Equipment: A frog nerve-muscle preparation is pinned in a chamber and bathed in a normal saline solution. The nerve is lying across two wires and covered with vaseline to keep it from drying out. The wires are connected to the stimulator. You view the preparation through the dissection microscope. Extraneous electrical noise is reduced by the aluminum foil shield. A manipulator carries the microelectrode, which records the membrane potential, processed by the probe amplifier and filter. The instrument controls are shown in the image below.
Impaling a muscle fiber: With the microelectrode positioned above the muscle, adjust the position knob to zero the potential on the monitor. Press the electrode test button to check the electrode resistance; the deflection on the monitor should be 10-40 mV. Now impale the muscle fiber: move the electrode tip over a muscle fiber (push and pull the 'joystick' on the manipulator) and then lower the electrode by turning the silver knob on the bottom of the joystick. It might be helpful to press briefly the tickler button, which will 'burn' a tiny hole in the membrane and assist impalement. Once the cell has been impaled, nearly all controls may be adjusted from the computer.
Computer program
'Froglab' is a Matlab program for use in a student laboratory exercise designed for recording, displaying, and analyzing synaptic potentials recorded with an intracellular micropipette from a frog nerve-muscle preparation. The program was written for a PC computer fitted with a National Instruments A/D card. The display is controlled with various buttons and sliders. For help, position the cursor over a button for a second, and a 'tooltip' will appear. Buttons with labels that begin with a lower case letter can be accessed from the keyboard (by pressing that letter). Controls common to all four modes are colored yellow and are located in the lower right corner. At the top right, the voltage is displayed. There are four modes of operation, selectable by clicking the radiobuttons at the top of the display:  1. Scope mode (red controls) is for oscilloscope (or chart paper) display. Sweeps are continuous (non-triggered). Nerve stimuli (continuous or trains) can be generated. Recordings can be dc or modified ac. 2. MEPP mode (green controls) is for recording miniature end plate potentials. Threshold for MEPP detection can be adjusted; captured MEPPs can be edited and are stored for later analysis. An amplitude histogram is displayed as MEPPs are captured. 3. EPP mode (blue controls) is for recording nerve-evoked end plate potentials. Stimulus trigger timing can be internal (trigger pulse to stimulator starts analog input) or external (trigger pulse from stimulator triggers analog input). The time window for peak EPP detection can be adjusted; captured EPPs can be edited and are stored for later analysis. An amplitude histogram is displayed as EPPs are captured. 4. Analyze mode (gray controls) plots histograms of MEPP amplitudes (with gaussian fit) and EPP amplitudes (with Poisson fit). The EPP fit can be altered with 3 sliders that modify mean MEPP amplitude, MEPP SD, and mean EPP amplitude. Minimum amplitude threshold to determine 'failures' is adjustable.
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1. Scope mode (red buttons)
The sweeps run continuously. Stimuli can be given, but they are not synchronized with the sweeps. Any button label that begins with a lower case letter can be accessed from the keyboard, by pressing that letter (e.g., 'a' for a/c mode). The following controls are on the left side of the display: Vmax slider determines the value of the voltage at the top of the display. The value is independently adjustable in all three recording modes (scope, mepps, epps). Vmin slider determines the value of the voltage at the bottom of the display. The value is independently adjustable in all three recording modes (scope, mepp, epp). a/c radiobutton (keyboard: a) toggles the display as D/C or modified A/C. In A/C mode, the average value of the most recently acquired data is subtracted before the data are displayed. The Vmax and Vmin slider values are separately adjustable in the A/C mode. The display is not truly an A/C (highpass filtered) recording. For example, a steady ramp of voltage will be changed to a sawtooth (with a period equal to the frequency at which a block of data is sampled). The following controls are below the display: 
STIMULATOR controls: The cont button (keyboard: c) toggles the stimulator on and off in the continuous mode at the frequency shown on the button. When the stimulator is on, the button turns bright red. The slider below this button controls the frequency of stimulation. The Hz radiobuttons on the left are for convenience and set the frequency to the number shown on the radiobutton. The single button (keyboard: s) delivers a single shock. The hold button turns red for one sweep, and the sweep during which the stimulus was delivered is displayed in black. The train button (keyboard: t) triggers one train (the number of shocks in the train are shown on the button) at a frequency and duration shown on the button below it. To change the train stimulus frequency or train duration, click this button. If the cont mode is on and the train button is pressed, the continuous mode is suspended while the train executes, and then the continuous mode resumes. It is not possible to program repeated trains; the train button must be pressed for each train. zoom button (keyboard: z) will enlarge a portion of the graph. After clicking the button (or pressing 'z'), draw out a rectangle in the graph; it will be enlarged to occupy the full screen. Alternatively, zoom can be turned on by clicking in the graph. This will zoom only the X axis, not the Y axis. The trace will be centered at the position of the cursor when the button was clicked. The width (time duration) of the display is determined by the 'zoom width' setting in the popup menu; default is 10 msec. To turn off zoom, click again in the graph, or click the zoom button, or press 'z'. Hold offset slider display shows the offset (in mV) between successively held sweeps. The value of the offset is set by the slider below the display. For example, if set to 1 mV (the default), each time a sweep is held (see hold on/off button above) it is offset 1 mV above the previously held sweep. dispTime radiobuttons (keyboard: d) allow the X-axis time limit to be set to a preset value (e.g., 0.5, 5, 60, or 600 seconds). Pressing 'd' on the keyboard advances to the next radiobutton (but ignores the last one), or loops back to the first. Yellow buttons. See Buttons Common To All Modes above. |
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1. MEPP mode (green buttons)
The following controls are on the left side of the display: Vmax slider determines the value of the voltage at the top of the display. The value is independently adjustable in all three recording modes (scope, mepp, epp). Vmin slider determines the value of the voltage at the bottom of the display. The value is independently adjustable in all three recording modes (scope, mepp, epp). Thresh slider. This sets the level of the threshold for MEPP detection (indicated by the horizontal red line). The threshold may also be set by clicking in the window. The Y value clicked becomes the new threshold. The following controls are below the display: 
Flatten 0 flattens the trace. First a smoothing routine (moving bin) is applied. The Flatten slider determines the number of points in the moving bin. The result of the smoothing is then subtracted from the raw data. To enter a particular value, click on the Flatten button. Keepit 0 button cycles between three modes: 0 means the peak detector is turned off. Click once (keepit=1) to turn on the peak detector; each detected MEPP is marked at its peak with a red asterisk, but the MEPPs are not saved. Click again (keepit=2) and the MEPPS will be kept. An inset histogram appears at the upper right corner and shows the distribution of peak amplitudes of kept MEPPs. The histogram is updated after each sweep is analyzed for MEPPs. Click again (keepit=0) to turn off the peak detector. Reset button erases all kept MEPPs. Yellow buttons. See Buttons Common To All Modes above. |
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3. EPP mode (blue buttons)
The following controls are on the left side of the display: Vmax slider determines the value of the voltage at the top of the display. The value is independently adjustable in all three recording modes (scope, mepp, epp). Vmin slider determines the value of the voltage at the bottom of the display. The value is independently adjustable in all three recording modes (scope, mepp, epp). Time window for measuring peak EPP is between the two vertical red lines. To move them, click in the window. Left click to move the left margin of the window (accepted only if the click is to the left of the right margin); right click (always staying to the right of the left margin) to move the right margin of the window. The following controls are below the display: 
STIMULATOR controls: The Stim on/off button toggles the stimulator on and off. When on, the button is red. Below this button the stimulus frequency is displayed. The Stimulus frequency slider below controls the frequency. Sweeps are captured and displayed continuously at the set frequency, whether or not the stimulator is on. At the bottom are two Trigger radiobuttons. These determine whether the stimulator is triggered from the program (Internal) or whether the program is triggered by the stimulator (External). In the external mode, the stimulator controls above are not visible. Also, no sweep appears, and the voltage displayed at the upper right of the screen is not updated until a trigger is received from the stimulator. In EPP mode, the stimulus rate should not exceed a few Hz. Show Avg button toggles on and off the display of the average of all kept sweeps. Keepit button toggles between two states (not 3 states, as in MEPP mode). When 0, no EPPs are saved; when 1, each EPP is saved (but see next sentence) and the peak voltage during the time that falls within the window (the 2 vertical red lines) is measured and saved. Note that EPPs are saved only if the stimulator is running (the stim button is red in internal mode, or the external mode is active). When EPPs are being kept, an inset histogram appears at the upper right corner and shows the distribution of peak amplitudes of kept EPPs. The histogram is updated after each stimulus, as is the average trace. Reset button erases all kept EPPs. DispTime radiobuttons allow the X-axis time limit to be set to a preset value (e.g., 0.05 or 0.2 seconds). |
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4. Analyze mode (gray buttons)
Two histograms are shown (see figure below). The larger shows the observed distribution of evoked EPPs (blue bars) and the calculated (expected) distribution of EPPs (curved red line). The calculation is made according to the Poisson distribution, using three observed values (average and standard deviation of observed MEPP amplitudes, and average of observed EPP amplitudes). The inset histogram shows the distribution of observed MEPP amplitudes (green bars); the red line is a Gaussian fit to the observed MEPP amplitude distribution. The vertical red line in the main graph shows the 'cutoff' for EPP failures. That is, an EPP smaller than this cutoff will be assigned a value of zero amplitude in the calculations, and put into the left hand bin. Move the cutoff by left clicking in the image. The number of failures (observed and calculated) are shown at the top left, and also indicated on the Y axis. The observed value of m (=avgEPP/avgMEPP) is also printed to the left of the display. The mean evoked EPP amplitude is shown on the X axis. The results of a chi squared test for fit of the expected to the observed data are shown on the left. A smiley face means the fit is good; a frowny face means not good (poisson curve does not fit the observed data). If no MEPPs or no EPPs have been acquired when the Analyze button is pressed, the data plotted are 'typical' data drawn from hypothetical experimental observations. The Ymax button allows the Y axis to be rescaled. At the bottom are four sliders (see figure below). One allows the number of bins into which EPP amplitudes are placed to be changed (default is 20 bins). The other three sliders show the values used to calculate the expected Poission distribution (the curved red line in the main graph). At the outset, each slider shows the observed value. The values can be changed by moving the sliders. To return a slider to its default (observed) value, move the slider all the way to the right. Move the sliders to 'massage' the fit - watch the chi squared value (left side of display); a lower value means better agreement between the Poisson prediction and observed data. The sliders are: Avg MEPP gives the average amplitude (mV) of observed MEPPs. SD MEPP gives the standard deviation (mV) of the observed MEPP distribution. Avg EPP gives the average amplitude (mV) of observed EPPs. 
Lab handout
Much of our knowledge about synaptic structure and function has come from studies of the neuromuscular junction. In today's laboratory, we will impale living frog skeletal muscle fibers with a glass micropipette and record action potentials and synaptic potentials in four different bathing solutions: normal physiological saline (no additions), Mg/low calcium, curare, and neostigmine. For each solution, we will discuss relevant clinical situations that are mimicked by the additions. For example, curare, which blocks post-synaptic receptors and leads to flaccid muscle paralysis, is used on poison darts to paralyze prey by Yanomamo Indian hunters in South America, is used often in surgery for muscle relaxation, and also mimics the effects of a disease, myasthenia gravis, in which antibodies block receptors in a similar fashion to curare. We will begin with a brief introduction to the lab, in which the preparation and equipment will be described, and the general procedures outlined. Then we will break into two groups; each group will work with one instructor on two setups. I. Observations A. Identify the components of the setup (micromanipulator, oscilloscope, stereo microscope, amplifiers, microelectrode, recording chamber with frog nerve-muscle preparation, instructor). B. Looking through the microscope: Identify muscle fibers, nerve branches and myelinated axons, red blood cells, pigment cells (black), parasites (if any), tiny pins, nerve in Vaseline filled trough (why the Vaseline?). (use different magnifications as needed.) C. Stimulate the nerve while looking through the microscope. Change the frequency of stimulation. At what frequency (approximately) do the single twitches fuse into a smooth tetanus? NOTE: Instructions in square brackets refer to software and stimulator settings. [Scope mode; adjust stimulus frequency slider; click stimulus on (button turns red) and off] II. Impalements Your instructor will demonstrate how to impale single muscle fibers and how to use the software. [Scope mode; turn Position knob on amplifier box to zero the voltage before impaling] A. Resting potential (mV)? B. Miniature end plate potentials (mepp's) present? [A/C radiobutton on] Approximate amplitude (mV)? Is this close to threshold for an AP? C. Action Potential (AP): Stimulate the nerve once. [Set DispTime to 0.1 second (click 0.1 radiobutton); depress the single toggle on the stimulator to get one shock] Does the AP overshoot zero mV? Look for a notch or inflection of the AP rising phase. What does it reflect? Did the electrode come out of the cell (resting potential to ~0 mV)? If so, why? Repeat part II a few times. III. Unknown solutions One preparation will receive the low calcium solution; the other will receive the curare solution. Before applying the solution, discuss the composition of each (i.e., the approximate concentrations of sodium, potassium, and calcium ions, and major anions, and curare). Discuss experimental observations that would permit you to distinguish the two solutions. Now do the experiment: Impale a muscle fiber and look for mepps. Then change the bathing solution (your instructor will show you how to use the perfusion system). Observe the effects of the unknown solution on mepps: Also observe the muscle through the microscope and stimulate the nerve with a single shock (depress the single toggle on the stimulator) - any contractions? Which solution is it? How can you rule out the other possibility? IV. Wait for paralysis The Mg/low calcium and curare solutions require 5-10 minutes to block synaptic transmission. Remove the electrode from the muscle fiber, and monitor the developing paralysis by stimulating the nerve every minute or so and watching the twitch through the microscope. When the twitching stops, the muscle is paralyzed. The two solutions (Mg/low calcium and curare) are discussed sequentially below. V. Mg/Low calcium preparation Impale a muscle fiber. A. Are there mepps? If so, are they normal amplitude? [Scope mode; Click A/C radiobutton] B. Stimulate the nerve at a frequency of about 1/sec and observe the end plate potentials (epps). Note amplitudes and rise times. [EPP mode; lift repeat toggle on stimulator; click Hold On/off button to store and display repeated epps; click Erase Hold button to erase] Compare the epp's with the mepp's. Can you observe discreet "steps" in the amplitudes of successive epp's? Are the "steps" about the size of a mepp? C. Quantitative analysis: Collect a series of mepps [MEPP mode; adjust Threshold for detecting peaks of mepps; click Keepit button twice to keep results] Collect a series of epps [EPP mode; stimulate repeatedly at about 1 shock per second; adjust window (vertical red lines) so the epp peaks fall inside; click Keepit button once to keep peak values.] Display the results of acquiring mepps and epps [Analyze mode] D. Clinical discussion {1. Hypocalcemia - IS OPTIONAL a. Signs/symptoms b. Mechanism c. Causes d. Rx e. Does the Mg/Low Ca solution mimic hypocalcemia? If not, why? } - end optional part 2. Botulism a. Signs/symptoms b. Mechanism c. Causes d. Rx e. Why is botulism relatively common in Colorado, compared to other places? 3. Myasthenic syndrome a. Signs/symptoms b. Mechanism c. Causes d. Rx e. Facilitation: Stimulate the Mg/Low Ca preparation at high frequency. [Scope mode; adjust stimulus frequency slider] What happens to the epp amplitudes? Does the muscle contract? What is the cellular mechanism of this facilitation? VI. Curare preparation Impale a muscle fiber. A. Are there mepp's? [Scope mode; click A/C radiobutton] B. Stimulate the nerve and observe epps. How do they compare with epp's in the Mg/Low Ca solution? [EPP mode] C. Two shock facilitation. Stimulate about once a second, with different intervals between the dual shocks [EPP mode; click Hold On; On the stimulator, move the Twin pulse knob down; change the Delay settings on the stimulator] How long does facilitation last? Mechanism of facilitation? Repeat the above a few times with different impalements. D. Clinical discussion: Myasthenia gravis 1. Signs/symptoms 2. Mechanism 3. Cause 4. Rx E. High frequency stimulation: Record control epps before a high frequency train, and follow the recovery from depression, which may overshoot to produce post-tetanic potentiation. Deliver a train of stimuli at high frequency. Record a few epps before the train at low frequency (1/10 seconds) and after the train. Then click the Train button to deliver a single train of shocks at 30-50 per second. [Scope mode; adjust stimulus frequency slider to 0.1 (click radiobutton); select Train parameters (frequency: 30-50 per second; duration 40-60 seconds)] What happens to epp amplitudes during a high frequency train of stimuli? Mechanism of synaptic depression? Does the amplitude recover when the high frequency stimulation ends? Mechanism of PTP? Significance of PTP? F. Anti-cholinesterase (Neostigmine). 1. What does acetylcholine esterase (AChE) normally do? Where is AChE located at the synapse? What do you predict will be the effects of blocking AChE? What would be the effect of a therapeutic dose of Neostigmine on a normal person? 2. Impale a muscle fiber in the curare solution and observe epp amplitudes. Then perfuse the chamber with the same solution to which some Neostigmine has been added. What is the effect? [EPP mode; Hold On] 3. Clinical effects of Neostigmine use. a. Overdose: What effect of overdose do you predict? Add an excess of Neostigmine to the bath (your instructor will demonstrate how). What happens to epp amplitudes? What is the mechanism of the effect? {b. Side effects of a therapeutic dose of Neostigmine. - IS OPTIONAL There are two major types of ACh receptors: Nicotinic and Muscarinic Where found in body? Activated by ACh and? Blocked by? Effect of blocking AChE c. What are the side effects of Neostigmine therapy in myasthenia gravis? Now what do you predict would be the effect of a therapeutic dose of Neostigmine on a normal person? Neostigmine doesn't cross the blood brain barrier. What would happen if it did? 4. Environmental hazards of anti-cholinesterases. a. Organophosphate insecticides. Signs of poisoning? What does SLUD or SLUDGE mean? b. Nerve gas. Signs after inhalation of nerve gas? Effect of making the nerve gas lipid soluble?} - end optional part |