BIO315 Laboratory Guide #4
NEURAL SUBSTRATES OF
CRAYFISH ESCAPE BEHAVIOR
A command neuron is a neuron for which activation is both a necessary and a sufficient condition for initiating a specific complete behavior. Behaviors under the control of such command neurons are generally stereotyped behaviors with very short response latencies, such as "startle" behaviors. Command neurons themselves are often very large "giant" neurons with high activation thresholds and rapid action potential propagation speeds.
In the crayfish, the initial stage of tail-flip escape responses is under the control of two bilaterally symmetrical pairs of such command neurons in the ventral abdominal nerve cord. Abruptly poking the head of a crayfish activates the paired medial giants. Each medial giant is a single neuron, with its cell body in the crayfish brain and a large diameter axon extending the length of the abdomen. The result of a single medial giant action potential is a tail-flip involving rapid sequential flexion of all abdominal segments. This propels the animal rapidly in reverse, away from the stimulus. In contrast, poking the telson (tail-fan) activates the lateral giants. Each lateral giant is actually a string of segmental giant neurons in the anterior three segments of the abdomen, connected by fast electrical synapses. An action potential in each lateral giant set results in a flexion of only the anterior segments of the abdomen. This actually flips the animal over, and a subsequent series of tail-flips propels the animal away in this new direction, again, roughly away from the stimulus.
In this lab you will implant simple wire electrodes in the vicinity of the ventral abdominal nerve cord of a live crayfish. This is a chronic preparation, in that the electrodes will remain in the animal and will subsequently be used to monitor neural activity while it is actively behaving. The specific goals of this lab are to 1) induce tail-flips by tactile stimulation and record concurrent activity of the medial and lateral giant command neurons and 2) establish a strength/duration curve for eliciting tail-flips via electrical stimulation of the giant command neurons.
LAB 4A. SETUP
A. Preparation of Recording Leads.
1) Cut two 15" lengths of fine "wire-wrap" wire.
2) Strip ½" on one end of each wire.
3) 2" in from the stripped end of each wire, strip a 1/16" bare region.
4) Set your two recording lead wires aside.
B. Implantation of Recording Leads
1) Snag a crayfish and secure both claws with rubber bands.
2) Dry it off with paper towels and KimWipes.
3) Mount it ventral-side up in the surgery tray, using rubber bands to hold it down. Secure the surgical tray in a larger tray full of ice.
4) Locate the 2nd and 3rd abdominal sternites and the medial blue line.
5) Cut off the 2nd and 3rd swimmerettes on each side, cutting close to the base of each.
6) Use a long needle to carefully bore a hole through the dorsal tergite just posterior to each of the four cut swimmerette stubs. Try not to shatter the tergite as you bore through it. You should have four holes when you finish.
7) Thread the stripped end of one recording lead wire through the eye of a short needle, and crimp it over.
8) Carefully (under a dissecting microscope) run the needle through the left tergite hole on the 2nd abdominal segment, in through the ventral cuticle, under the medial blue line, out through the ventral cuticle, and through the right tergite hole.
9) Pull the needle and lead wire through until the bare region is centered under the blue line (just above the ventral nerve cord).
10) Remove the needle and repeat steps 7, 8, and 9 for the 3rd abdominal segment using the other recording lead wire.
11) Double check and adjust each lead so that the bare region is centered under the blue line on the abdomen. Apply a small drop of super-glue to each side of each location where a recording wire passes through the dorsal tergite. You may have to first very carefully dry each glue site with a KimWipe twisted to a point. Try not to glue adjacent tergite segments together.
12) Allow the super-glue to dry for at least 15 minutes.
13) Using fine scissors, cut off only the short end of each of the two recording leads very close to outer surface of the tergite.
14) Apply a drop of nail polish to the outer tergite surface on each side, where each recording lead passes through. This will reinforce the glue bond and water-seal the cut wire ends. Again, try not to seal adjacent tergite segments together.
15) Allow 15 minutes for the nail polish to dry. While the polish is drying, tear several 6" x ½" strips of aluminum foil. Very carefully wrap the two recording leads together in these strips of foil and crimp it down to a small diameter, forming a single cable. This will provide an electrical shield for these recording leads and keep them from getting tangled. Extend this foil shield from within 1½" of the crayfish abdomen to within 1 inch of the other end on the recording wires.
16) Strip ½" from the free end of each recording lead.
17) When the nail polish is dry, remove the restraints on the crayfish and carefully turn it over.
18) Using super-glue, attach a short piece of ¼" tubing to the dorsal surface of the carapace, just in back of the head. There is a nice central groove that the tubing can rest in. This usually takes several glue applications and a good deal of patience. When the glue is dry, seal with nail polish. During this process the animal can be loosely restrained by leaving it in the surgical tray, but covering the tray.
19) When the tubing is thoroughly dry and firmly attached to the crayfish, thread the recording cable with its foil shield through the tube from posterior to anterior. Continue to carefully pull the cable through until there is just enough slack left (~ 1½") to allow the abdomen to freely flex. If you leave two much slack, the walking legs and/or claws will become entangled and tend to pull out the recording leads.
20) Unbind the claws of the crayfish, return it to its home cage, cover the cage, and add tap water. Make sure that there is enough free cable inside to allow the animal to move around without pulling on its recording leads.
21) This chronic implant preparation is good for several weeks. Don't even think about what happens if the crayfish tries to molt - it isn't pretty.
LAB 4B. RECORDING
I. BEHAVIORALLY-EVOKED ACTIVITY:
TAIL-FLIPS
A. Model 1700 AC Amplifier
This amplifies the signal coming from the crayfish and filters out noise. Confirm the following settings on this amplifier:
Power ON (switch lights up)
Amplifier #1 or #4
low cutoff knob 10Hz
high cutoff knob 5 kHz
gain knob x 100
mode switch REC
notch switch IN
B. Stereo Amplifier
This sends the amplified crayfish neural activity to speakers so that you can monitor it. Confirm the following settings in this order:
volume knob ½ way up
power ON (red light comes on)
mono button in (mono)
tone knob midway
balance knob midway
source knob Tuner
C. Hooking Up the Crayfish
1) Visual inspect your implanted crayfish to make sure that neither of the leads has become broken or dislodged.
2) Suspend the crayfish by its recording "cable" in the aquarium so that it is fully immersed, but not touching the bottom.
3) Clip the amplifier leads to the two recording lead wires, making sure that they do not touch.
4) Clip the foil shield of the crayfish cable to the ground of the gray amplifier cable.
5) Turn on the 1700 amplifier.
6) If there is excessive electronic noise, consult the instructor.
D. PowerLab/Computer
1) Turn on the PC and PowerLab box.
2) Open the Application template called "Snippymeister". Locate the following screen icons and "controls" and confirm the following setup conditions:
a) confirm that a single channel is displayed
b) on control panel at right:
INPUT A Ch 1 Range: 500mV
INPUT B Off
TIME BASE 4kHz Samples:512 Time: 10 msec
3) On the PC display pull down SETTINGS; choose SAMPLING . . .
set MODE to REPETITIVE; SOURCE to USER
click OK to close SAMPLING. . . window
4) Hit the START button at the lower right.
E. Recording
Much of the functional recording will be done by adjusting settings "on the fly". The
general procedures are as follows:
1) Repeatedly poke the head of the crayfish until you get a strong tail-flip response. Is this response accompanied by a large action potential, visible on the monitor and audible on the speaker? If so, set for single sweep, triggered by the neuronal response itself (the instructor will help you set this up), then try to capture the waveform of the action potential.
2) If you are successful, record several medial giant action potentials. How uniform are they in size and time-course?
3) Repeat this process for pokes to the telson (tail-fan). Can you behaviorally distinguish the form of this tail flip from the tail-flip in response to head stimulation? Can you elicit and record lateral giant action potentials? How uniform are they in shape and amplitude? Do they differ from medial giant APs?
II . ELECTRICALLY-EVOKED ACTIVITY:
THE STRENGTH/DURATION CURVE
Because the giant axons are driving the tail-flip, electrical stimulation of the ventral
nerve cord should elicit tail-flip responses. For this part of the lab you elicit tail-flips with threshold-level stimuli - i.e. stimuli which are just strong enough to elicit tail-flips.
Each square-wave electrical stimulus is defined by its amplitude or strength in volts and its duration in milliseconds. At threshold , the relationship between stimulus amplitude and stimulus duration is a negative one. As stimulus amplitude is increased, the duration necessary to evoke a threshold-level response decreases. For a wide range of physiological systems, threshold stimulus amplitude and duration are related by the following formula:
V=R(1+C/t)
For this formula V = threshold stimulus amplitude and t = threshold stimulus duration. R is a constant called rheobase and is defined as the minimum amplitude necessary to evoke a response for an indefinitely long duration stimulus. C is a constant called chronaxie and is defined as the threshold stimulus duration corresponding to a stimulus amplitude of twice rheobase.
For this part of the lab you will:
1) determine multiple stimulus amplitude and duration pairs at threshold
2) use these data to plot an experimental curve of stimulus amplitude as a function of duration
3) experimentally determine constant values for both rheobase and chronaxie
4) use these two values to plot a theoretical curve of stimulus amplitude as a function of duration
5) compare your experimental and theoretical curves to see how well they agree
A. Data Collection
1) Make sure that your electronic stimulator is plugged in and turned on. Set the two MODE switches to Regular and Off, and the two POLARITY switches to Normal and Mono. Set the DURATION knob and switch to 1 millisecond (10ms X 0.1). Set the VOLTS knob and switch to 1 volt (10 volt X 0.1).
2) Disconnect your crayfish from the amplifier and connect the leads to the output cables of the stimulator. The instructor or lab assistant can help you do this. Have the instructor or lab assistant double-check your stimulator settings before proceeding.
3) While closely watching the crayfish, deliver a single stimulus pulse by depressing the MODE switch in the direction of "Single". The red MONITOR light should briefly flash.
4) If the stimulus evokes a tail-flip, reduce the stimulus voltage (amplitude) and stimulate again. Continue reducing the stimulus voltage until you find the minimum voltage necessary to evoke a tail-flip for a duration of 1 msec. Alternatively, if the stimulus fails to evoke a tail-flip, incrementally increase the voltage until you get one. You should not have to go higher than 10 volts.
5) Record your threshold stimulus duration and voltage (e.g. 1 msec x 1.2 V) as your first data point.
6) Repeat steps 3-5 for successively longer durations in .25 msec increments. At some point you will find a minimum voltage, below which even a very long stimulus pulse (>2 msec) will fail to elicit a tail-flip. This absolute minimal voltage is Rheobase (R). Record this value.
7) Now work in the other direction from your original data point. Repeat steps 3-5 for successively shorter durations, in .1 to .05 msec increments. As the stimulus pulses get shorter you will have to increase the stimulus voltage. You will not be able to go below .02 msec. In any case DO NOT EXCEED 10 VOLTS.
8) Finally, set your stimulus voltage to twice your rheobase value. Determine the corresponding duration at threshold. Record this duration in msec. as Chronaxie (C).
B. Data Analysis
1) Enter your experimental data into Excel as three adjacent columns. The first column "Duration" will contain your measured duration values. The second column "Experimental Threshold" will contain your measured voltage values. The third column "Calculated Threshold" will contain values calculated using the formula Vq=R(1+C/t). In this formula t will be read from the Duration column and R & C the constant values for rheobase and chronaxie which you recorded in steps A6 and A8 above.
2) Produce a dual plot of the experimental and theoretical curves, using the Scatterplot option in the Excel graph wizard. Add lines for Rheobase and Chronaxie.
3) Did your experimental strength-duration curve have the expected shape? How well did it match the calculated strength-duration curve?
III. WRITE-UP FOR THIS LAB
The write-up for this lab should include a brief introduction, a presentation of any data that you gathered for part I above, the well-labeled graph specified in part II above, and a discussion of what your results mean.