In this laboratory, traditional (old) and new equipment will be set up to demonstrate:
(1) principles of recording
(2) limitations solved by more modern equipment

History
(1) With the smoked kymograph, the frog nerve-muscle preparation would use a stylus to scratch into paper that was covered with soot using a wing-tipped bunsen burner
(2) the record was then dipped in shellac for preservation

For measuring Voltage, we have the Volt meter. Try it.

The "polygraph" displays voltage as a function of time

On the amplifier, the following will be demonstrated:
(1) position
(2) gain [mV/cm]
(3) time constant
(4) filter

Trace on physiograph shows 1 mV calibration pulse:
(1) square wave [DC]
set mV/cm to 10 and multiplier to 10 (that comes to 1 mV/cm)
(2) low-pass [high-cut-off] filtering ["filtering Hz"]
(3) high-pass [low-cut-off] filtering ["time constant"]

For the sake of completion, the bottom of this sheet shows the circuitry and math for RC filtering, and a few of you may be interested in this, though I certainly will not hold you responsible for this.

I have set up some "electrodes" that can be gripped in each hand to get an EKG (ECG = electrocardiogram).
(1) set mV/cm to 2, multiplier to 10, time constant to 0.3 and Filter Hz to 3.
(2) doesn't look as good as the computer EKG, does it?
(3) vary the settings to see why I suggested these starting values

Several students in the Fall, 2004 lab got pretty nice EKG's from the polygraph, and here is a good one.

The oscilloscope
(1) graphs voltage as a function of time
(2) as a blue dot with a green afterimage
(3) here, the sweep is started by external input on the time base amplifier [right]
(4) the gain is set on the differential amplifier [middle] (Volts/division)

Problems with the oscilloscope (solved by more modern computers)
(1) how to save image:
here the Grass (brand) camera is shown, and a blue filter captures the blue dot
(2) how to get the response in the center of the screen
here the Grass (brand) stimulator synch out (delay) and the delay feed the oscilloscope's time base
(3) I am showing you a tank for developing 100 foot rolls of film with dark room chemicals
Inside there are spools for the film.

Microelectrodes (micropipettes) [Other researchers call these "needles" and use them to inject DNA for transformations.)

We will make micropipettes
(1) We use a Narishige PD-5 (Tokyo) horizontal puller with controls for an early magnet, a heater, and a late (stronger magnet).
(2) The heater glows red while the first magnet pulls gently.
(3) A microswitch with a shim detects the melt and the early pull to kick in the harder pull.
(4) After the second pull, two electrodes are made.

We will fill micropipettes
(1) Over the history of micropipettes, many tricks have been developed to get the very narrow tip to fill. Currently, a capillary tube with an inner filament has magic filling properties.
(2) First you back fill the butt end a little with a spinal tap needle.
(3) The electrolyte is carried to the tip. Then, you can finish back filling the elecrode with the syringe.

We will test micropipettes
(1) On the electrometer (microelectrode amplifier), position, electrode check and capicity compensation will be demonstrated.
(2) You will be provided with a micromanipulator to insert electrode (in this case simply into a grounded block of agar)
(3) We will need to introduce some simple features of the Powerlab and the T-chart program, selecting a channel, setting the range, and starting and stopping a record. (Many more features will be demonstrated in later labs.)
(4) Then we can test the electrode resistance and demonstrate the "ringing" (used by some single cell electrophysiologists to get the electrode into cells) that comes from overcompensation of the capacity.

I work on Drosophila, recording extracellularly in the eye using the ERG (electroretinogram), and here is a picture of a fruit fly with an electrode in its eye.

Here is another example of electrophysiology used in my research. Here is an outline of my work on rescue from blindness in a mouse model of Batten's disease using stem cells, and these data indicate that the stem cell injected eye has a better ERG than the sham injected eye.

Text

Most of the apparatus discussed below will be demonstrated, but only the most fundamental is on the web hyperlinks (above). After an initial demonstration the groups of lab partners will rotate to get their hands on these most fundamental pieces of apparatus in several separate work-stations. While trying to be clear and concise (below), I do not hesitate to introduce the students to the jargon electrophysiologists babble.

One of the main properties of life defined in the introductory chapter of any introductory biology book is "excitability." There is no better example in biology than in nerve and muscle cells of animals. As the pedagogy of texts gets more and more advanced, the "cartoons" (diagrams) get further and further removed from the real world. Here, in this laboratory, I want to show you how biologists measure electrical signals (and not to re-hash what is known about electrophysiology).

Way back before electronics, work could be done on the frog nerve-muscle preparation. The nerve could be jolted, and the muscle connected to a stylus. A kymograph was fitted with a piece of paper that was coated with soot from a yellow bunsen burner flame. The stylus scratched a record onto the smoked kymograph drum with the speed of rotation providing time on the X-axis, and the record was preserved with shellac.

A hobbyist or an electrician would measure Voltage with a Volt meter. Bioelectric signals are smaller than the 1.5 V flashlightlight battery (but not all that much smaller). So the "voltmeters" we use must be sensitive enough to measure mV. This is done with amplifiers, and so, in many respects, the input of the physiologist's voltmeter is a differential amplifier to measure voltage difference (say, from inside a cell relative to the extracellular fluid, ground).

Perhaps more important than the greater sensitivity for physiologists is the need to measure Voltage (on the Y-axis) as a function of time to monitor fast changes. The "polygraph" achieves this by drawing a line and rolling the paper out under the line (at various speeds) to put time on the X-axis. If the signal is recorded directly, that is called DC. Although DC is usually interpreted to mean "direct current" (as opposed to alternating current), here it means "direct coupled." If a signal is fast but your ability to see the signal is obscured by slow signals, then a filter composed of a circuit with capacitance and resistance (RC) provides high-frequency-pass (low-cutoff). If fast noise is interfering with recording, then a different type of electronic filter is high-cutoff (low-pass).

The oscilloscope sweeps a dot across the screen at various speeds controlled by the time base amplifier to put time on the X-axis. A stimulator can be used to feed pulses into the oscilloscope for demonstration. The rate of stimulation and the voltage output can be varied. To keep the record on the screen, the position knob and the gain (sensitivity) can be varied. To put the signal in the middle of the screen horizontally, the delay (on the stimulator) measures the time between starting the oscilloscope sweep and the output pulse from the stimulator. The stimulator's "synch out" feeding the time base on the oscilloscope controls this.

The oscilloscope screen needed to be photographed to produce a permanent record. A camera runs film from a roll. I used a blue filter to capture the dot on the oscilloscope (and not get a blurred photograph from the green afterimage of the dot). But then I needed to develop film in a developing tank with chemical developer, fix, rinse, etc.

Nowadays, the face of the computer is used as if it were the face of the oscilloscope or the polygraph paper (and that is what we will use to test electrodes, below). An interface (the Powerlab) is used on the input, and all the functions like gain (sensitivity) and time base are on the computer program. A position knob is provided as "input offset on an additional bridge amplifier. Permanent records are achieved by saving files to disk, Traces can be zoomed, and put into montages using PhotoShop. In short, given enough computer savvy, lots of things can be done easily that used to be difficult.

We will pull fine tipped microelectrodes. These are so small they can be inserted into individual cells. They are filled with a concentrateed salt solution. It would take several paragraphs to say how this used to be done and how hard that was. Eventually, it was found that an inner tube helped in the filling, and, since nobody understood why, I called this "magic glass." 25 years ago, I bought a kilo of magic glass from Germany and that'll probably last me the rest of my life. We need to cutt off pieces the right length, then make and fill electrodes.

Using a micromanipulator, we will put a filled electrode into a grounded block of agar. The amplifier used for microelectrodes has that essential position knob to bring the trace into range. The resistance is checked with the electrode check and it might be10 megOhms. There is a knob for capacity compensation. Because of the high capacitance of glass and the high resistance of the electrode, the electrode itself makes a low pass filter, and the aplifier gives the researcher the opportunity to fudge the signal by adding just the right amount of high pass filtered signal to make a square wave square. If this were not done, fast signals (for instance, an action potential might be only 1 ms) would be filtered out by the high cut off inherent in the electrode's properties.

Last year, there was a quiz, and here it is along with the answers:

Your lecture professor briefly mentioned that intracellular recording electrodes were typically filled with potassium chloride. Why not sodium chloride?

A simple answer to this question is that KCl would more approximately match the high KCl inside cells. Many people had this idea but got confused in how they worded this.

You've filled a microelectrode and you're ready to get started. Typically, you can't just hook a glass microelectrode up to an oscilloscope to record an action potential. State one of the two properties of a microelectrode that force you to use a special microelectrode amplifier.

The high resistance through the fine tip and the high capacitance of glass makes the electrode a low pass filter and it would miss the very fast signals you want to record. Many people said that small (mV range) signals required amplification, and although that is not "one of the two properties of a microelectrode that force you to use a special microelectrode amplifier, I gave half credit."

Your lab professor is showing you a voltmeter, such as your lecture professor showed you as Fig. 5-37. Why can't you use such a device to measure a neural signal? (i.e. Why do you need a chart recorder, a polygraph, an oscilloscope, or a computer instead?)

You need to record fast signals. Again, so many students indicated lack of sufficient sensitivity that I gave half credit, in this case, reluctantly

Pretend the face of the oscilloscope recording an action potential is a graph in an algebra class. What is on the X axis?

Time, typically in ms

What is on the Y axis?

Voltage typically ranging from at least ­p;70 mV to +55 mV

Suppose you were trying to record an ECG (EKG, electrocardiogram). For the purposes of this question, you need to know: (1) there are 5 components, called P, Q, R, S, and T, (2) all 5 occur during each heart beat, (3) resting pulse is typically 72 beats per minute, and (4) from the beginning of the P wave to the end of the T wave might be typically 3/4 of a second. The amplifier of the polygraph is set to DC. Your subject cannot sit still, and the trace keeps going off scale top and bottom every 5 or 10 seconds. What should you do electronically to give a line that stays on scale?

To filter out slow "drift" and still pick up fast changes, use a high pass filter. Some people wanted to change the scale (lower sensitivity) and that would work, though the EKG would be small, so I gave credit.

How do you make fine tipped microelectrodes from glass tubing that is about 1 mm in diameter?

Basically, you melt the glass and pull it apart. I gave half credit for answers pertaining to filling rather than making


This page was last updated 9/10/04

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