BIO325 Laboratory Guide #19 (2024)
SENSORY PROCESSING II:
CRAYFISH STRETCH RECEPTORS
falls under category
A
In this lab you will explore the neural encoding of sensory input by sensory receptors in the crayfish abdomen. In each segment of the abdomen the superficial extensor muscles send axons from just two pairs of muscle receptor organs (MROs) into a pair of robust nerves which coalesce close to the dorsal carapace. These MROs are stretch receptors which monitor the degree of bending of the abdomen and inform the crayfish central nervous system of this.
So what specifically do the MROs encode? At first glance, it might seem that all the “rest-o-crayfish” needs to know is the position of the tail. The simplest way to encode this would be to have each MRO fire a continuous AP train whose frequency increases as the tail is bent more and more sharply. This kind of “position” monitoring would constitute a purely “tonic” response. This would be perfectly adequate for keeping track of the posture of the tail. In fact, your major record in this lab will demonstrate a remarkably simple linear relationship between sensory input (mechanical bending of the tail) and neurally encoded output (frequency of action potentials) over a wide range of bendings.
However, the real world poses two additional challenges. The first is that any sustained, unchanging stimulus often becomes irrelevant to the organism, essentially part of the “background”. The neural mechanism for accomplishing this is called “adaptation” and takes place right at the level of the receptor. The result of adaptation is that the AP firing rate in response to a sustained constant degree of bending diminishes slowly over time. The MRO1 unit on each side of each abdominal segment is a “slowly adapting, tonic” position sensor.
The second challenge to the crayfish is that it needs to be able to respond rapidly to sudden changes in posture. The neural mechanism for this is a receptor that fires only in response to abrupt changes in tail posture, and then immediately shuts off. This constitutes a “phasic” response pattern. The MRO2 unit on each side of each segment is just such a “rapidly adapting, phasic” acceleration detector.
These nerves turn out to be remarkably simple to access experimentally; two minutes, a pair of scissors, and your thumb are all that are required to expose the nerve roots for recording. Activating the receptors is also extremely simple; you just bend the abdomen. As in previous labs you will contact the sensory nerve root with a suction electrode. The nerve roots are remarkably robust and impervious to injury. The action potentials you will record are huge in amplitude and very readily distinguished from background noise. This laboratory exercise closely follows Crawdad Lab #10. Read through the guide for this lab to get an introduction to crayfish muscle stretch receptors.
I. RECORDING PREPARATIONS
A. Crayfish Surgery
1) Review the CD video guide for Crawdad laboratory #10.
2) Chill a crayfish and cut off the abdomen. Using a needle, make a small hole in the center of the telson and attach a thread.
3) Cut along both sides of the abdomen, cutting through each dorsal tergite near its ventral edge. Remove all of the tissue adhering to the ventral cuticle of the abdomen. Use your fingertip to remove the mass of muscle adhering to the dorsal surface of the abdomen, as in the video. This should leave just the superficial extensor muscles and associated segmental nerve roots attached to the dorsal cuticle. in each segment.
4) Pin the tail dorsal side down into a large Sylgard tray. Pin only the rostral end of the tail. Immerse the tail in cold crayfish Ringer's solution.
5) Position the tail under the dissecting microscope and locate the cut ends of the sensory nerve roots in each segment.
B. Recording Setup
1) Turn on the PC and open the Scope program. Set it up for recording from input channel A only. Set the channel for monopolar recording, set the Range to 50 mV, and activate AC and line filters. Set up Scope for repetitive sampling at 20-50 msec/sweep and the maximum sampling rate.
2) Turn on the Model 1800 AC amplifier. Make sure that the amplifier ground is connected to the common cage/MacLab ground. Turn on the amplifier. Set the left amplifier channel to Rec and x1000 gain. Turn on the 60Hz notch filter and set the Low- and High-cutoff filters to 100 Hz and 5 kHz, respectively.
3) Turn on the audio amplifier. Make sure that the Mono switch is out and the selector is set to Tuner (this corresponds to directly monitoring the output of the 1600 amplifier to the MacLab inputs).
4) Trace all of the connections of your recording setup and make sure that you understand what each is for.
5) Lower the suction electrode into the Ringers bath and perform a noise check to make sure that the recording noise is < 20 mV.
II. RECORDING FROM SENSORY NEURONS
A. Slow- and Fast-Adapting Receptors
1) Find a suitable nerve root and suck it up into the suction electrode. Make sure that you suck up the center of the root and not its torn end. The nerve should be completely quiet when the tail is relaxed. To hold a tight seal, you may have to apply a SMALL dab of Vaseline to the end of the electrode after the nerve is sucked up. It will also work best if the nerve you choose is towards the rostral (head) end of the animal.
2) Gently pull on the thread. This should produce repetitive firing of a single axon, corresponding to the slow-adapting MRO1 receptor. Firing rate of this cell should vary roughly with the degree of bending of the tail.
3) If you hold a constant tension in the thread, then tweak it with a probe or pair of forceps, you should see intermittent firing of the larger, fast-adapting MRO2 receptor.
Q1: What is the difference in the response properties of the MRO1 and MRO2 receptors?
Why should the crayfish have stretch receptors with these two different response
properties, i.e. of what advantage is it?
B. Stretch Coding in the MRO1 System
1) Set Scope to a time base value of 100 msec/sweep.
2) Position the second micromanipulator and attach the thread to it so that advancing the horizontal drive flexes the tail. Carefully adjust the tension in the thread just to the point at which action potentials are produced. Immediately record a single sweep on Scope. Repeat this process several times.
Q2: Is there a minimal firing frequency for the MRO1 receptor under constant stretch conditions? Based on what you know about action potential generation and refractory periods, why should this be? In other words, why won't a neuron fire indefinitely slowly?
3) Now systematically advance the micromanipulator a half millimeter at a time, recording a single Scope sweep at each point. Notice that the firing rate increases as the tail is flexed more.
Q3: Is the relationship between flexion of the tail and MRO1 firing rate a linear one? Is there some maximal firing rate?
Measure initial interspike periods (in msec) from each of your Scope sweeps. Produce an Excel spreadsheet with tail flexion (in mm), interspike perion (in msec) and, and firing rates (in spikes/sec). Again, the firing rates in spikes/sec can be calculated by dividing each interspike period (in msec) into 1000 (msec/sec).
Data Sheet Item #3b:
From your Excel spreadsheet produce a scatterplot of tail flexion in mm vs. MRO1 firing rate in Hz (spikes/sec).
C. Sensory Adaptation in the MRO1 System
1) Repeat the above experiment with Scope set to record multiple (32-64) sweeps per sample with a .5 second interval between sweeps.
2) You should notice that, if the tail is held at a constant degree of flexion, the firing rate of the MRO1 neuron drops off over time. This is adaptation.
Q4: Of what advantage to the animal is adaptation in sensory systems?
Data Sheet Item #4a:
From your Scope traces Produce an Excel spreadsheet with time, flexion, interspike period, and firing rate data in labeled columns.
Data Sheet Item #4b:
From this Excel speadsheet produce a plot showing the rate of firing as a function of elapsed time after stimulus onset for each of several fixed amounts of flexion. In other words your plot will have time in seconds on the X axis, firing rate on the Y axis, and several lines plotted. Each line will correspond to a particular amount of flexion of the tail.
III. SHUTTING DOWN
Complete the following steps before leaving the lab:
1) Make sure that you have saved all of your data to the hard drive, then quit Scope. Turn off the PowerLab box.
2) Turn off the amplifier and the stimulator.
3) Flush out the suction electrode with distilled water, then air.
4) Make sure that both the microscope and fiber-optic lights are turned off.
5) Make sure that both micromanipulators are magnetically secured to the steel plate.
6) Return all solutions to the refrigerator in 104 and store all crayfish parts in the freezer.
IV. PREPARATION OF THE LAB DATA SHEET
Your data sheet should include all FOUR of the items described in the boxes above. Make sure that the axes of all of the graphs and print-outs are labeled and calibrated. You should certainly discuss your results and the answers to the questions with your partners and others in the lab. However, please work independently when you prepare your data sheet.
The writeup
for this lab
falls under category
A