Start here: email your info to Henry Lester
Description, goals (from the Caltech catalog)
Instructors and their co-ordinates
Master Schedule of Experiments
Need help on the concepts or techniques?
Prepare now for Independent Projects (Week of March 2)
Bi/CNS
161
Cellular and Molecular Neurobiology Laboratory.
9 units (0-9-0); second term. Prerequisite: Bi 150 or
Instructor's permission. Experiments on the
molecules of membrane excitability--ion channels,
receptors, and transporters. Students synthesize
mRNA in vitro for these molecules from cDNA clones
and inject the mRNA into Xenopus oocytes. Students
then perform electrophysiological experiments on the
oocytes, including voltage-clamp recording of
macroscopic currents and patch-clamp recording of
single channels. Students analyze the data to reveal
quantitative biophysical concepts. Graded pass/fail.
Given in alternate years; offered 1998/99.. Instructor:
Lester
Times: Tuesday and Thursday PM, 1 PM ******sharp******
to 6 PM, 60 Beckman Institute
X2406
Student |
|
status |
Option |
Rig |
| Stephen Shepherd | <mailamber@caltech.edu> | Jr | Bi/CS | 1 |
| Svjetlana Miocinovic | <sm@ccox.caltech.edu> | Jr | Bi/CS | 1 |
| Kayla Smith | <kayla@cco.caltech.edu> | Jr | Bi | 1 |
| Rory Sayres | <sayres@its.caltech.edu> | Jr | Bi | 1 |
| Eugene Pivovarov | <evgueny@cco.caltech.edu> | Gd3 | Phys | 2 |
| Luis Vazquez | <vazquez@its.caltech.edu | Gd1 | Biol | 2 |
| Josh Maurer | <jmaurer@its.caltech.edu> | Gd3 | Chem | 2 |
| Ryan Simkovsky | <ryans@cco.caltech.edu> | So | Bi | 3 |
| Melinda Turner | <mlturner@its.caltech.edu> | So | E&AS:CNS | 3 |
| Brent Kious | <kious@its.caltech.edu> | Jr | Bi/SES | 3 |
| G. Bjorn Christianson | <bjorn@vis.caltech.edu> | Gd1 | CNS | 4 |
| Ofer Mazor | <mazor@its.caltech.edu> | Gd1 | CNS | 4 |
| Ania Mitros | <ania@goethe.klab.caltech.edu> | Gd1 | CNS | 4 |
| Javier Perez-Orive | <javierpo@caltech.edu> | Gd1 | CNS | 4 |
| Matt Paul | <mattpaul@ugcs.caltech.edu> | Jr | E&AS | 5 |
| Gerard Paul Vigil | <paulvig@its.caltech.edu> | Sr | SES | 5 |
| Minoree Kohwi | <minoree@its.caltech.edu> | Sr | Bi | 5 |
| Urie Eden | <eden@cco.caltech.edu> | Sr | Math/CNS | 6 |
| Patrick Drew | <notpat@ugcs.caltech.edu> | Sr | Biol | 6 |
| Ethan Snyder-Frey | <ethan@ugcs.caltech.edu> | Sr | Bi/CS | 6 |
| Gabriel Miller | <gabriel@caltech.edu> | Sr | Bi/Ch | 6 |
TA's:
|
|
|
|
|
|
|
(Chemistry) |
injections |
|
|
|
Professional Staff |
|
|
|
X6469 |
(Physics) |
Single channels |
|
|
X2032 |
(Biology) |
|
|
|
X6822 |
(Biology) |
|
|
The Axon Guide for Electrophysiology
and Biophysics
Note: you don't need to follow this link from the Rig computers.
The Axon Guide is on the Desktop.
Ecitatory Ligand-induced Channels
| Tuesday
Thursday |
cRNA injection | Recording technique | Data acquisition/
analysis |
Drugs/
Solutions |
Boondoggles |
|
|
Mouse AChR abgd subunits |
2-electrode clamp,
"slow" and "fast" |
Chart recorder
(also used for all subsequent weeks) CLAMPEX/ CLAMPFIT |
ACh | George S. away |
| January 19,21 | m2-muscarinic receptor; GIRK1+GIRK2 | 2-electrode clamp
"slow" |
CLAMPEX/
CLAMPFIT |
ACh, atropine,
Hi-K |
|
| January 26,28 | Shaker H4 K+ channel; Shaker IR channel | 2-electrode clamp
"fast" |
CLAMPEX/
CLAMPFIT |
HAL away Monday (N.Y.) | |
| February 2,4 | Na channels, Shaker IR | 2-electrode clamp
"fast"; 2-electrode current-clamp recording |
CLAMPEX/
CLAMPFIT |
Normal Ringer;
10% Na Ringer |
2/2 Marcus away |
| February 9,11
(Note next row also) |
More action potentials | 2-electrode current-clamp recording | CLAMPEX/
CLAMPFIT, and chart recorder |
Normal Ringer | 2/9 Henry at a funeral |
| 5HT2C receptors | 2-electrode clamp;
fura-2 optical recordings |
Chart recorder;
Axon Imaging Workbench |
5-HT 100 nM | ||
| ACh receptor-channel blockers | |||||
| February 16,18 | AChR and mutants
Inward rectifiers |
patch | CLAMPEX/
FETCHAN |
ACh, QX-222 | HAL away all week (Baltimore) |
| February 23,25 | Slowpoke | patch | CLAMPEX | HAL away
Thursday (D.C.) |
|
| March 2,4 | Projects: to be arranged | To be arranged | |||
| March 9,11 | March 9: Project Experiments
March 11: Project oral reports |
To be arranged | |||
| March 16 | Notebooks and project reports due. |
2. Keep a conscientious lab notebook for your data and analyses. Submit it on March 16, 9 AM.
3. Give a seminar on March 11.
Each group should generate a proposal for semi-independent experiments to be performed during the week of March 9. Your proposal should focus on Xenopus oocytes injected with RNA and studied with one of the techniques we will already have used (voltage clamp or patch clamp) or with another technique that you might like to learn. Examples of other techniques would be radioligand binding, tracer flux, or dye measurements of intracellular Ca.
You may reorganize your groups if everyone is in agreement.
Your project should be a real experiment, not a duplication of something we have already done, although you may do something different with an RNA we have previously studied (*). The # sign denotes cRNAs for which site-directed mutations have been made and studied. You might want to design an experiment around such mutants.
A preliminary one-page summary of your experiment should be given or Emailed to H. Lester (Lester@Caltech.edu) by February 15. You should develop your idea yourself, in consultation with Henry, or with other students or T. A.'s. We will then discuss and refine your idea together. A major purpose of this project is to teach you to use the original literature efficiently and intelligently. You may also want to consult the books available on reserve or the information on Henry's web page.
Please note that, although we do in principle have these reagents available, it may take a while to check them out and generate new cDNAs and/or oligos so that you can make RNAs. I'm also willing to phone my friends and beg for some interesting clone that's not on the list. So please allow us to plan effectively by talking with us in advance!
7-helix receptors
Serotonin 5HT1C
Serotonin 5HT1A#
muscarinic M1, M2
Thyrotropin releasing hormone
beta2-adrenergic*
mu, delta, and kappa opioid
Voltage-gated channels
Shaker*
Shaker IR (inactivation removed)*
MBK
Kv3
Shaw
Arabidopsis (yes, a plant channel!)
Na IIA#
SkM2 cardiac Na channel
Na beta subunit
Cardiac Ca channel
Ca-activated K channel
Slowpoke*
Inward rectifier channels
ROMK1*#
IRK1
GIRK1 through GIRK4 (G protein gated inward rectifier)#
phosphorylation-activated channel
cystic fibrosis transmembrane conductance regulator (CFTR)*#
neurotransmitter-gated channels
Muscle ACh (alpha, beta, gamma, delta subunits)*#
Neuronal alpha2, alpha4, alpha7, alpha8, beta2#, beta4# subunits
NMDA receptor
5HT3 serotonin receptor
P2X2, P2X3, P2X4,
P2X7 ATP-gated channels
electrogenic transporters
GAT1 GABA transporter#
GLY1 glycine transporter
Serotonin transporter
Na/glucose transporter
cyclic nucleotide-gated channels
retinal
olfactory, subunit 1
olfactory, subunit 2
Tissue-derived mRNA:
Your proposal could be an informed "fishing expedition" to look for
interesting responses from these RNAs, which presumably encode proteins
expressed by these rat tissues. Your proposal should cite previous studies
with these RNAs and suggest a specific experiment.
brain
olfactory epithelium
heart
liver
kidney
Name
email
Campus address
phone contact
Status and option (i.e., grad 3rd year, physics)
Neuroscience experience (Bi 150, etc)
Best afternoons and hours to attend the lab; list conflicts
Level of certainty to take the course (0=no; 1= definitely)
Your own data:
paste this template into your email program,
fill in the blanks, and send it to Henry
Lester
| Name
Campus address phone contact Status and option (i.e., grad 3rd year, physics) Neuroscience experience (Bi 150, etc) Best afternoons and hours to attend the lab; list conflicts Level of certainty to take the course (0=no; 1= definitely) |
Cleanup
Rinse the pipette-filling syringes ($20 apiece !!)
rinse solutions from tubing and chamber
empty vaccum reservoir
clean up any 3 M KCl
turn off oscilloscope and Geneclamp
cap on chart pen
oocytes down sink