Behavior, Neuroanatomy, Genomics: what can we learn from mouse mutants?

Our primary goal is to to generate excitement for and exposure to laboratory techniques used in neuroscience research. In addition we wanted to introduce them to the wealth of information available in genomics data bases that can be used as a powerful tool in solving scientific questions.

The specific goals of this sequence of labs are to use several sets of tools to determine:

a. how the mutation affects the behavior of the mutant when compared to the wild type,

b. anatomical differences between the brains of the mutant and the wild type by comparing Nissl and Golgi stains of both groups,

c. identify the motor mutant from the list of 5 motor mutantcandidates provided at the outset using Jax Mice website and Mendelian genetics,

d. the molecular basis of the mutant using genomics databases and the literature.

Our Neuroscience major at Wellesley College comprises 3 core courses at all 3 levels. The 100 level is designed to excite and motivate students about Neuroscience, the 200 level builds and provides a firm foundation for continuing on to upper levels and 300 level 'capstone' experience. In designing this sequence, our goal was to have students take the 200 level course as sophomores allowing time for them to focus on upper level courses. Hence this 200 level course currently has only one requirement, the 100 level course.

This 5 week module was developed as a new lab sequence for our 200 level course "Neurons, Networks, and Behavior". Each lab is 3.5 hours long with a maximum of 12 students/lab.

This lab sequence could also be used in an intermediate level cell or in a molecular biology course.

Lab 1A: Behavior.

A series of behavioral tests which can be used to compare mutant animals with normal wild type controls. This battery of tests can be used to determine some of the less obvious changes, such as problems with balance and problems with reflexes. Lab 1A (Microsoft Word 516kB Jun18 09)

Lab 1B: Neuroanatomy: Nissl and Golgi stains.

Cerebellum - Golgi stain by NEUR 200 '09 students Details

Instructions to section (coronally) and stain the mutant and control brains using the Nissl stain. Nissl stains RNA so cell bodies stain blue/purple. Golgi stains the entire cell membrane black, yet only stains ~1 in 500 cells. Golgi sections (sagittal) of both the mutant and control brains are prepared in advance by the instructor.

Lab 1B (Microsoft Word 56kB Jun18 09)

Lab 1C: Neuroanatomy: Comparison of mutant and control brains.

Start by reviewing neuroanatomy with a short quiz. Followed by time to answer questions about the molecular genetics worksheet from Lab 1A (p. I.10). Then determine if there are any anatomical differences between the mutant and the control brain.

Lab 1C (Microsoft Word 8.1MB Jun18 09)

Lab 1D: Introduction to Genomics: defining our mutant.Tutorial and introduction to the following data bases:

Jax Mice web site describes mouse strains and gives information on traits of mice:
http://jaxmice.jax.org/
MGI - Mouse Genome Infomatics. A resource of Jax Mice which gives comprehensive genetic information about the mice mutants:
http://www.informatics.jax.org/
ECR browser - Evolutionary Conservation of Genomes. This database aligns genes between different species giving a graphical output that color codes introns, exons etc.:
http://ecrbrowser.dcode.org/

ECR browser Details

NCBI Map Viewer - Map Viewer allows you to view and search an organism's complete genome, display chromosome maps, and zoom into progressively greater levels of detail, down to the sequence data for a region of interest:
http://www.ncbi.nlm.nih.gov/mapview/maps.cgi?ORG=mouse&CHR=6&BEG=63206851&END=64616273&MAPS=ideogr%2Ccntg-r%2CugHs%2Cgenes&thmb=on
TMHMM - Prediction of transmembrane helicies in proteins. This is a probability based algorithm that uses the hydrophobicity of amino acids to determine which regions of the gene are transmembrane. The graphical output is very helpful.
http://www.cbs.dtu.dk/services/TMHMM/

TMHMM output http://www.cbs.dtu.dk/services/TMHMM/ Details

The Basic Local Alignment Search Tool (BLAST) finds regions of local similarity between sequences.
http://blast.ncbi.nlm.nih.gov/Blast.cgi

Lab 1D (Microsoft Word 287kB Jun18 09)

We then have a workshop where each pair of students makes an oral presentation on a different aspect of the project.

The final assignment is to write a results and discussion section. Students are asked to select another of the motor mutants and use the genomics data bases to investigate this gene as described above.
assignment (Microsoft Word 41kB Jun18 09)

• For more information about the mutant used please contact the authors (we want to keep it a secret from the students!). We chose the mutant because it is expressed in the heterozygote form - hence 50% of the offspring will be mutant and 50% wildtype. This is a robust model since mutants are not vulnerable to a short life span. The breeding scheme is mutant males with wildtype females. This heterozygote expression is the identifying feature that distinguished the mutant from the other motor mutants.
  
• It is helpful and cost effective to be able to house mice and it is preferable to be able to breed a mutant mouse colony in an animal care facility.
  
• Behavioral equipment was made 'in house' from easily obtained supplies (for more details, contact the authors)
• Mice have to be sacrificed by perfusion for sectioning.
• It is necessary to have a Vibratome or equivalent for sectioning the brains and compound and dissecting microscopes.
• Mice brains were pre stained with the Golgi Kit (MTR Scientific FD Rapid GolgiStain Kit). This takes ~ 2 weeks lead time to fix, section, stain and mount. Note that this stain is toxic, so pay close attention to the instructions that come with the kit.
• It is recommended that the instructor becomes very familiar with the Genomics web sites listed in Description above.
• A computer lab is helpful for the genomics section of the lab series.
• Students should come to lab prepared by reviewing fundamentals of molecular genetics (see handout, Lab 1A, p.1). Also in preparation for the histology lab, students are required to review their neuroanatomy (see handout, Lab 1C, p20-29)
• The Allen Atlas is a wonderful resource for illustrating in situ hypridization data: http://developingmouse.brain-map.org/data/Grid2.html
• Also Brain Explorer is an animated look at the brain, nerve transmission, and brain disorders: http://www.brainexplorer.org/
• References link from MGI output is extremely useful for students.
• FASTA gives an output of amino acid sequence - students get confused with the letter references to amino acids and also need help distinguishing this sequence from DNA sequence.

See attached assignment sheet in teaching materials above in description.
(1) Knowledge assessments:

in lab quizzes (ungraded) on cell biology and neuroanatomy
statistical analyses of behavioral data
sharing lab data on neuroanatomy
in class discussion regarding mutant choice
workshop with oral presentations on different aspects of the project
literature style final paper (results and discussion) with grading rubrick (posted above).

(2) Attitude and belief (and/or affective) assessments:

end of project survey (posted above)
Student Evaluation Questionnaire and end of semester faculty evaluations.
questionnaire. assessment (Excel 11kB Jun23 09)

(3) Performance assessments:

as part of the final paper, students are expected to work through the entire genomics sequence, described in Lab1D, using another mouse mutant.

See web sites in attached files.