pCLAMP workshop
Jeffrey Tang, PhD
2013©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Axon Conventional Electrophysiology Family• pCLAMP software• Clampex-data acquisition• Clampfit-Data analysis• Axoscope-Data continuous
monitoring• Digitizer• Digidata 1440A-Analog-to-Digitalconverter• Patch-clamp amplifiers• Axopatch 200B
• MultiClamp 700B
• AxoClamp 900A

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Flow of recording signal
Amplifiers
Biological signals:
current and voltage

Axopatch 200B

Axoclamp 900A MultiClamp 700B

DigitalConversion

Digidata 1440A

Digitizer
Software©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

pCLAMP 10
SoftwareElectrophysiology rig©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Measurement Techniques• Extracellular voltage recording• Single unit and field potential recording in brain or brainslices, electrocardiagrams, encephalograms andoculograms• Voltage clamp• Voltage is held constant and the current passing throughthe cell membrane is measured• Patch-clamp recording• Two electrode voltage clamp• Voltage is held constant through one electrodes andcurrent is measured with the other electrode• Current clamp• Current is held constant and the corresponding membrane
voltage of the cell is measured• Intracellular sharp electrode recording• Measurement of action potentials• Bridge balance for the pipette resistance• Discontinuous clamp• Amplifier divides its time between passing current andrecording voltage• Ion-selective electrodes and electrochemistry• Measurement of small changes in ion, neurotransmitterand hormone concentrations in tissues and cells©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

MolDev Axon CEP Products

The position of Molecular Devices• Market leader• Large install base (17,000)• Axon brand•over 25 years• High quality

ultra low noise amplifiers• High speed, low noise
digitizers•• Academic research labs
• Pharmaceutical/biotechnology drug
discovery labs
• >10K amplifiers sold• >10K digitizers sold• >10K copies of pCLAMP• Reliable•

Lasting in a good condition
for many years• Technical support•

Professional assistance©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Customer support activities• Scientific conferences• Society for Neuroscience• Biophysical Society• FENS• Online webinars• Getting the Most Out of
pCLAMP series• pCLAMP Workshops atuniversity• Duke University• University loaner
program/Support training
courses• Cold Spring Harbor marine
biology lab• Woods Hole marine biology lab• Australian Course in Advanced
Neuroscience (ACAN)• Patch clamp workshop in
Singapore• More…

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

A Walkthrough of Protocol Editor in
Clampex©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Golden Tips that help
• Study manual• Study manual again• Ask someone who uses pCLAMP• Clampex tutorial• Online HELP
• Knowledge base• Webinar tutorials• Technical Support©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Agenda• Acquisition Mode
• Inputs• Outputs• Trigger• Statistics• Comments• Math• Waveform• Stimulus©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Feature Highlights• Acquisition modes• Scope trigger• Holding level overrides• Digital OUT holding pattern• Stimulus file
• Digital bit pattern• Pre-sweep train• P/N leak subtraction• User list
• Membrane test between sweeps©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Telegraphs• Clampex can receive and incorporate arrangeof “telegraphed” amplifier settings as thevariable gain, lowpass filter, whole-cell
capacitance compensation.

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Telegraph setting in AxoPatchTM 200B

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Telegraph setting in AxoPatchTM 200B

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Telegraph setting in MultiClampTM 700B

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Lab Bench setting in AxoPatchTM 200B

Voltage Clamp

1. To define signals for each digitizer channel
2. To set units and scale factors©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Lab Bench setting in AxoPatchTM 200B

Current Clamp

1. To define signals for each digitizer channel
2. To set units and scale factors©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Lab Bench setting is telegraphed in
MultiClampTM 700B

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Protocol Editor

Protocol Editor:
is a the “central” for protocol writing.
©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Acquisition Mode?

What? Signals
How fast? Sampling rate
How long? Sweep duration©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Mode/Rate• Passive Acquisition• Gap-free• Fixed-length• Variable-length• High-speed oscilloscope• Stimulus Acquisition• Episodic stimulation©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Gap-free Mode

• Data are acquired continuously• No gaps in data file• single-channel or minis recordings©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Fixed-length Events Mode

• Constant data segment above the thresholdand pre-trigger portion are recorded• Action-potential spikes or other constantwidth events recordings©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Fixed-length Event Mode

Threshold

SignalPre-trigger length©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Variable-Length Events Mode
• Data above the threshold, and for pre- andpost-trigger periods are recorded.
• Variable recorded segment• “Bursting" data recording©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Variable-Length Events Mode©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

High-Speed Oscilloscope Mode

• Like an oscilloscope• Data above the threshold, and for pre- andpost-trigger periods are recorded.

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Passive acquisition modes
Check this option to acquire data until the disk is full, or until
acquisition is stopped manually

the duration of the data acquisition. in megabytes,
and in hours, minutes and seconds
Specify the sampling rate of theanalog-to-digital conversion
Enable Configure / Automatic Analysis in Clampfit tohave new files automatically open in Clampfit.

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

High-speed Oscilloscope Mode

Check this option to save the raw sweeps andaverage sweep©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Episodic Stimulation Mode

• Provide a command waveform and record
responses, in fixed-length sweeps• Analog waveform, holding level and/or digitalpulses are outputs• Special features include pre-sweep trains, online
leak current subtraction, online event detection
and statistics, and an on-line derived-mathchannel.

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Episodic Stimulation Mode

Recorded
Current

CommandVoltage©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Episodic Stimulation Mode

Specify a delay inseconds before the trial
begins.

Specify how many runs to execute in the trial, up to
10,000. Runs are always averaged, with just the
average run being saved to the data file.

Set the number of sweeps in each run,up to 10,000.

Enter the sweep duration in seconds, The
maximum number of samples is 1,032,258
per sweep per channel.

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Episodic Stimulation Mode---Holding
Periods
Two "holding" periods at the start and endof each sweep, each 1/64th of the total
sweep duration.

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Episodic Stimulation Mode
1.
2.

Specify the time (in seconds) from thestart of one sweep to the start of the next.
The time entered must be greater than
the time it takes to complete a sweep.
This includes the time for P/N Leak
Subtraction and Pre-sweep Trains.

1 st sweep

2 nd sweep

Start-to-Start Interval©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Inputs©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Q&A• Q: Why the selected input name in the Lab
Bench does not show up in the scope
window?
• A: You need to select the input name in the
Input tab of the Edit Protocol.

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Outputs

Voltage-clamp
Current-clamp
Check to specify one digital output
channel to go high (and stay high)during the entire length of a trial©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Digital Out Holding Pattern©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Holding Level Overrides©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Holding Level Overrides©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Holding Level Overrides©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Q&A• Q: How do I change the command voltage formore than 200 mV• A: You need to change the scale factor andexternal sensitivity of analog output signal on
the Lab Bench©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Scale Factor/ External Command
Sensitivity©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Scale factor/Output©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Q&A• Q: I want to perform current clamp experiment. How do Ichange the command voltage to current• A: You need to change the signal unit and scale factor on the
Lab Bench.

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Trigger• Immediate• As soon as you choose Acquire / Record or
Acquire / View Only, or press the equivalent
toolbar button, the trial starts.

• Digitizer START Input• To trigger the start of a trial from anexternal device.

• Space Bar• Start the trial with a toolbar press or mouseclick.

• Line Frequency• Only for series 132x Axon digitizers• synchronized with the mains line frequency(typically 50 or 60 Hz).

• Software Message• To trigger the trial from the other program©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Trigger• Internal Timer• Determines acquisition according to thelength of the sweeps and Start-to-Start
Intervals and any Conditioning Trains or
P/N Leak Subtraction• First Acquired Signal• Once a threshold in the signal from thefirst Analog IN Channel is crossed.

• Digitizer START Input• To trigger the start of a trial from anexternal device.

• Space Bar• Acquisition is started by pressing thespace bar

• Line Frequency• Only for series 132x Axon digitizers• Synchronized with the mains line
frequency (typically 50 or 60 Hz).

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Scope Trigger• A 5 V TTL trigger signal from a digitizer
output when the signal reaches thethreshold.
• The rear panel TRIGGER OUTPUT BNC (Digidata
1322)• The front panel SCOPE BNC (Digidata 1440)©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Scope trigger
scope

Trigger

DD1440A©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Stimulator

Hysteresis• To prevent signal noise activating
false triggers, you can adjust thehysteresis settings to reduce
trigger sensitivity.
• Time hysteresis• This field adjusts the amount of time that thesignal has to be under the threshold level (asdetermined by the Level Hysteresis setting
below) to re-arm the trigger.

• Level hysteresis• This field adjusts the distance that the signal
must be under the threshold in order to re-arm
the trigger (subject to the Time Hysteresis©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Time hysteresis
rejectsignalnoise• Adjust the time hysteresis to reduce
sensitivity to avoid the false triggers.

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Threshold-Based Statistics• Percentage above threshold• Display the percentage of time that the signal is above thethreshold value• Event frequency (Hz)• Display the frequency of threshold-crossing• Mean open (ms)• Display the average time that the signal is above threshold.

• Mean closed (ms)• Display the average time that the signal is below threshold.

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Threshold-Based Statistics©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Statistics©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Statistics
B

B

1©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

1

Statistics
B
B

1

20%

50%

80%

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

1

Rise

Decay

Rise

Decay

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Q&A• How many search regions can be applied during dataacquisition?
• How?

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Comments• The comments can be written automaticallyto every data file
• The comments are stored with data fileinformation in the file header, viewed from
File / Properties, and can also be displayed in
the Data File Index.
©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Comments©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Math• Allows arithmetic calculation between two
analog input signals• Calculate on-line ratios, products, sums anddifferences.
©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Math©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Waveform

112 mV92 mV
72 mV

100 ms

0 mV©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Epochs©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Default waveforms

Step:

Ramp:

Pulse
train:©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Default waveforms

Biphasic
train:

Triangle
train:

Cosinetrain:©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Stimulus File©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Stimulus File©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Stimulus File©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Stimulus File©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Stimulus File©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Action Potential Waveform©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Digital Outputs©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Digital Bit Pattern

Digital bit pattern (#3-0): 0101

DO#3 DO#2

1 High, single TTL (5V)0 Low, No single TTL (5V)
an asterisk,* A pulse train of TTL (5V)

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

DO#1 DO#0

Digital Bit Pattern---Single Pulse
Digital bit pattern (#3-0): 0101

DO#0:
DO#1:
DO#2:
DO#3:

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Digital Bit Pattern---Train Pulse©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Digital Bit Pattern---Train Pulse©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Digital Bit Pattern©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Intersweep Holding Level©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Intersweep Holding Level---Use lastepoch

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Intersweep Holding Level---Use
Holding©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Alternative Waveform

CMD 0
CMD 1

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Alternative Waveform

CMD 0
CMD 1

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Alternative Waveform©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Alternative Digital Outputs

DO# 0

DO# 1

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Alternative Digital Outputs©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Pre-sweep Train• A pre-sweep train consists of repeatedsquare waveform pulses• Conditioning trains• No acquisition occurs©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Pre-sweep Train©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

P/N Leak Subtraction• Leak subtraction corrects for the passive responses byapplying test subsweeps before or after the main
stimulus waveform• The technique is called P/N subtraction, in which N
subsweeps each 1/Nth of the amplitude of the main
stimulus waveform are applied.
• Example: Number of subsweep (N)=4, the stimulus
waveform (P)= 80 mV, the pulse of eachsubsweep=80/4=20 mV©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

P/N Leak Subtraction©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

User List
• Customizing output features• Analog• digital• Overrides the generalized settings made elsewherein the Protocol Editor.

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

User List---Holding Level
Sweep 1: -50 mV
Sweep 2: 65 mV
Sweep 3: -20 mV
Sweep 4: 100 mV
Sweep 5: 29 mV

User List

You can set the arbitrary delta values
for the waveform holding levels.
©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

User List---Epoch Duration
Sweep 1: 22 ms
Sweep 2: 44 ms
Sweep 3: 100 ms
Sweep 4: 75 ms

You can set the arbitrary delta values
for the waveform sweep durations.
©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

User List---Change in Parameters• Epoch A–J level• Epoch A–J duration• Epoch A–J digital pattern• Epoch A–J train period• Epoch A–J train pulse width• Time between sweep starts (s)• Inactive analog OUT holding level• Digital intersweep holding level• Number of P/N subsweeps• Number of pulses in pre-sweep
train• Pre-sweep train baseline duration(ms)• Pre-sweep train baseline level• Pre-sweep train step duration (ms)• Pre-sweep train step level• Post-train duration (pre-sweep
train) (ms)• Post-train level (pre-sweep train)©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Lists of parameter values• The list of values for the A Epoch level might be:• -50, 65, -20, 100, 29• The list of values for the A Epoch duration might be:• 22, 44, 100, 75©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Membrane Test Between Sweeps

1st sweep

1s

2nd sweep

1s

1s

3th sweep

1s
100 ms

100 ms1s
100 ms= Membrane test

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

100 ms

pCLAMPTM 10 data acquisition software: Key features review©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Key features of Clampex 10• Online Statistics• Sequencing keys
• User list
• Membrane test between sweep• Protocol editor• LTP assistant• Stimulus file
• Triggering external devices©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Online Statistics• Measure various parameters of evoked events suchas peaks, slopes, areas, and rise time
• Available in episodic and oscilloscope modes• Up to 8 search regions©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Outward signal
Peak

Inward signal
Baseline

*
Slope
Slope

Baseline*
Example:Excitatory postsynaptic potential (EPSP)

Peak

Example:Excitatory postsynaptic current (EPSC)
Population spikes
Field EPSP

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Peak Current (pA)0

Tetanus

Slope (pA/ms)

Time (min)Baseline

Slope*

Tetanus0

Peak©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Time (min)

Peak

EPSC

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Rise slope©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Click “Help” forfeature description©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Sequencing Keys

• Sequencing keys command allows you associate events, or asequence of event, with a keystroke.
•
•
•
•
•
•

Set various digital outputs
Change the holding levels
Insert a comment tag
Start a Membrane Test
Load or run a protocol
Display a prompt• Use sequencing keys to link one event to another, and run anexperiment in an “automated” fashion.

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

An experimental protocol
START
Membrane test
Protocol A
Drug A/
Digital Output “0” ON

Protocol B
Prompt/Wait for 5 s

Protocol C
Membrane test
END
©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Sequencing Keys
• The key strokes and tool button identifiers used for sequencing keys
are:
• <Alt + 0> through <Alt + 9> (i.e. hold down the Alt key and press therelevant numeral)• <Alt + Shift + 0> through <Alt + Shift + 9>
• <Ctrl + 0> through <Ctrl + 9>
• <Ctrl + Alt + 0> through <Ctrl + Alt + 9>
• <Ctrl + Shift + 0> through <Ctrl + Shift + 9>

• 50 sequencing keys can be triggered in one time.

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Set sequencing keys
START

Ctrl 1

Membrane test

Ctrl 2

Protocol A

Ctrl 3

Drug A/
Digital Output “0” ON

Ctrl 4

Protocol B

Ctrl 5

Prompt/Wait for 5 s

Ctrl 6

Protocol C

Ctrl 7

Membrane test

END
©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Sequencing Keys
• Only the key for the first element needs to be
triggered to start off the entire sequence.

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Run a protocol in an automated fashion
START
Ctrl 1

Membrane test

Ctrl 2

Protocol A

Ctrl 3

Drug A/
Digital Output “0” ON

Ctrl 4

Protocol B

Ctrl 5

Prompt/Wait for 5 s

Ctrl 6

Protocol C

Ctrl 7

Membrane test

END
©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

User List
• Provide a way of customizing one of a range of
analog and digital output features.
• It overrides the generalized settings madeelsewhere in the Protocol Editor.

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

User List
• With User List you can select one of a range ofparameters offered, to configure on a sweep-bysweep basis.
• This function allows you to set specific values foreach sweep in a run. You can also set a sequenceof values and have this repeat.

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Change in Epoch holding levels
Sweep 1: -50 mV
Sweep 2: 65 mV
Sweep 3: -20 mV
Sweep 4: 100 mV
Sweep 5: 29 mV

You can set the arbitrary delta values
for the waveform holding levels.
©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Change in Epoch holding levels
Sweep 1: -20 mV
Sweep 2: 100 mV
Repeat…

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Change in Epoch durations
Sweep 1: 22 ms
Sweep 2: 44 ms
Sweep 3: 100 ms
Sweep 4: 75 ms

You can set the arbitrary delta values
for the waveform sweep durations.
©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Change in Epoch durations
Sweep 1: 22 ms
Sweep 2: 75 ms
Repeat…

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Parameter to Change• Epoch A–J level• Epoch A–J duration• Epoch A–J digital pattern• Epoch A–J train period• Epoch A–J train pulse width• Time between sweep starts (s)• Inactive analog OUT holding level• Digital intersweep holding level• Number of P/N subsweeps• Number of pulses in pre-sweep train• Pre-sweep train baseline duration (ms)• Pre-sweep train baseline level• Pre-sweep train step duration (ms)• Pre-sweep train step level• Post-train duration (pre-sweep train)(ms)• Post-train level (pre-sweep train)©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Examples: List of Parameter values• The list of values for the A Epoch level might be:• -50, 65, -20, 100, 29• The list of values for the A Epoch duration might be:• 22, 44, 100, 75©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Change in Epoch holding levels©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Change in Epoch durations©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Membrane Test Between Sweeps©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Membrane Test
• Access resistance, Ra• Membrane resistance, Rm• Membrane capacitance, Cm• Time constant, Tau• Holding current, Hold©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

1st sweep

1s

2nd sweep

1s

1s

3th sweep

1s
100 ms

100 ms1s
100 ms= Membrane test

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

100 ms©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

.sta©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Summary

Online statistic measurement

Membrane Test Between
Sweeps

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Stimulus File
• Stimulus Files are used when the Waveform Editor
cannot create the desired waveform.

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Default waveforms

Step:

Ramp:

Pulse
train:©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Default waveforms

Biphasic
train:

Triangle
train:

Cosinetrain:©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

How to create an action potential
waveform?

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Advanced Tips for Protocol Writing in the Clampex Data
Acquisition Module©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Whole cell and Extracellular Recordings

Electrode 1:
Intracellular

Electrode 2:
Extracellular

Presynaptic
Stimulation

Presynaptic
Stimulation

Postsynaptic
current

Postsynaptic
fEPSPsPresynaptic
cell

Postsynaptic
cell©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Pair-pulse stimulation

50 ms

Application: Synaptic plasticity, release probability
in brain slice preparation©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Pair-pulse Stimulation: Presynaptic afferents

Electrode 1:
Intracellular

Electrode 2:
Extracellular

Presynaptic
Stimulation

Presynaptic
Stimulation

Postsynaptic
current

Postsynaptic
fEPSPs

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Pair-pulse stimulation

50 ms©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Train stimuli
10 Hz

1 sec

Application: Synaptic plasticity, LTD
in brain slice preparation©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Train stimuli-10 pulses

10 Pulses©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Train stimuli-50 pulses

50 Pulses©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Summary• Telegraphing/Lab Bench settings• Writing protocols for• Pair-pulse stimulation/Digital Output• Train stimulation©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Tetanus Stimulation protocol

100 HzPresynaptic stimulation

1sec

10 secPostsynaptic holding 0 mV
potential-80 mV

Application: Synaptic plasticity, LTP/LTD
in brain slice preparation©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Tetanus Stimulation and Postsynaptic
Depolarization

LTP:
Pre-stimLTD:

Post-stim

Pre-stim

100 HzPresynaptic stimulation
1sec

10 secPostsynaptic holding 0 mV
potential-80 mV©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Post-stimTetanus Stimulation and Postsynaptic
Depolarization©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Spike timing protocol

Current
Clamp

Current
Clamp

Presynaptic
stimulation
Postsynaptic
stimulation

10 ms

Application: Synaptic plasticity, LTP/LTD
in brain slice preparation and culture cell©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Dual Whole-cell Patch-clamp Recordings

Current
Clamp

Postsynaptic
cell©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Voltage
Clamp

Dual Whole-cell Patch-clamp Recordings
Presynaptic
Stimulation

Postsynaptic
current

Presynaptic cell
(Current-clamp)©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Postsynaptic cell
(Voltage-clamp)

Spike timing protocol: EPSP precedes Action
Potential

10 ms later

DendriteAll about the “Timing”:
The change of synaptic efficacy of thepostsynaptic cell depends upon
Postsynaptic cell
1. the timing of the incoming inputs
2. the excitability of the postsynaptic cell (under Current Clamp)©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Spike timing protocol: EPSP precedes Action
Potential

Current
Clamp

Current
Clamp

Presynaptic
stimulation

Postsynaptic
stimulation

10 ms©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Spike timing protocol: EPSP precedes Action
Potential

10 ms©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Spike timing protocol: Action Potential
precedes EPSP

10 ms later

Dendrite

Postsynaptic cell
(under Current Clamp)©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Spike timing protocol: Action Potential
precedes EPSPCurrent
Clamp

Current
Clamp

Presynaptic
stimulation

Postsynaptic
stimulation

10 ms©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Spike timing protocol: Action Potential
precedes EPSP

10 ms

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Action Potential Analysis in Clampfit©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Frequent Ask Questions in Action Potential Analysis• How can I measure the frequency of action potentials?
• The baseline of my recording is drifting. It is hard to analyze thespikes.
• How do I sort the spikes?
• My recording is too noisy. It is hard to retrieve the spikes from thenoises?
• When I do the event search, how can I avoid the large spike ofstimulation artifacts?
• How do I perform a phase plot of dV/dt vs V in Clampfit?
• How do I calculate the cardiac action potential duration at 90%
repolarization (APD90%)?

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

What we learn today• Baseline adjustment• Event Search• Event sorting• Noise/Event rejection• Spike alignment• Combine trace• Phase plot
• Action potential analysis©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Features in Clampfit• Baseline adjustment• Manual baseline adjustment• Search event• Event Detection/Threshold Search• Event sorting• Event Detection/Threshold Search• Spike alignment• Time shift• Phase plot/Combine trace• Arithmetic• Action potential analysis• Statistics©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Baseline adjustment

Drifting baseline

Stable baseline©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Event Search

Trigger level
Baseline level

If an event crosses the trigger level, the event will be accepted.

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Event Sorting• Extract events from a gap-free file and transform them into
sweeps.

Gap-free files©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Sweep files

Noise/Event Rejection• Event rejection based on amplitude• Noise rejection based on short duration• Event rejection based on event length©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Noise/Event Rejection based on amplitude

RejectRejection level
Trigger level
Baseline level
If an event crosses the rejection level, the event will be rejected.

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Noise/Event Rejection based on too short duration

AcceptRejectNoise duration in ms can be set to reject noise spikes.

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Noise/Event Rejection based on event duration

RejectTrigger level
Baseline level

You can select Min allowed duration to eliminate short events.
You can select Max allowed duration to discard events that are too long.

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Peak Alignment©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Combine traces+

Trace in File A=

Trace in File B©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Trace combined

6/20/2013

Pg. 171

Phase Plot
• Plot dV/dt versus V©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Cardiac Action Potential Analysis• Resting membrane potential(RMP)• Action potential Amplitude(APA)• Action Potential Duration(APD)• Max Slope of depolarization(Vmax)APA
Vmax

APD50%APD90%
RMP©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Event Detection/Threshold Search©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Set category• Each category represents a
unique search criterion. There
are up to 8 different
categories• The positive- and negativegoing describe the location ofpeak relative to baseline.
• Action potential is a positivegoing event©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Bring markers• Bring markers• Baseline marker• Trigger marker

Trigger marker

Baseline marker©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Re-arm
Start ofan event

End ofthe event
TriggerBaseline

End ofthe event

Trigger

Re-armBaseline©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Event Rejection

R

R

R

Rejection level
Trigger level
Baseline level©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Noise Rejection

Event Start and End
without Noise rejection©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Event Start and End
with 5 ms Noise rejection

6/20/2013

Pg. 179

Pre-trigger and Post-trigger

5 ms

20 ms

Trigger

TriggerBaseline

Baseline©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Event Rejection

TriggerBaseline

R

R

R
R

R

R
TriggerBaseline©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Accept, Reject, Suppress, Accept Tag

A

A

R

R

S

S

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

AT

AT

AT

Event States

A

A

R

R

S

S

AT

AT

Event Detection/Show Suppressed Events©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

ATPhase Plot

Select the trace

Transfer trace

ColumnArithmetic
cC=diff(cB)©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Plot dV/dt vs V

Summary• Baseline adjustment• Event Search• Event sorting• Noise/Event rejection• Spike alignment• Combine trace• Phase plot
• Action potential analysis©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Analysis of Synaptic Events with theClampfit Data Analysis Module©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Agenda•Postsynaptic miniature currents (EPSCs)
50

0Pre-drug

-50

IN 0
(pA)

•

-100

-150

Minis02_Predr

-200
12.5

13

ug.abf13.5
Time (s)0-50Post-drug

-100

IN 0
(pA)

•

-150

-200

-250
34

• Are there differences in the amplitude distribution?
• Post-synaptic mechanism• Are there differences in the frequency?
• Pre-synaptic mechanism©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

34.5
Time (s)35

Minis03_Postdrug.
abfStrategy

Create a template fromone of the data files

Perform a template search on a data
segment before addition of compound…

…and on a dataset after
addition of compound©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Strategy

Create cumulative histograms of
the peak amplitudes…

…and the inter-event intervals.

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Strategy

Perform a statisticalanalysis to determine
whether there aredifferences between
the two datasets.

p = 15%
K-S = 7%p < 10-6
K-S = 43%

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Clampfit Features Discussed• Event Detection > Create Template• Event Detection > Template Search• Move Cursors efficiently• View > Zoom > Between Cursors• Analyze > Arithmetic• Format > Column > Rename• Format > Rename Sheet• Analyze > Histogram• Efficiently creating a graph using X-Y pairs• Editing a graph• Analyze > Kolmogorov-Smirnov Test
• Analyze > Basic Statistics©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Creating a Template

Zoom in to
the first
few
seconds[Shift] + [Click]brings the cursors©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Creating a Template
Enclose an event with
cursors 1 and 2Continue adding events
to generate asmooth template

When done, savethe template©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Delimit the Analysis Region

Full-scalethetime axis,

and send cursor 1…

…“Home” to the startof the trace©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

unlockcursors 1
and 2,

Delimit the Analysis Region

Doubleclick on
Cursor 2,

send it to
30 s,…and zoom theregion we aregoing to analyze©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Template SearchStart Template Searchandload the newly createdtemplate©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Template Search

Define the measurement
region.

Set taus and analysis
region.

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Template Search©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Defining “Sanity-check” Graphs

This graph Indicates theabsence of an overall trend(run-up or run-down ofamplitude or frequency).

This graph suggests that
there are not two (or more)classes of events with clearly
distinct amplitude.

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Copying the Pre-compound Results to a
New Sheet©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Computing the Absolute Peak AmplitudecBH = cH * (-1)
yields the absolutepeak amplitude

Double-click on thecolumn header torename©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Creating a Cumulative Peak Histogram©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Repeat the Template Search After
Compound Addition

Starting Template Search with a
new file erases all results on
the Events sheet (but not
Sheet 19 )©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Post-compound Results

The post-compound data fileafter completed analysis with“sanity check graphs”.

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Repeat Post-processing Steps
Copy the results to Sheet 20,
rename the sheet and column H
Compute theabsolute peakRename the otherrelevant columns

Create a post-compound
cumulative histogram©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Normalizing the Histogram Values
Normalize the Bin Count columnson the Histogram sheet

Rename the two new columns

cE = norm(cB)
cF = norm(cD)©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Creating a Combined, Normalized
Cumulative Histogram
Make the first “BinCenter” column an X
columnMake the “Norm Pre
Peak” column a Ycolumn©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Define the twocolumns as an XY pair

Creating a Combined, Normalized
Cumulative Histogram
Repeat for the second “BinCenter” column and “Norm
Post Peak”

Create a new graphand double-click on it©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Creating a Combined, Normalized
Cumulative Histogram
Modify Graph titles, axis
titles and plots for an
appearance as shown©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Kolmogorov-Smirnov Test

Copy both “Abs Peak”
columns

to Sheet 18

Apply the KolmogorovSmirnov Test
©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Kolmogorov-Smirnov Test Results
15% probability that the tested
samples are from the same
universe => no statisticallysignificant difference

The Kolmogorov-Smirnov Statistic
value corresponds to the greatest
Y difference between thehistograms:©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Creating Histograms for Inter-event
Intervals
Create Histogram(x2)

cK = norm(cH)
cL = norm(cJ)©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Creating Histograms for Inter-event
Intervals
Define X-Y pairs(x2)

Create a graph
Modify the graph©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Kolmogorov-Smirnov Test for Interevent Intervals
Copy Pre and Post
IEI columns to
Sheet 18

Be sure to Append,
not Replace theresults©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Kolmogorov-Smirnov Test Results
p<10-6 => highly statisticallysignificant difference between the
IEIs recorded in the two conditions

The Kolmogorov-Smirnov Statistic
value again corresponds to thegreatest Y difference between thehistograms:©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Retain the Kolmogorov-Smirnov Results

Copy the results ofthe two K-S tests
to Sheet 17©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Basic Statistics for Peaks and Interevent Intervals©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

Summary• Are there differences in
the amplitude
distribution?
• No.
• Post-synaptic
mechanisms areunaffected.

• Are there differences in
the frequency?
• Yes.
• Pre-synaptic
mechanisms are
upregulated.

©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

p = 15%
K-S = 7%p < 10-6
K-S = 43%

Clampfit Features Discussed• Event Detection > Create Template• Event Detection > Template Search• Move Cursors efficiently• View > Zoom > Between Cursors• Analyze > Arithmetic• Format > Column > Rename• Format > Rename Sheet• Analyze > Histogram• Efficiently creating a graph using X-Y pairs• Editing a graph• Analyze > Kolmogorov-Smirnov Test
• Analyze > Basic Statistics©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

End
Thank You©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.

www.moleculardevices.com©2012 For research use only. Not for use in diagnostic procedures. Trademarks mentioned herein are property of Molecular Devices, LLC or their respective owners.