Stereology & Morphometry

Overview

Dr. Siwek’s lab is a component of a program project grant that is examining the effects of perinatal manipulation of dietary choline and folate. The work on this project (now entering its 11th year) is distributed among a number of different laboratories at BU, Duke University and VA Medical Centers in Bedford, MA and Durham, NC. The project has molecular biological, behavioral, nutritional, electrophysiological, genetic, and morphological components.

An important observation, made early in the projects history, is the fact that when the diet of pregnant mice is supplemented with choline in the chow and water, their offspring show a marked improvement in their performance in certain behavioral tasks (e.g. spatial memory). Because this effect is permanent, it suggests that there may be morphological changes that are associated with the improved behavioral performance.

As the neuroanatomical core for this project the work in Dr. Siwek’s lab is examining the brains of these animals to try and identify morphological differences between control, choline supplemented and choline deficient groups. We are focusing our attention on the hippocampus and basal forebrain because of the critical role they play in learning and memory.

A powerful technique that has recently become more accessible is Unbiased Stereology. This method is used to make accurate estimates of size, number and volume in complex three-dimensional structures. In very simple terms, the method utilizes a systematic random sampling paradigm that means the objects of interest (neurons, plaques, glia etc) all have the same likelihood of being assessed and are not being subjected to human or methodological bias. By clearly delineating the boundaries of the structure or region being studied one can measure a known fraction of the total and use those measurements to compute an estimate that approaches the real value. Clearly in some cases, it is convenient to physically count or measure structures in total, but obviously there will be other cases where the counting or measuring is just too ponderous to undertake. That is where unbiased stereology becomes a powerful tool.

In addition to the choline research, Dr. Siwek has a long collaborative history with the Sleep Research Laboratory, directed by Dr. Subimal Datta. The primary concerns of this lab are the cellular and neurochemical mechanisms of REM sleep, the cellular basis of the brainstem PGO wave generation and the mechanisms underlying the cognitive functions of sleep. Work in this lab uses multidisciplinary techniques (Anatomy, Physiology, Pharmacology, Biochemistry, and Behavior) to study the basic mechanisms and functions of sleep. The specific goals of this work is to;

1. Understand how single cells of the brain and their specific chemical signals (neurotransmitters and neuromodulators) are organized at the level of population ensembles to determine the global behavioral states of waking and sleep;
2. Study the neurophysiological and molecular mechanism of state-dependent learning and memory consolidation; and
3. Study the consequences of altered brain development in the behavioral states of wake-sleep cycle. Dr. Datta received number of national and international awards for his contribution to the sleep research.

Publications

Publications from the lab are varied and due to the nature of the core function, much of the data included in the publications of other members of the project.

McKeon-OÕMalley, C; Siwek, DF. Jeffrey A. Lamoureux, Christina L. Williams and Neil W. Kowall (2003) Prenatal Choline Deficiency Decreases the Cross-Sectional Area of Cholinergic Neurons in the Medial Septal Nucleus. Brain Res. 977: 278-83

Clement AM, Nguyen MD, Roberts EA, Garcia ML, Boillee S, Rule M, McMahon AP, Doucette W, Siwek D, Ferrante RJ, Brown RH Jr, Julien JP, Goldstein LS, Cleveland DW. (2003) Wild-type nonneuronal cells extend survival of SOD1 mutant motor neurons in ALS mice. Science, 302: 113-117

Albright CD, Siwek DF, Craciunescu CN, Mar MH, Kowall NW, Williams CL, Zeisel SH. (2003) Choline availability during embryonic development alters the localization of calretinin in developing and aging mouse hippocampus. Nutr. Neurosci., 6: 129-134

Methodology

The principal methodology utilized is unbiased stereology. Stereology is a statistical method for estimating the morphological properties of three-dimensional structures from a number of two-dimensional images (e.g. histological material). Estimates include: volume, area, surface area boundary length, population size and numerical density among others. At the core of the technique is the idea that measurements are made using a systematic random sampling paradigm, so that neither human of methodological bias can skew the results.

Estimates are generated using a library of “probes” that are specifically designed to reveal a specific characteristic (length, number etc). A probe is applied to the tissue being studied using a random sampling scheme that helps prevent bias’ imposed by shape orientation and so on. In the past theses analyses were performed manually from drawings or photographs using transparent overlays and tedious mathematical calculations. The advent of powerful personal computers has changed that.

Our lab employs a sophisticated software/hardware suite made by MicroBrightField, Inc. (Colchester, VT) called StereoInvestigator. The system consists of a Microscope with a computer controlled stage and focus (Z-axis) encoder. A high-resolution digital camera mounted to the microscope produces the image of the tissue on the computer monitor. The software generates a graphical overlay of the particular probe being used and not only moves the stage as needed, but encodes the positional data for all three axes.

In some analyses, the image produced by the camera and monitor my not be suitable. For example if one were trying to count neurons but did not want to include glia in that count, the native optics of the microscope are preferable. In this case a second option for the analyses is a small (1″ diagonal) SVGA monitor, that projects the software interface into the microscope, so that it appears as an overlay on the tissue as it is seen through the microscope.

People

Dr. Donald Siwek, PhD
Dr. Siwek has served as the Data Manager for the Boston University Alzheimer’s Disease Center (1995 Ð 2001) and is member of the Gross Anatomy Teaching Staff. Interests include neurobiology of sleep, neuronal morphology, imaging and image analyses, stereology, gross anatomy and astronomy.

Dr. Neil Kowall, MD
Dr. Kowall is a neurologist and the director of the Bedford GRECC and the Director of the Boston University Alzheimer’s Disease Center. His interests include neurodegenerative disease and the role of oxidative damage in those diseases.

Dr. Jan Krzysztof Blusztajn, PhD
Dr. Blusztajn is Professor of Pathology and Laboratory Medicine and Research, Professor of Psychiatry. He is the PI for the “Aging Of Brain–Effects Of Perinatal Nutrition” grant that funds the Stereology Lab. His interests are in the role of membrane lipid-derived second messenger molecules as possible modulators of cell transformation, and the regulation of the expression of the cholinergic phenotype, i.e. of the genes coding for proteins involved in the synthesis (choline acetyltransferase) and the storage (the vesicular acetylcholine transporter) of the neurotransmitter, acetylcholine.

Dr. Subimal Datta, PhD
Dr. Datta’s interests include: 1. understanding how single cells of the brain and their specific chemical signals (neurotransmitters and neuromodulators) are   at the level of population ensembles to determine the global behavioral states of waking and sleep; 2. studying the neurophysiological and molecular mechanism of state-dependent learning and memory consolidation; and 3. studying the consequences of altered brain development in the behavioral states of wake-sleep cycle.