presence of the Cre recombinase, and one gets a variety of outcomes, resulting in the production of a red, blue or green label. When more than one copy of the Brainbow cassette exists within a cell, the primary tones can be mixed, providing more possible color combinations. This provides a powerful platform for studying neuronal morphology and cell movements. In the January issue of Cold Spring Harbor Protocols, Alex Schier and colleagues offer Multicolor Brainbow Imaging in Zebrafish. This protocol translates the system for use in zebrafish, which offer the advantages of easy visualization of transparent embryos and efficient generation of labeled subjects.

 

New Imaging Manuals Available

For those looking to add to their arsenal of laboratory techniques, Cold Spring Harbor Laboratory Press has just released a new series of Imaging manuals.

I had a hand in putting these books together, and I’m always pleased when we manage to publish books that I know I would have found incredibly helpful in my previous incarnation as a bench scientist. These two hit home as I was a postdoc in an imaging lab. While that was ten (10!!!) years ago, it’s almost shocking to realize that there weren’t any comprehensive lab manuals out there that really covered the whole of bio-imaging, from the basics of optics to the most current, bleeding-edge techniques. Consider that problem solved, courtesy of series editor Rafa Yuste.

The new series spins off from a previous set of publications. In 2000, CSHL Press published Imaging Neurons, based on a CSHL laboratory course. The book was a few years ahead of its time, and the methods had really caught on by the time the sequel, Imaging in Neuroscience and Development was released in 2005. Five years later, and there’s been far too many new applications developed to fit into one volume, hence the release of the new series.

Imaging: A Laboratory Manual is the flagship of the series. It offers all the basics: optics, confocal, multi-photon, lasers, cameras, staining cells, etc. The manual goes on from there though, through labeling and indicators to advanced techniques like photoactivation, light sheet imaging, array tomography, fast imaging, molecular imaging, superresolution imaging and every acronym you can think of (FRET, FLIM, FRAP, FIONA, PALM, STORM, BiFC, AFM, TIRFM to name a sampling). If you have a microscope in your lab or if you spend any time in your local imaging center, this is the book you need.

Imaging in Developmental Biology: A Laboratory Manual is the second book in the series, just released. We old-school developmental biologists used to have to look at fixed sectioned specimens taken from different time points, and try to piece together the big picture of what was really happening as an embryo developed. New techniques have revolutionized our understanding of dynamic processes, as they allow for real-time imaging, often over the entire course of an organism’s development. Like the preceding volume, the book starts with the basics, methods for visualizing development in laboratory standard model organisms (C. elegans, Drosophila, zebrafish, Xenopus, avians and mouse) and then step by step brings the reader to the cutting edge of imaging technology.

The supplemental movies from both books are freely available through Cold Spring Harbor Protocols. Look for a third volume in March, on Imaging in Neuroscience, which will offer an astounding 90-plus chapters for analyzing every aspect of the nervous system in detail.

 

Superresolution Microscopy: PALM

How do you see something smaller than the wavelength of light itself? Fluorescence microscopy is the most common optical technique used for visualizing cellular functions. The latest techniques allow labeling of specific organelles and proteins with molecular precision. But conventional microscopy cannot resolve objects closer than 200 nanometers at the focal plane. Many subcellular structures and groups of proteins occur on the 10 nanometer scale. A true understanding of cellular physiology requires new superresolution methods.

Of these methods, PALM (Photoactivated Localization Microscopy) provides the highest shown resolution in biological samples (approximately 10 nanometers) and allows for the assessment of individual molecules. In the December issue of Cold Spring Harbor Protocols, Oregon Health Science University’s Haining Zhong presents Photoactivated Localization Microscopy (PALM): An Optical Technique for Achieving ~10-nm Resolution. The article provides an overview of the basic principles of PALM, its implementation and the potential applications in neuroscience.

 

Imaging C. elegans Development

New imaging technologies have revolutionized the study of developmental biology. Where researchers once struggled to connect events at static timepoints, imaging tools now offer the ability to visualize the dynamic form and function of molecules, cells, tissues, and whole embryos throughout the entire developmental process. In order to observe development over time, it is necessary to grow the embryos of laboratory model organisms on the microscope stage, and keep them as healthy and in as natural a state as possible. Methods for culturing and imaging the embryos of model organisms are featured in the December issue of Cold Spring Harbor Protocols.

Caenorhabditis elegans has been a key organism for understanding cellular differentiation and development. The fate of every one of the worm’s somatic cells has been mapped out, and its short developmental time, transparent shell, and nonpigmented cells makes C. elegans an ideal subject for imaging studies. Timothy Walston from Truman State University and Jeff Hardin from the University of Wisconsin-Madison provide An Agar Mount for Observation of Caenorhabditis elegans Embryos, an easy way to prepare live C. elegans embryos for microscopic visualization. The method involves embedding the embryo in agar to hold it in place,providing a fixed orientation for consistent imaging. Embryos prepared this way are amenable to both light microscopy and confocal microscopy. As one of our featured articles, the protocol is freely available to subscribers and non-subscribers alike.