Association of alcohol consumption with brain volume in the Framingham study.

BACKGROUND: While adults who drink low to moderate amounts of alcohol have lower rates of cardiovascular disease than other adults, the effect of alcohol on the brain is less clear. There is evidence that drinking large amounts of alcohol is related to brain atrophy. It is uncertain what the effects of low to moderate consumption might be. OBJECTIVE: To determine whether consumption of smaller amounts of alcohol negatively affects brain volume or is protective in reducing the well-documented age-related decline in brain volume. DESIGN: Total cerebral brain volume (TCBV) was computed, correcting for head size. Multivariate linear regression models were used to evaluate the association between 5 categories of alcohol consumption (abstainers, former drinkers, low, moderate, high) and TCBV, adjusting for age, sex, education, height, body mass index (calculated as weight in kilograms divided by height in meters squared), and the Framingham Stroke Risk Profile. Pairwise comparisons were also conducted between the alcohol consumption groups. PARTICIPANTS: A total of 1839 subjects from the Framingham Offspring Study who had magnetic resonance imaging of the brain between 1999 and 2001. RESULTS: Most participants reported low alcohol consumption, and men were more likely than women to be moderate or heavy drinkers. There was a significant negative linear relationship between alcohol consumption and TCBV (r = -0.25; P < .001). This relationship was modified by sex, with alcohol consumption having a stronger association with TCBV in women than in men (r = -0.29 vs -0.20). CONCLUSIONS: In contrast to studies on cardiovascular disease, this study found that moderate alcohol consumption was not protective against normal age-related differences in total brain volume. Rather, the more alcohol consumed, the smaller the total brain volume.

Sectioning of brain tissues.

INTRODUCTIONA fixed brain, although harder than the brain in its natural state, is still not hard enough to cut into very thin slices. To make uniform, thin sections, the brain must be frozen. A wide variety of sectioning devices are available, each with their own requirements. With a microtome, for example, a very sharp knife is drawn manually across a brain that has been frozen to a chilled platform using dry ice. After each cut, the knife is lowered mechanically by a preset amount, giving consistent sections of a required thickness. A drawback of this instrument is that the temperature of the platform can fluctuate, thus causing changes in the quality of the sections. Other instruments, such as a cryostat, maintain the platform-mounted brain and the knife inside an enclosed refrigerated compartment. The temperature is kept at -20°C, and electronics are used to control the speed of the knife and the thickness of the sections. Some models can be set up to make a series of sections automatically.

Perfusion of brain tissues with fixative.

INTRODUCTIONIn many experiments it is necessary to section the brain to determine the location of a treatment (lesion or electrode) or to look at the histology of the brain using various staining techniques. Because the texture of the brain is so soft (often likened to soft cheese), it must be "fixed" before it can be removed from the skull. A fixative is a chemical that cross-links the molecules of the tissue, rendering it hard and preserving the tissue. This protocol describes a method for perfusing the brain with fixative (specifically, it describes how to perfuse a rat brain; slight modifications may be needed for different animals). A relatively simple gravity feed and the pumping mechanism of the heart is used to get fixative into the brain. A cannula is placed in the heart, or directly in the ascending aorta, of a deeply anesthetized animal. Blood is flushed out with saline first, and then with a fixative. The choice of fixative is often important if a specific staining technique is to be used, especially in immunocytochemistry, because the fixative can interfere with the staining sensitivity.

Dissection of rat brains.

INTRODUCTIONIn this protocol, the perfused brain of a rat is separated from the surrounding tissue and post-fixed in a formalin/sucrose solution in preparation for freezing and sectioning. Dissection should be done as soon as possible after perfusion to prevent desiccation of the brain. This procedure can also be used to dissect fresh (non-perfused) brains, for example, for Golgi-Cox stain or neurochemical assays.

Subbing slides.

INTRODUCTIONWhen mounting sections on a slide, it is essential that the slide be ultraclean. To ensure that the section sticks to the slide, slides can be covered with a thin layer of gelatin. The process of cleaning and gelatin-coating is called "subbing." Several boxes of slides should be subbed at a time and stored, covered, in the refrigerator until they are needed. Subbed slides can also be purchased commercially.

The nissl stain: a stain for cell bodies in brain sections.

INTRODUCTIONThe Nissl stain is used widely in many research labs to examine the overall morphology of the brain. It is often used to verify the location of a lesion or an electrode.

Testing spatial and nonspatial learning using a morris water maze.

INTRODUCTIONAnimal studies of memory have used many different types of mazes and tasks, where the animal is required to learn the demands of the test and then perform correctly until a predetermined performance criterion is reached. Traditional studies have relied on water or food deprivation to motivate the animal to do the test using food or water rewards. In 1984, Richard Morris introduced the water maze. The maze consists of a large cylindrical tank of water with a hidden platform; the animal has to swim until it finds the platform. The animal generally uses cues outside the maze (extramaze cues) to develop a spatial map of the environment and guide its performance. This maze avoids the problems associated with food or water deprivation, because the animal's goal is to find the hidden platform so that it can stop swimming. Many types of memory can be studied by varying the visual cues, the placement of the platform, and the starting point of the animal. This protocol describes the use of the water maze to test rats in two behavioral tasks: a spatial task and a nonspatial, visual task.

Observational methods used to assess rat behavior: neurological testing.

INTRODUCTIONThe rat is the most frequently used animal for behavioral experiments, and several methods can be used to assess behavioral changes that result from modifications to its nervous system. Objective neurological tests can be used to establish a "behavioral baseline" for a normal animal, and to study the effects of drugs or a lesion on behavior. Neurological tests should be administered much like a doctor would administer a battery of tests after a trauma, with objective observation, precise documentation, and repeated measurements by the same experimenter. This protocol outlines a series of neurological tests designed to assess reflex movements in the rat. Male or female rats may be used, although data should be analyzed separately to determine if sex differences exist.

Observational methods used to assess rat behavior: general activity.

INTRODUCTIONThe activity-inactivity continuum is an important parameter of behavior, and quantification of overall locomotor activity in the rat should identify it as a naturally nocturnal animal. Disruptions in nocturnal activity can be caused by damage in visual inputs to the brain or damage in the hypothalamus. Many commercial devices are available to measure activity automatically; some can be integrated with a computer to allow overnight monitoring in the absence of an observer. A less sophisticated but still accurate method of measuring activity is to create a home-made activity chamber by replacing the bottom of a box with Plexiglas or by marking lines on the bottom of a clean rat cage so that the observer can record rat activity by noting when the lines are crossed, while simultaneously recording other behaviors. Activity in rat pups can be observed as soon as they are 10 days old using smaller activity chambers. This protocol describes the construction of a home-made activity chamber and how to measure four activities: locomotion, rearing, circling, and grooming.

Observational methods used to assess rat behavior: behavioral patterns.

INTRODUCTIONThis protocol describes a method for quantifying observation of normal rat behaviors in a controlled environment and in a defined area. It is necessary to have precise definitions of the behaviors to be quantified and a reliable procedure for quantifying them. The observation room should be quiet, and it should be equipped for light or dark observations (rats cannot see red light so dark observations can be made using a red light). Ideally, the observer should be separated from the animal by a one-way screen. If such a screen is not available, the observer should stand or sit in a constant, unobtrusive position and make as few movements as possible.

Observational methods used to assess rat behavior: general principles.

INTRODUCTIONThe behavioral approach to solving neuroscience questions, unlike cellular and molecular approaches, is difficult to define and is therefore sometimes considered a less-disciplined approach. Methods describing behavioral procedures are often thought to be crude when compared to the precision of physiological or molecular methods. This apparent lack of precision results from the problems inherent in the study of behavior--deciding what to measure among the large number of variables, understanding that behavior is the final output of a vast array of neurochemical and electrophysiological changes, designing reproducible experiments that have appropriate controls, and most importantly, interpreting results. Conflicting results from similar protocols have led to prolonged debates and controversies in interpretation of behavioral data; therefore, it is necessary to strive for a high degree of precision through the cultivation of excellent handling and observational skills and thorough documentation strategies. This article is an introduction to several methods used to measure and assess normal behavior in a rat, including suggestions about how to make objective behavioral observations. Behavioral tests can be used to establish a "behavioral baseline" for a normal animal, to assess the effects of drugs or a lesion on behavior, and to determine whether transgenic animals have a normal behavioral repertoire. It should be kept in mind that, along with the intended effect, experimental manipulations often have unintended side effects, and behavioral tests can also be used to determine some of the less obvious changes.

IFEL TOUR: A Description of the Introduction to FUN Electrophysiology Labs Workshop at Bowdoin College, July 27-30, and the Resultant Faculty Learning Community.

The workshop "Introduction to FUN Electrophysiology Labs" was organized by Patsy Dickinson (Bowdoin College), Steve Hauptman (Bowdoin College), Bruce Johnson (Cornell University), and Carol Ann Paul (Wellesley College). It took place July 27-30 2006 at Bowdoin College. There were fifteen participants, most of whom were junior faculty at college and universities around the country. This article describes the workshop content, the incorporation of lab exercises at home institutions, and the faculty learning community that has resulted from the workshop.

Exploring neurogenesis in crustaceans.

Plasticity, learning and memory, and neurological disease are exciting topics for students. Discussion around these subjects often results in the consideration of the role of neurogenesis in development, or its involvement in a potential cure for some diseases. We have therefore designed a lab that allows students to experimentally examine how the rate of neurogenesis can be altered by environmental factors. Neuronal cell division in crayfish is identified with fluorescently-labeled BrdU and quantified using conventional or confocal microscopy. Recent studies indicate a conservation of mechanisms that control neurogenesis from insects and crustaceans to mammals. Yet the use of invertebrate models such as crayfish or lobsters has advantages over mammalian models. Invertebrate nervous systems have a simpler organization and larger, identifiable neurons - qualities that make such preparations easier for students to manage. This lab offers many opportunities for student designed experiments and discovery-oriented learning by exploring factors that regulate neurogenesis such as environment, hormones and light. This article illustrates our first experience with the lab, using an experiment designed by our students. We include ideas for expansion of this model and suggestions for avoiding potential pitfalls. It is written in the form of a scientific paper, reporting on a single student experiment, to aid as a teaching tool for future classes.