Integrative activities to study the evolution of nervous system function

Chris Wallace, Whitman College
Ginger Withers, Whitman College

Summary

The next generation of neurobiologist needs to be ready to put gene sequences into a functional context at the level of cells, neural systems, and ultimately behavior of the organism. Comparative genomics offers a powerful opportunity to engage neurobiology students in integrative thinking because it necessarily involves considering nervous system functions in the broadest evolutionary context, and raises questions that lend themselves naturally to multiple levels of analysis.
This multi-week series of laboratory exercises gives students a chance to become familiar with, and apply genomics analysis tools to explore the hypothesis that specialized nervous system functions will have a "genomic signature."
Week 1: Learn to use basic genomics tools to address a question relevant to neurobiology. Show more

Students begin the first session familiar with the ionic basis of the action potential such that they are ready to deconstruct a variety of action potential phenotypes into expression of subtypes of ion channels. A computer-based exercise introduces them to basic genomic tools (BLAST & Genbank searches) and provides data insight into the relative evolutionary conservation of sodium and potassium channels.

Week 2: Game of Integration. Building on the concept that electrical excitability of neurons is conferred by specific gene products localized to specific intracellular locations, the second week examines the integral role of glial-neuronal interactions in nervous system signaling in the mammalian brain. Show more

This is intended as an open-ended exercise, but we designed a "game of integration" modeled after a grant proposal to scaffold student inquiry. Students work in groups to brainstorm about candidate gene products they would expect to be clustered in association with myelination or the tripartite synapse, building on background information from class and journal club. An emerging literature is enhancing resources for investigating neuron-glia interactions and illuminate new questions about the phylogenetic distribution of glia in nervous systems (e.g. apparent convergent evolution of myelin in invertebrates).

Weeks 3 and 4: Comparative Evolution Project and Poster Session. Students now use their candidate genes identified in Week 2 as a probe for studying the evolution of a neuron-glial relationship by performing comparative genomic analyses.

During the poster session, teams present their project findings to the class and invited "external reviewers" consisting of professors with expertise in genetics/molecular biology, evolutionary biology, and comparative anatomy. This exposes students to scientific debate as they, we, and our departmental colleagues interpret data from multiple perspectives.
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Groups collaborate to make informal posters to present both the mouse-based integrative analysis and the comparative genomic analyses. Faculty colleagues with expertise in molecular biology, evolutionary biology and comparative physiology are invited to the session to emphasize that the students are engaged in real science.
This exercise is intended to develop iteratively across multiple classes, like a grant would across multiple renewals. Making examples of the work of the last class improves scaffolding, sharpens thinking and allows the next class to dig deeper into the key questions. It is also becoming easier to teach integration from genome to behavior and from cnidarians to humans as models of this kind of analysis begin to appear in the
literature. While this exercise is framed in terms of comparative "neuro-gliomics," it can be readily adapted to explore the genomic signature of comparative evolution of a variety of neural processes (e.g., FOXP2 and language, NMDA receptor and experience-dependent plasticity or PAR proteins and neuron polarity) appropriate to the instructor's expertise and research interests.

Biology | College Lower (13-14), College Upper (15-16)

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Learning Goals

The learning goals for the project are:
-To use GenBank to find sequence information, use other free genomics tools to address questions about that sequence

-To actively combine molecular, structural and physiological approaches to address a specific question about nervous system function.
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- Use primary literature searches (PubMed) and bioinformatics tools (BLAST, GenBank), to develop and test hypotheses about the molecular/cellular mechanisms of the particular aspect of the nervous system under study
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- To use comparative analyses to test hypotheses about the evolution of the nervous system
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-To more accurately model how science is done using an open-ended project in which students develop questions to test, and begin to test them. Poster presentations, combined with scientific discussion/debate with multiple professors who have expertise in molecular genetics and evolutionary analyses will help students synthesize and interpret their findings.

Context for Use

The complete module is designed to be implemented in an upper level elective neurobiology course for biology majors. It is compartmentalized in such a way, however, that it can be scaled appropriately to meet the level of the course. More advanced students could spend a greater amount of time on the exploratory-based parts of the project, whereas entry level students can focus more on developing integrative skills that play levels of analysis off one another, using more closely guided exercises. It also offers opportunities for an expanded wet lab component that could follow as a second part of the course, or as independent study. Although designed for Biology majors, our class typically includes juniors and seniors with diverse backgrounds (e.g. including students with other majors like psychology, anthropology and art, some of whom have never used GenBank or BLAST). One strategy, should this be the case, is to pair students with complementary experience and backgrounds.

Description and Teaching Materials

Projects associated with the module
Week 1: An introduction to genomics in the context of electrophysiology
In the first week of the project, students learn to apply genomics analyses to problems in neurobiology by comparing the number of putative sodium and potassium channel genes in multiple species (e.g. mouse and Drosophila). They then reconcile how this sequence diversity can account for the distinctive properties of action potentials in different regions of the brain, and across organisms.
-Conceptual development: specific functions of the nervous system, e.g. action potential properties, will have "genomic signature"
-Skills development: using Blast, GenBank, extracting information from sequence data
Materials to attach: do sheet, instructions for how to use genomics tools, references for relevant literature

Week 2: Planning the "Neurogliomics" project
We broke the planning phase of the project into 2 parts this week, a "Game of Integration" and "Predictions about Nervous System Evolution".

I. Game of Integration
Elements of the game: Neurodollars (models a real proposal with budgetary constraints, but money also attaches value, helps students think about the cost of science, and gives them some fun material), tool cards (structures their thinking as they are challenged to account for multiple levels of analysis in the experiments they propose)

Rules of the game. Students are divided into teams of 3, given $100,000 Neurodollars, a set of tool cards, and tasked to outline a grant proposal to:
1. define a specific neuron-glial cell interaction
2. identify candidate gene product(s) that would be involved in that interaction
3. outline a diverse set of experiments that test the function of this gene in the mouse.
Teams must use a variety of tool cards to study their proposed neuro-glial interaction experimentally, but are limited in budget.

Tool cards. Tool cards give possible experimental approaches from molecular to behavioral approaches, and include estimated costs for those types of experiments. Sample tool cards are shown below, and an attached file lists the categories of approaches we provided (in bold) with suggested applications underneath.

Game of Integration Tool Cards (Acrobat (PDF) 53kB Jul10 09)

Neurodollars. The purpose of these bills is to break the process of integrative analysis into a series of concrete steps so that it is easier for students to develop systematic cross-disciplinary strategies for understanding gene function. Each bill represents a key step of integrating across levels of analysis with increasing denominations reflecting a progressive synthesis of data from genomic to organismal fitness in terms of behavior or cognition. On the face of each bill is a scientist whose work has bridged levels of analysis framed by images representing the transformative influence of their work.

Value of bill Level of analysis
$1000 "Read genome as DNA sequence"
$5000 "Reconstruct molecular machinery from DNA sequence"
$10000 "Localize expression to within cells of nervous system"
$20000 "Determine gene product(s) function in nervous system"
$50000 "Determine relevance to cognition & behavior"

program project phase 1 template (Acrobat (PDF) 47kB Jul10 09)
II. Predictions and Discussion about Nervous System Evolution
Students are given cards that have schematic illustrations of the nervous systems of different organisms and work in teams to predict an evolutionary tree based on their anatomical plan.
Class discussion follows to debate order of tree. Instructors present data based on anatomical vs. molecular data, and allow time for further discussion.
Set up for next week with anatomical data that suggests some organisms don't have glia, images that show increase in #glia/neuron with organism complexity

Supplementary Materials to attach:
Organism cards--Cartoons of n.s. plans?
Published Phylogenetic trees based on physical characters vs. molecular
Table of phylogenetic diversity of glia

Week 3. Comparative Evolution project: Test for evolution of the neuron/glia partnership
Students work with genes they identified last week in the Game of Integration to plan and execute a comparative genomics analysis to test for their gene across species.

They then identify what they think are key next steps in their investigation (e.g. tests to confirm presence and localization of their gene product in simple vs. more complex nervous systems)

And to begin to assemble their data into an informal poster that presents their findings.
Materials attached:
Summary instructionsSummary instructions for comparative genomics analysis (Acrobat (PDF) 52kB Jul10 09)

Proposal Phase 2 matrixprogram project phase 2 template (Acrobat (PDF) 37kB Jul10 09)

Week 4. Synthesis and Presentation Students make an informal poster that summarizes their data and interpretations, and present in a discussion session with other members of the class and outside faculty with expertise in genetics, evolution and comparative anatomy.

Teaching Notes and Tips

Motivation for developing this module
This project developed out of a series of discussions we had as co-instructors of this upper level biology elective about how to better 1) anchor the course in an evolutionary context appropriate to its place in a biology curriculum, 2) engage students in cross-disciplinary integration aimed at accounting for brain function in cellular and molecular terms, and 3) model the process of science experimentation by giving students entrée into an open-ended project that goes beyond textbook knowledge. Comparative genomics struck us as an ideal approach because it offers a fresh and unbiased approach to nervous system diversity based on a common thread of molecular evolution. In addition, because many of the tools in comparative genomics are free and readily available on the web, students have unprecedented access to state-of-the-art resources, and unlimited opportunities for experimentation.

Tips for Implementing the Module
A. Background preparation
The project as we carry it out is very open-ended, but much of our course is designed to prepare students for such an open-ended exercise. We've identified key themes that thread through the semester to give the class multiple opportunities to learn core material as well as an introduction to big ideas for which the field of neuroscience does not yet have an answer and so are not included in the textbook. In this particular example, where students are asked to identify and test for genes that they predict would be involved in an evolving neuron-glial partnership, we focus these preparatory themes:

A1. Social cell biology

Students study the anatomical relationship between neurons and glia (e.g. using immunocytochemistry, and classic neuroanatomy stains like Golgi and Nissl), and publically available electron micrographs such as those from Kristen Harris' synapse website: http://synapse-web.org/lab/lab.stm

Students read and discuss review and primary literature papers that address the metabolic, physiological, and evolutionary relationships between neurons and glial cells
Materials provided--(example references provided below).

A2. Gene product localization and microdomains

We also emphasize that identifying genes is not sufficient to understand their function. Toward that end, students read papers that describe specialized protein localization in polarized neurons, and at synapses. In the lab, they do immunostaining in cultured neurons to test whether channel proteins, transporters and synaptic machinery has a uniform distribution, or a specialized localization. This helps to prepare students to think about what kinds of gene products might serve specialized functions in neuron-glial physiology.

B. Challenges to anticipate in this open-ended project:
B1. Integrating genomics into a Neurobiology course.

Some students will have a preconceived bias that these kinds of tools do not belong in this course. We try to dispel this notion by emphasizing the importance of using multiple levels of analysis to address questions in neurobiology. We try to be explicit about the importance of animal models and a comparative approach in understanding the nervous system.

We also found that it is very important to introduce genomics tools in a canned exercise, where there are clear predictions, and interpreting the data are more straightforward. This was the rationale for developing the action potential/ion channel exercise in Week 1.It is important to be explicit at the beginning of the semester about what the project will involve, and to prepare students that there is no "right answer," and in fact there are likely to be multiple interpretations.

B2. Challenges to doing "real science" (which does not necessarily have a right answer, or a single conclusion) in the classroom:
We have found that most students are not familiar with genuine scientific debate, and so one of our goals is to help them to understand that genuine debate doesn't mean they (or we) got the answer wrong. It is fundamental that they understand that there are multiple approaches to address an experimental question. In addition, there are multiple interpretations of data.

In addition, students have different comfort levels with debate. To help facilitate discussion, we have intentionally made the poster sessions very informal. Emphasis is on process and content, not form.

Assessment

Assessment Disclaimer: Assessment of this project is currently under development. Any instruments, descriptions of measurements, or summaries of assessment described in this website have not been vetted by a trained educational assessment expert, and should be regarded as preliminary.

In comparing student responses before and after completing this module, we found students were more likely to propose using multiple experimental approaches, and were more likely to propose using genomics tools to solve problems in neurobiology. Based on these data, we conclude that this project helped students to think more integratively across levels of analysis (e.g. behavior, neural circuits, cell/molecular, genomic).

In their evaluation of this module, one student wrote "At the time, I found some of these activities to be very frustrating. But in retrospect, they really pushed me to think more critically about neurobiology and enhanced my overall understanding of social cell biology."

Assessment goals include evaluating the student's knowledge base, their ability to apply knowledge to develop hypotheses, and to plan and execute experiments that test these hypotheses, and their beliefs about the value of comparative approaches, evolution and application of tools from other disciplines (e.g. genomics) to understand brain evolution and nervous system function.

I. Knowledge Assessments. To assess student understanding of foundation material, we identified basic principles covered in the course, e.g. the how diversity of voltage-gated ion channels contribute to the rich variability of action potentials in the nervous system, how different types of cells contribute to neuronal function, ability to generate accession numbers.

Examples of Knowledge Assessments:
--completion of "do-sheet" assignments and projects in lab (requiring running blast search, and evaluating data obtained in these searches),
--questions on exams (records of performance not kept this year)
--Ability to define the tripartite synapse at the functional level in the mouse
--survey questions

II. Performance Assessments. To assess performance, we designed measurement instruments with the goal of testing how well the students are able to integrate across different levels of analysis to generate and test new hypotheses, as well as assessing ability to carry out assignments to generate accession numbers and conduct genomics searches independently.

Examples of Performance Assessments:
1. Pre and post assessment surveys that asked students to propose experiments relating to channel diversity, and identify neuron-glial interactions
2. Performance on phase one of the "program project" including ability to
-- define a specific neuron-glial cell interaction (in mouse) that will study during the project
--search the literature to identify candidate genes that are likely to be involved in this process
--ability to propose appropriate experiments
--ability to propose multiple experiments that span levels of analysis
3. Performance on phase two of the "program project" (test for evolution of the tripartite synapse)
4. performance on poster presentations and ability to engage in discussion with a panel of faculty.
5. Additional performance assessments embedded within the course included integration pieces in final writing/image/data based portfolio

III. Assessment of beliefs--we developed three themes that we tested with survey questions administered pre- and post-project:

Theme 1. goal to assess beliefs about the legitimacy and value of deconstructing physiological function into specific gene products

Theme 2. goal to assess beliefs about evolution of the nervous system

Theme 3. goal to assess attitudes about value