General Daily Timeline*
8:00 – 9:45 AM Breakfast & Lecture, B100
10:00 – 12:30 AM Molecular Lab, B169
12:30 – 1:00 PM Lunch (courtyard, Beckman entrance)
1:00 – 3:00 PM Electrophysiology Lab, B169
3:00 – 3:30 PM Tea Time
3:30 – 5:00 PM Electrophysiology Lab, B169
5:00 – 6:00 PM Electrophysiology Analysis, B100
6:00 – 7:00 PM Dinner and faculty presentations, B100
*General schedule will be altered from time to time as noted below.
Acknowledgments
The instructors of SIN 2007 gratefully acknowledge the
generous support of the following groups,
without which the course would not have been possible.
BioVision
Carl Zeiss MicroImaging, Inc.
Molecular Devices
Quest Scientific Instruments Inc.
Sutter
Instruments
Olympus America Inc.
Technical Instruments
Nikon Instruments
Improvision / Perkin Elmer
Department of Molecular and Cellular
Physiology
Syllabus
Monday, September 10
8:00 – 8:30 AM Welcome and orientation
John
Huguenard, Ph.D.
8:30 – 10:00 AM Lecture 1: Introduction to Neuronal Cytology and
Architecture
Craig
Garner, Ph.D.
o
Neuronal Anatomy of
Neuronal Cells
o
Protein synthesis and
membrane trafficking
o
Cytoskeleton
o
Membrane
specializations
Ø
10:15 – 12:30: Molecular
Lab
o
Overview of
experiments Joy & Lynette
o
MB-Expt 1 Tagging the C2 domain of PKC with GFP
o
MB-Expt 1A Digesting plasmid DNA/Pour 0.8% agarose gel
o
MB-Expt 1B Run and Isolate DNA from agarose gels
§
Discuss DNA
quantification and Ligation theory
Ø
12:30-1:00
PM: Lunch
Ø
1:00-2:30
PM Lecture 2: Basic Electronics – The bedrock of electrophysiology
John Huguenard, Ph.D.
o Ohm’s law
§ While Ohm’s law is simple, a thorough understanding of it and its implications is an absolutely necessary underpinning for understanding everything electrophysiological
o RC circuits
§ Understanding the behavior of an RC circuit is central for all electrophysiology. Cell membranes all operate on the principals of an RC circuit.
o Cable properties;
§ The understanding of basic cable properties is important for understanding all electrical behavior of cells that occurs at some spatial or temporal distance from an originating signal. Cable properties are best understood in context of RC circuits.
Ø
2:30-3:00 PM:
Tea Break
Ø
3:00-6:00 Electrophysiology
Lab
o EP-Expt 1: Dry lab. Electrophysiological recordings.
§ Using the Multiclamp Amplifier, voltage vs current recordings.
§ Developing stimulus protocols with Clampex
§ Analyzing the results, Clampan
Ø
6:00 to 7:30
Dinner and Faculty Talk
o
Tony Ricci, PhD
Tuesday, September 11
8:00-9:45 AM Lecture 3:
Introduction to molecular biology
– from gene to protein
Richard Reimer, M.D.
o gene structure
§ nucleotides, DNA and RNA –how strings of bases form single and double stranded polynucleotides
§ introns and exons – what is splicing how does it occur
§ coding and non-coding regions – converting RNA to protein
o manipulating DNA
§ cDNAs – how are they made
o expression vectors – their components (MCS, promoters and polyadenylation sites, selection (for bacteria and mammalian cells))
§ restriction endonucleases and ligase – how to subclone
· specificity of enzymes
· compatible cohesive ends
· blunting restriction sites
§ PCR – its many uses in manipulating DNA
· oligo design
· introducing mutations
· TA subcloning
Ø
10:00-12:30
Molecular Lab
o
MB-Expt 1B Qiaquick isolation of DNA from gel slices
o
MB-Expt 1B Gel analysis of DNA fragments:
quantification of DNA concentration
o
MB-Expt 1C Ligate fragments in vector
o
MB-Expt 1D Transform Bacteria/ plate on LB-agar/Kan
plates
o
DEMO ON STERLE
TECHNIQUE AND PLATING BACTERIA part 1
Ø
12:30-1:00
PM: Lunch
Ø
1:00-1:45: Introduction to recording techniques
Shaul Hestrin,
Ph.D.
Ø
1:45-3:30: Electrophysiology Lab
o EP-Expt 2 : Learning to Patch Clamp, immature brain slices
§ Making seals, breaking in
§ Voltage clamp recordings
§ Analysis of VC results
Ø
3:30-4:00 PM:
Tea Break
Ø 4:00-6:00 PM: Electrophysiology Lab
Ø
6:00 to 7:30
Dinner and Faculty Talk
o
Jamie Zeitzer, PhD
Wednesday, September 12
8:00-9:45 Lecture 4: Excitability
John
Huguenard, Ph.D.
Ø From the properties of individual ion channels to the behavior of whole cells containing a repertoire of ion channels
o Voltage-independent conductance
o Voltage-dependent conductances
o Time-dependent conductances
Ø I / V plots
o development of an understanding of the properties of an excitable cell using the I / V plot as a teaching device
§ What are equilibrium potentials?
· Why are they important?
§ Why is the resting potential the resting potential?
§ Where is the threshold for an action potential?
§ What are the maximum voltages a cell can reach?
§ Resistance of the cell membrane
o The true ability to “read” an I / V plot is an important skill even for the non-electrophysiologist who want to follow electrophysiological seminars.
Ø
10:00-12:30
Molecular Lab
o
DEMO on Bacterial
sterile technique part 2.
o
MB-Expt 1E Pick colonies/ inoculate 5ml LB/Kan tubes
o
Home work: Designing
restriction digest to identify positive clones
Ø
12:30-1:00
PM: Lunch
Ø
1:00-3:00
Electrophysiology Lab
o
EP-Expt 3: Passive and active properties of
neurons
§
Acute brain slices
§
Whole cell current clamp recording
§
Assessing input resistance, membrane time
constant
§
Action potential generation
Ø
3:00-3:30 PM:
Tea Break
Ø 3:30-5:00 Electrophysiology Lab
o
EP-Expt 3: Passive and active properties of
neurons, continued
Ø 5:00-6:00 Electrophysiology Analysis
o
membrane time constant, capacitance, resistance
o
Action potential waveform, spiking patterns, I/O
relationships
Ø
6:00 to 7:30
Dinner and Faculty Talk
o
John Huguenard, Ph.D., Neuroscience Program Orientation
Thursday, September 13
Ø 8:00-9:45 AM: Lecture 5: Presynaptic Mechanisms
Shaul
Hestrin, Ph.D.
o Synapse anatomy
o Basic release properties
§ calcium dependence
§ time dependence
§ vesicle theory
o Probabilistic release
§ Minis and quanta
§ Binomial statistics and Poisson statistics
Ø
10:00-12:30
Molecular Lab
o
MB-Expt 1F Perform Plasmid mini-preps
o
MB-Expt 1G Digest plasmids overnight
Ø
12:30-1:00
PM: Lunch
Ø
1:00-3:00
Electrophysiology Lab
o
EP-expt 4: Slice preparation: Spontaneous PSCs
§
heterogeneity
§
voltage dependence
§
underlying receptors
(if time available, modulation)
Ø
3:00-3:30 PM:
Tea Break
Ø 3:30-5:00 Electrophysiology Lab
o
EP-expt 4 Slice preparation: Spontaneous PSCs, continued
Ø 5:00-6:00 Electrophysiology Analysis
o
Membrane time constant, capacitance, resistance
o
Action potential waveform, spiking patterns, I/O
relationships
Ø
6:00 to 7:30
Dinner and Faculty Talk
o
TBD
Friday, September 14
Ø
8:00-9:45
Lecture 6: Postsynaptic Mechanisms
Shaul Hestrin, Ph.D.
o Postsynaptic receptor activation
o Synaptic Integration
o EPSP-spike coupling
Ø
10:00-12:30 PM: Molecular Lab
o
MB-Expt 1H Analyze restriction pattern on Agarose gels
o
MB-Expt 2D
o
MB-Expt 2E Photograph Fixed and Stained Neurons
Ø
12:30-1:00
PM: Lunch
Ø
1:00-3:00
Electrophysiology Lab
o
EP-expt 4: Slice
preparation: Evoked synaptic responses
§
Excitatory and inhibitory
components
§
Voltage dependence
§
Synaptic integration
Ø
3:00-3:30 PM:
Tea Break
Ø
3:30-5:00 Electrophysiology
Lab
Ø 5:00-6:00 Electrophysiology Analysis
o
Isolation of excitatory and inhibitory
conductances
o
Components of the excitatory response, NMDA vs
nonNMDA
o
Shunting inhibition
Ø
6:00 to 7:30
BBQ & wiffle ball
Monday, September 17
Ø 8:00-9:45 AM Lecture 7: Channels –
the biological nature of resistors
Merritt Maduke, Ph.D.
Ø This lecture will build upon the previous in that it gives a biological description of the resistors in the RC circuit.
o It will extend the understanding of biological RC circuit behavior in that it will deal with the unique electrical properties of ion channels
§ Conductance, permeation and selectivity
§ Voltage dependent gating
· activation
· deactivation
· inactivation
Ø
10:00-12:30
Neural Imaging or Electrophysiology Lab
o
Each group of 2-3
students will spend one day at 4 of 6 stations
o
Each group will be
assigned to present a final report on 1 station
o
The following 6
Stations will be available all week
1)
Craig Garner: Live
imaging of synaptic proteins, imaging immunoreactivity
2)
Tony Ricci: Hair Cell
physiology
3)
Ricardo Dolmetch: Live
imaging of Ca2+ entry and membrane trafficking
4)
Shaul Hestrin:
Neocortical inhibitory circuits
5)
Rich Reimer:
Purification of a recombinantly expressed vesicular neurotransmitter
transporter
6)
John Huguenard: Neural
oscillations in thalamic networks
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Group assignments |
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Monday |
Tuesday |
Wednesday |
Thursday |
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A. Craig |
1 |
2 |
3 |
4 |
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B. Tony |
2 |
3 |
4 |
5 |
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C. Ricardo |
3 |
4 |
5 |
6 |
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D. Shaul |
4 |
5 |
6 |
1 |
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E. Rich |
5 |
6 |
1 |
2 |
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F. John |
6 |
1 |
2 |
3 |
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Ø
12:30-1:00
PM: Lunch
Ø
1:00-3:30
Neural Imaging or Electrophysiology Lab
Ø 3:30-4:00 PM: Tea Break
Ø 4:00-6:00 Neural Imaging or Electrophysiology Lab
o In electrophysiology lab the last hour will be dedicated to analysis of the days results
Ø
6:00 to 7:00
Dinner and Faculty Talk
o
Craig Garner, PhD
Tuesday, September 18
Ø
8:00-9:45
Lecture 8: Calcium Signaling
Ricardo Dolmetsch, Ph.D
o Voltage-dependent calcium channels
§
Subtypes
·
Genetics
·
Physiology
·
distribution
o Calcium channels and neuronal signaling to the nucleus
Ø
10:00-12:30
Neural Imaging or Electrophysiology Lab
Ø
12:30-1:00
PM: Lunch
Ø
1:00-3:30
Neural Imaging or Electrophysiology Lab
Ø 3:30-4:00 PM: Tea Break
Ø 4:00-6:00 Neural Imaging or Electrophysiology Lab
Ø
6:00 to 7:00
Dinner and Faculty Talk
o
Stefan Heller, PhD
Wednesday, September 19
Ø
8:00-9:45
Lecture 9: Hodgkin, Huxley and beyond.
Tony Ricci,
Ph.D.
o Understanding the molecular basis of action potential generation, as correctly inferred by Hodgkin and Huxley's analysis of electrogenesis of squid giant axon
o How action potentials and excitability is dictated by variability in underlying conductances
Ø
10:00-12:30
Neural Imaging or Electrophysiology Lab
Ø
12:30-1:00
PM: Lunch
Ø
1:00:3:30
Neural Imaging or Electrophysiology Lab
Ø 3:30-4:00 PM: Tea Break
Ø 4:00-6:00 Neural Imaging or Electrophysiology Lab
Ø
6:00 to 7:00
Dinner and Faculty Talk
o
o
Assignments for Friday Presentations
o
Outline of Power Point presentations of
DATA
§
Objectives
§
Rationale
§
Methods and Results (from your group)
§
Conclusions
Thursday, September 20
8:00-9:45 Lecture 10: Cellular Mechanisms of Synapse
formation
§
Craig Garner, Ph.D.
o
Principles of synaptic
structure, assembly and function
o
Synaptogenesis
Ø
10:00-12:30
Neural Imaging or Electrophysiology Lab
Ø
12:30-1:00
PM: Lunch
Ø
1:00-3:30
Neural Imaging or Electrophysiology Lab
Ø 3:30-5:00 President’s Reception for New Graduate students.
Ø
5:00-6:00 Data preparation for Friday presentations
Ø
6:00 to 7:00
Dinner and Faculty Talk
o
Isabella Graef
Ø
7:00-9:00 Data preparation for Friday presentations
Friday, September 21
Ø
8:00-9:45
Lecture 11: Role of ion channels in behavior
Miriam
Goodman, Ph.D.
Ø
10:00-12:00
Student DATA presentations part 1
Ø
12:00-12:45 PM:
Lunch
Ø
12:45-6:00
Student DATA presentations part 2
Ø
6:00 to ?
o
BBQ and Party!