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Each lab has a diagram showing how to connect the recording and stimulating equipment required for that lab. When considering how equipment is connected, think of the flow of information from the recording site to your display devices. Electrical current flows from the biological tissue through your electrode to the amplifier and from the amplifier to your display devices (oscilloscope, computer, and audio monitor). This principle applies whether the recording is extracellular or intracellular and whether or not a stimulator is used.
The biological signals you will record are very small, of the same order of magnitude as the many stray electrical signals present in any building. For example, extracellularly recorded action potentials are in the μV range, and intracellular action potentials and synaptic potentials are in the mV range, while stray electrical signals are often in the tens of mV. Since we are interested only in the biological signals, we consider the other signals to be interference or noise and try to prevent them from appearing in our recordings. Thus we want a high signal-to-noise ratio. This goal can be achieved in three ways, listed in order of preference:
Getting a large signal is primarily an issue for extracellular recording. For the best recording with a suction electrode, the nerve must fit snugly in the electrode tip. A tip much larger than the nerve allows an action potential’s current to flow to ground through the saline (a lower resistance path) instead of through the electrode to the amplifier (a higher resistance path), thus reducing the signal you can record. If the tip is too large for the nerve, you can suck the nerve into the tip and then push the tip against a nearby deeper structure (such as a muscle) to seal the opening. If this does not help, ask your instructor for more suggestions.
The Faraday cage and steel plate both shield your recording setup from stray electric fields. Connect them together and connect one of them to the ground point of the oscilloscope or amplifier. Be sure that a ground wire is in the saline bath and connected to ground.
There are several different types of electrical interference. Any object that conducts electricity can bring noise into the recording if it is not grounded. Use alligator clip cables to connect the microscope to the cage to ground it. The manipulators should already be grounded through their magnetic bases, but if noise persists after all other equipment is grounded, try grounding the manipulators too. When using a suction electrode, keep the syringe and tubing inside the Faraday cage. Finally, when doing anything that requires you to bring your hand into the cage, touch a grounded object (such as the steel plate) with the other hand to ground yourself. Remember that your body is also an electrical conductor and potential bearer of noise. Figure C.1 is a simple flowchart to follow when attempting to reduce noise.
If you have a good recording and have effectively taken steps to reduce noise, you should not need to use filtering to get a clean recording. This is particularly true of intracellular recording. You should usually turn filtering off on the intracellular amplifier because it distorts action potential waveforms and offers little benefit (Figure C.2).
In extracellular recordings, filtering can be useful. Most electrical noise is 60 Hz, the frequency of alternating current in power lines (50 Hz in Europe). An extracellular amplifier usually has a notch filter for 60 Hz, and you should usually have this filter turned on. In addition, your amplifier may have settings for high-pass and low-pass filters. A high-pass (low cutoff) setting of 100 to 300 Hz may help and will not hurt. Because action potentials are high frequency events, using the low-pass (high cutoff) filter will distort action potential waveforms as well as reducing high-frequency noise. Experiment with settings of 5 to 20 kHz, going below 5 kHz only if noise is severe. Notice that as you decrease the low-pass (high cutoff) setting, the action potentials in your recordings become smaller and less sharp (Video C.1, Filter Effects).
The most important setting on the extracellular (AC) amplifier is gain. This is the amount by which the signal coming from the electrode is amplified. Most amplifiers offer gains of 100×, 1000×, and 10000×. The 1000× setting is correct for most of the recordings in these labs. If the recording is poor, you may see some improvement by increasing the gain, but the noise level will also increase. If the recording saturates, giving flat tops to spikes instead of rounded spiky tops, try decreasing the gain.
Some amplifiers offer a switch for Stimulation vs. Recording mode. In Stimulation mode, the amplifier is effectively turned off and just passes a stimulus (from a stimulator or computer) through to the electrode. Stimulation mode also protects the amplifier from electrical noise when the electrode is not in place. Keep the amplifier in Stimulation mode until ready to record, then switch to Recording mode.
An intracellular (DC) amplifier usually has two choices of gain, 1× and 10×. This is set by choosing which of two output jacks to use. For these labs, use the 10× output.
The DC level of the amplifier’s output is set by a knob labeled DC Offset or Position. When the electrode is placed into the saline bath before recording, adjust this knob until the output of the amplifier is zero. On some amplifiers, this knob is coupled with a switch for + or −, which determines whether turning the knob makes the output more positive or more negative.
When the Electrode Check button is depressed, the amplifier measures the resistance of the electrode by injecting a 1 nA current through the electrode and recording the resulting voltage change. By Ohm’s law, 1 mV of output from 1 nA of current indicates an electrode resistance of 1 MΩ. If the amplifier has a digital readout, you can read the resistance directly; otherwise you can measure it from the oscilloscope or connect the amplifier’s output to a digital voltmeter. Be sure to turn Electrode Check off before starting to record.
The next set of controls is for injecting a stimulus current through the electrode. The only knob you will use here is the Current or Current Level knob, which is coupled with a switch for polarity (Hyperpolarize, Depolarize, or Off). When not stimulating a cell, be sure to turn this switch to off. Any knobs labeled DC Balance, Stimulus Cancel, or Transient should be turned all the way off (counterclockwise).
Finally, there is a set of controls for capacitance compensation. On most amplifiers, the knob should always be turned all the way off. There should also be a pushbutton labeled Buzz or Cap. Override in this area. This button can be used to get an electrode into a cell when its tip is right at the membrane. See the lab instructions for details.
Although there is a great deal of variation in oscilloscope design, all have certain basic features in common. Every oscilloscope simply gives you a plot of voltage vs. time.
Each input channel has a setting for scale (V/div), input mode (AC/GND/DC), and position. The scale knob sets the y-axis of the display in volts per vertical division. The lab instructions suggest appropriate settings for this value. The input mode determines whether changes in the DC value of the input are shown. For extracellular recording, this should always be set to AC. For intracellular recording, it should start set to DC so you can see the resting potential. Once the recording is made, you may want to set it to AC and change the scale setting to better see activity. Finally, the position knob allows you to determine where zero is shown on the screen. Set the input mode to GND and move the trace with the position knob. Be sure to set input mode back to AC or DC before continuing.
The x-axis scale of the screen is set by the time scale knob (s/div). See the lab instructions for suggested settings. In general, you want a fast time scale (0.2 to 1.0 ms/div) to see details of action potentials and a slow time scale (0.5 to 2.0 s/div) to look at a sequence of events.
Finally, the trigger settings determine when a trace is drawn on the screen. When set to Auto (or Line), the trace is continually redrawn. This is how you will usually use it. When set to Norm (or Trig), the oscilloscope waits until an event occurs on the selected channel before starting a trace. This setting is useful when you want to synchronize the oscilloscope with a stimulus. In that case, connect the stimulus to an oscilloscope channel, select that channel as trigger input, and put the oscilloscope into triggered mode. You may need to adjust the Level knob until your stimulus triggers the oscilloscope.
The computer software that works with A/D boards is even more variable than oscilloscopes. This section covers only the basics common to all such systems.
First, you will need to select a sampling rate. This determines how often the computer samples the voltage from your recording. The faster it samples, the more accurately it can represent a waveform (Figure C.3). However, a fast sampling rate requires more memory and limits the total amount of data that can be collected. To accurately represent the EOD of an electric fish, a high sampling rate (100 kHz) is required because the signal itself is a brief event. For most intracellular and extracellular recordings, a sampling rate of 20 to 40 kHz gives an good waveform without taking too much memory.
Second, you will need to specify a gain for the A/D board. Most accept a range of ±10 V maximum. Even after amplification, the signals that you send to the computer are usually much smaller than that. To get the best representation of a waveform, you can set the range of the A/D board to ±1 V (which may instead be specified as a gain of 10×).
Like the oscilloscope, the computer’s data acquisition can be triggered by an external event. Again, you will need to specify a channel for trigger input and a trigger level. You may also be able to specify a pretrigger delay, allowing the triggering event to appear centered in the display.
There is a great deal of variation among commonly used stimulators, and many labs are replacing electronic stimulators with computer software. However, most stimulators and software have settings comparable to those described here. Your instructor can explain the specifics of your equipment. See Figure C.4 for a summary of stimulator settings.
Every stimulator should have a switch for single pulses vs. pulse trains (labeled Single vs. Train or Repeat). When set to Single, the stimulator produces one pulse when activated. This pulse has the duration set by the Duration knob, is delayed by the amount set by the Delay knob, and has the amplitude set by the Voltage knob.
Many stimulators have a switch for single pulses vs. pulse pairs (labeled Regular vs. Twin or Double). When set to Twin, the stimulator produces a pair of pulses when activated. Each pulse has duration and amplitude as described above, but the Delay knob now controls the interval between the onsets of the two pulses.
When set for Repeat or Train stimuli, the stimulator produces a series of pulses, each with duration and amplitude as described above. The interval between pulses in the train is set by the Frequency or Rate knob; the Delay knob usually has no effect. Some stimulators also allow you to set the total duration of the train, while others produce the train for as long as the button is held down.
The stimulus isolation unit (SIU) is an important component of the setup for extracellular stimulation. An electronic stimulator or computer-generated stimulus can bring noise into a recording, add a large DC offset, and add a large stimulus artifact (see below) that can obscure the biological response to be recorded. Much of this noise results from stimulus current traveling through the ground of the recording electrode. With an SIU, however, most of the stimulus current travels directly from the positive pole of the stimulator, through the tissue to be stimulated, to the negative pole of the stimulator without going through the ground of the recording electrode. Some stimulators and computer A/D boards have built-in SIUs, so you may not be aware of this piece of equipment. However, if you are using an external SIU, you will need to set the stimulus amplitude on the SIU rather than on the stimulator itself.
Most stimulators have a monitor output that reproduces the timing and duration of the stimulus pulses. This output is independent of the amplitude setting, SIU, and response of the preparation. It can be used to trigger oscilloscope or A/D board and to monitor the stimulator output on an oscilloscope or computer.
In Lab 10, Plant Action Potential, and Lab 6, Synaptic Plasticity, you use an extracellular stimulus to cause a response that you record intracellularly. Although most of the stimulus current should go from the positive pole of the SIU through the tissue to the negative pole of the SIU (see above), there are always voltage spikes in the recording at the onset and offset of the stimulus. This is a stimulus artifact, which can often obscure part of the signal of interest.
There is no way to completely eliminate stimulus artifact, but you can minimize its disruption in two ways. First, strive for good isolation between stimulating and recording electrodes. That allows you to use the smallest possible stimulus to elicit a response. For example, when stimulating through a suction electrode, this means getting a good connection between the nerve and the electrode tip. The advice given below for extracellular electrodes applies equally to recording and stimulation. When stimulating through posts, as in the Plant Action Potential lab, this means keeping the three wells of the recording chamber electrically isolated with Vaseline. Second, use the briefest stimulus that will elicit a response. Because the biological response always follows the stimulus after a delay, using a brief stimulus prevents the artifact from overlapping the response. The trade-off is that a shorter stimulus requires a higher voltage. If you have a good electrode connection, you should be able to reduce the stimulus duration to 1 ms by increasing the voltage. If the electrode connection is not so good, you may require a stimulus of 5 to 10 ms.
To use a suction electrode, lower its tip into the saline bath and suck saline up to the level of the wire inside the tube. The saline will provide electrical contact between the nerve and the amplifier. The external wire of the electrode must also rest in the saline to complete the circuit. Now place the electrode tip on the nerve and suck a small loop of the nerve into the tip. You should now have a recording. If not, check for the following problems.
The troubleshooting suggestions below apply to the plastic or glass suction electrodes described in the instructor’s supplement. If you are using a different type of electrode, not all of these suggestions will apply. If these suggestions do not help, you may need to replace the electrode; ask you instructor for assistance.
Your instructor will show you how to use the electrode puller to produce two microelectrodes from a small glass tube. Place the two electrodes, sharp end up, in a 10 ml beaker with a small amount of 3 M KCl. After a few minutes, the tips of the electrodes should have filled with KCl (Figure C.5). Use a fine syringe to fill the rest of the electrode with KCl (Video C.2, Electrode Filling). It does not matter if there are some bubbles in the electrode, as long as there are none right at the tip. (There is a glass fiber inside the electrode that wicks the recording solution past most bubbles.) Put the electrode in the electrode holder, place its tip in the saline, and measure the electrode resistance. Press the Electrode Check button on the amplifier and check the voltage change. Each mV of change indicates 1 MΩ of electrode resistance.
The resistance of an electrode is an indicator of its sharpness and shape. Greater resistance results from a smaller opening at the tip and a longer, thinner shank leading up to the tip. Different tissue types require different electrode resistances and shapes; see lab instructions for specific recommendations. You can often start with an electrode 10 to 15 MΩ above the suggested resistance. It will usually break back to a more usable and lower resistance when entering tissue such as muscle.
Glass microelectrodes are delicate and easy to break. You will often not be aware of having broken an electrode and will not be able to tell that it is broken without checking the resistance. When using the electrode, you should check the resistance periodically between recordings, since the tip can break when the electrode is being moved between recording sites. A resistance of zero means the tip has definitely broken. Sometimes, the resistance after a recording is higher than before. Increased resistance usually means that the tip has become clogged. This can often be fixed by pressing the Buzz button on the amplifier. If the resistance increases by only a few MΩ, it is not a serious problem; you can probably continue to use the electrode. However, if the resistance increases by more than 5 to 10 MΩ, discard the electrode.
If the potential recorded from an electrode is unstable even before a recording, or if you cannot get a consistent resistance reading from the electrode, this may indicate a problem with the electrode holder or the ground electrode. Ask your instructor to check them.
The electrode tip is usually hard to see, which makes it difficult to place just above a muscle fiber or directly pointed at a particular snail neuron. Consult your instructor for ways to make the electrode tip more visible.
The material presented here is intended to get you started with your experiments and to help you minimize problems with your recordings. For more complete discussion of electrophysiological recording techniques, refer to the resources listed below. Answers to many general questions concerning electrophysiological recording techniques can be found at http://en.wikipedia.org/wiki/Electrophysiology.