Toolbox/Lecture 17

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WIKIPEDIA BIO154/254: Molecular and Cellular Neurobiology

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This page is part of the BIO154/254 Experimental Toolbox!

Contents


Lecture 17 Model Systems

C. elegans vulva formation

Drosophila spermatogenesis

Lecture 17 Techniques

Cantab Paired Associates Learning

Cambridge Neuropsychological Test Automated Battery Paired Assocation Learning is a type of visual memory test that characterizes episodic memory. (www.cantab.com). There are six boxes displayed around the edge of the screen. In random order, the boxes will display different, unique patterns. The patient is then shown the same patterns in a box in the center of the screen, and must recall where they saw each pattern (in which box).

Human genetics

The difficulty, obviously, is that we cannot study human genetics directly. We cannot manipulate who mates with whom. However, there are certain strategies for studying human genetics, particularly in relation to disease. Twin studies can suggest the relative role of genetic and environmental factors in inducing the onset age or severity of disease. For instance, a twins study suggested that genetic factors do not play a major role in causing sporadic PD that begins after age 50. Also, study of pedigrees is useful for tracing familial history of genetic diseases, for instance X-linked inheritance disorders. Additionally, transgenic animal models (mouse, fly) can be used to elucidate the molecular pathways that lead to diseases.

T-maze

The T-Maze is used to measure rat learning. The rat is placed at the bottom of the "T," and it is allowed to choose either the right or left branch of the T. A reward is waiting at one of the branches. When this process is repeated over days, the rat (hopefully) learns and remembers to which side to run. In the Chapman article (1998) referenced in class, the rat's task was to learn to alternate branches to find the reward. That is, the sugar-water reward would consistently be located at the other arm than the rat chose previously.

Reversal learning

Radial water-maze

The radial water maze, or the radial arm water maze, is an adaptation of such classic learning apparati for rodents as the T maze and the dry radial arm maze. The maze consists of a large, round basin of water with metal wall inserts that form lanes, or arms, from a central open area. One arm is chosen as the target arm, in which an escape platform is placed that allows the mouse to climb atop it, thus exiting the water, and rest. The result is a maze with the navigating area appearing like an asterisk, although the apparatus can be changed to include more or less arms. There are several advantages to the radial arm water maze over other mazes, namely, that it has "the spatial complexity and performance measurement simplicity of the dry radial arm maze combined with the rapid learning and strong motivation observed in the Morris water maze without requiring foot shock or food deprivation as motivating factors." (Alamed et al., Nature Protocols 2006)

The radial arm water maze is used to test spatial learning and working memory. Typically, experimenters train mice to navigate to the target arm by inserting mice into a start arm and maintaining the same target arm during training. During the experimental trials, the target arm is moved from its original position. In Morgan et al., Nature 2000, the radial arm water maze was used to test the restoration of spatial learning ability in TG2576 x PS1 double-transgenic mice, a mouse model of Alzheimer's Disease, via amyloid-β vaccination. The findings suggested that such vaccination might be an effective therapeutic approach to treating Alzheimer's dementia.

Longitudinal study

A study done over a period of time, involving observing/examining certain variable(s) in a set group of study subjects. Also called a diachronic study. The opposite of a cross-sectional/synchronic study which is done at one point in time (e.g. to learn about a certain disease's prevalence and distribution within a population at one time), rather than with the passage of time. In the last lecture, we learned that recent longitudinal studies have shown that PD may occur more acutely. Using F-18-Dopa to image dopamine terminals of clinically unaffected twins of PD patients suggests that disease progression is rather acute, with a 5-10 year span from normal imaging results to abnormal to the onset of clinical symptoms.

ELISA

ELISA stands for Enzyme-Linked Immunosorbent Assay. This technique uses antibodies (type of protein) as extremely specific analytic reagents to detect and quantify the amount of antigen (a substance that when introduced into the body, stimulates the production of an antibody. Antigens can be proteins, toxins, bacteria, foreign blood cells, cells of transplanted organs, etc.). Here, most likely, we are dealing with protein antigens.

Overall Method:

  1. Enzyme: You have an enzyme which reacts with a colorless substrate to produce a colored product.
  2. Antibody-Enzyme Complex: The enzyme (from part 1) is covalently linked to a specific antibody that recognizes a target antigen.
  3. Antibody-Enzyme Complex + Antigen = Reaction: If the antigen is present, the antibody-enzyme complex (from part 2) will bind to it, and the enzyme part of the antibody-enzyme complex (from part 2) will catalyze the reaction generating the colored product.
  4. What does the reaction (i.e. getting a colored product) mean? The presence of a colored product indicates the presence of the antigen (see part 3).

Why do people use the ELISA method?

  1. It is fast and convenient.
  2. It is sensitive--can detect less than 10^(-9) grams of a protein.
  3. It can be performed with either polyclonal (mixture of different antibodies that bind to the same antigen) or monoclonal (identical antibodies cloned from a single antibody-producing cell, used to bind to one type of antigen) antibodies, but using monoclonal antibodies yields more reliable results.
  • Note: We can use polyclonal and monoclonal antibodies to a specific antigen because antigens have different surface markers/binding sites that specifically recognize different types of antibodies.

There are several types of ELISA. Here are two popularly used types.

  • Indirect ELISA: used to detect the presence of antibody
    • Indirect ELISA is basis of test for HIV infection.
  • Sandwich ELISA: allows both detection and quantification of antigen
  1. Antibody for a particular antigen is absorbed to bottom of a well.
  2. Antigen (or blood/urine containing the antigen) is added to the well and binds to the antibody.
  3. A second, different antibody (this second antibody is linked to an enzyme, whereas the first antibody was not linked to an enzyme) to the antigen is added. Remember from the note above that different antibodies can bind to the same antigen because the antigen has different surface markers that recognize and bind to these different antibodies. Basically in this step, you are adding the antibody-enzyme complex (described in the Overall Methods above) to the well.
  4. A colorless substrate is added to the well. The enzyme part of the antibody-enzyme complex (from part (3) above) will react with the substrate and produce color. The rate of this color formation is proportional to the amount of antigen present (because the enzyme is linked to the second antibody which is linked to the antigen).

Suppressor screen

A suppressor screen is a highly sophisticated type of genetic screen that uncovers information about a known gene or mutation. A suppressor screen starts with a known mutation and then finds second-site mutations that suppress the mutant phenotype. An enhancer screen, on the other hand, will find a mutation and enhance its effects.

A suppressor screen has two main advantages. Firstly, the screen identifies new genes that are often involved in the same biological processes as the gene of interest. This allows for the pathways of the mutation to be further elucidated and understood. Secondly, and most importantly, the suppressor screen isolates genes that could potentially have invisible phenotypes, and therefore would not show up on a basic screen. A suppressor screen, however, is sensitive enough to detect such "silent" genes.

Generally, it is easier to isolate suppressors of weak, or partial loss-of-function mutations, as opposed to strong loss-of-function mutations or null mutations. Two types of suppressors are usually found by performing a suppressor screen: informational suppressors and functional suppressors. Functional suppressors act through mechanisms that usually help elucidate the processes dictated by the gene of interest. Informational suppressors typically include gene products that suppress the mutation though a genetic mechanism. Other informational suppressors include mRNA degradation mutants and protein-degradation mutants.

Immunotherapy

Immunotherapy is a broad class of therapies that uses the natural molecules of a body's immune system to either treat or prevent diseases. There are many types of immunotherapies, the most common form being anti-microbial vaccinations. Other kinds of immunotherapies do exist, such as cancer immunotherapy, where the patient's immune system is stimulated to kill tumor cells, and allergy immunotherapy, where increased dosage of injected allergens leads to a desensitization response.

In the context of the class, immunotherapy was explored as a treatment for Alzheimer's disease. Schenk et al (Nature, 1999) found that young mice predispositioned to get AD did not develop beta-amyloid (Ab) plaques when injected with Ab over a period of time and older mice that already had plaques showed retarded plaque growth following Ab injection. The reason this therapy works is that injection with Ab causes the body's immune system to synthesize high titer anti-Ab antibodies. These antibodies then bind to Ab, causing its clearance from the brain and the prevention of plaques. The result is improved learning and memory. However, human trials were curtailed in 2002 after some patients showed inflammatory responses.

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