BIO325 Laboratory Guide #12 (2024)
ACTION POTENTIALS IV:
ACTION POTENTIALS IN THE SNAIL BRAIN
falls under category
A
In this lab you will be recording from individual neurons in the circumesophageal ganglia ("brains") and buccal ganglia of two species of freshwater snail, Lymnea stagnalis and Helisoma tivolis. This is a very challenging lab; both the surguical preparation and recording techniques are more difficult and demanding than any work you have done so far.
The surgical isolation will involve four basic steps. First you will anesthetize the snail, then peel away the anterior shell, then pin out body, then expose and remove the viscera, then locate and remove the brain and buccal ganglion, then pin out the ganglion in a recording dish, then remove the enveloping connective tissue via a protease treatment.
You will record with standard glass microelectrodes, as you did with the crayfish superficial flexor muscle cells. However, there are a few twists. First snail brain neurons are much smaller and your electrodes must therefore be as sharp as possible. Electrode resistances of 20-50 Mohms will be idea, and anything less than 10 Mohms will not be useful. Second, you will need to be able to pass current through your electrode, as well as recording from it. This adds a whole new set of amplifier knobs to twiddle before your electrode and amplifier are ready for cell penetration and recording. In addition to the DC balance and capacitive compensation with which you are familiar, you will also have to both monitor and compensate for the injected current pulses. This is necessary in order to be sure that recorded events are due to voltage responses of the cell and not just passive behavior of the electrode. If you find this to be onerous, just be grateful that you can both stimulate and record from a single electrode, i.e. that you don't have to hit and hold the same tiny neuron with two separate recording and stimulating electrodes. Third, you will be looking for a variety of simultaneously occurring neuronal potentials, including resting potentials, postsynaptic potentials, generator potentials, and action potentials, as well as a variety of possible spontaneous activity patterns including quiescence, repetitive spiking, and spiking in bursts. Fourth, you will have to adjust your recording techniques and experimental design "on the fly" based on the observed spontaneous and induced activity patterns of each cell from which you record.
If this sounds daunting, that's because it likely will be. However, success will have its own rewards. Across the class we will, hopefully, see a broad range of potentials and activity patterns, involving active cellular channels and conductances that go way beyond Hodgkin and Huxley. We will see postsynatic potentials (PSPs) and use injected currents to reveal the channel reversal potentials, and hence the ions that mediate these. We will see action potentials that depend on both sodium and calcium inward currents. We will see cells which have no stable resting potential and either produce simple repetitive APs, or bursts of APs riding on an underlying slow oscillation of the membrane potential. We may be able to induce activity changes by applying neuroactive chemicals which act as neurotransmitters, channel agonists (mimics), channel antagonists (blockers), or neuromodulators. We may see some phenomena which we will not be able to explain or account for. And we may see some entirely new phenomena which no one has seen or reported on before.
For this lab you will loosely follow closely the instructions for recording from Helix aspera neurons, as described in Lab #5 of the Crawdad manual. Previewing the video clips of surgical and ganglion preparation techniques will also be extremely valuable and save you a lot of time.
I. SETUP
A. Snail Surgery
Please handle ALL of the surgical instruments used in this lab with extreme care, especially the very fine Vanna scissors and Dumont forceps. These are expensive, irreplaceable, and will likely be irreparably damaged if they are dropped or allowed to rust.
The tiny Minuten pins took some time and effort to manufacture "in house". Try NOT to lose them. Keep them imbedded in the Sylgard of the chamber when not in use, and rinse the chamber after each use.
1) Choose either a Lymnaea or Helisoma snail.
2) Watch the appropriate dissection video at least twice. The surgical procedures for the two species are similar, but not identical and conform loosely to the sequence below.
3) Anesthetize the snail by placing it in 5% EtOH or 10% Listerine for 5-10 minutes.
4) Carefully peel back and chip away the shell from the head of the snail using iris scissors, fine forceps, and/or your thumbnails. Continue until the head and mantle are completely exposed.
5) Use straight pins to pin out the snail in a large Sylgard-lined Petri dish, filled with standard Snail Ringer's. Make sure that the snail is pinned ventral side down. Place one pin though the center of the body and visceral mass posterior to the mantle, then one pin through the head to either side of the antennae. Fold the mantle back from the head and pin it on each side.
6) Using the smallest iris (Vanna) scissors, make a shallow midline dorsal cut through the mantle and up to the anterior end of the head. Reflect the outer body wall back and pin it to each side.
7) Remove the overlying reproductive organs.
8) Locate the midline upper digestive system, with the multi-lobed brain wrapped around the esophagus and the anterior buccal mass. The easiest way to identify the brain is by following the white fibrous connectives leading into it from all sides.
9) Cut through the esophagus posterior to the brain, carefully pull it out through the brain, and pin it out in front, so that the ventral surface of the buccal mass is now exposed.
10) Locate the tiny two-lobed buccal ganglion on the exposed ventral surface on the buccal mass. Again, the easiest way to find the ganglion is by following the white fibrous connectives leading into it from all sides.
11) Prepare one of the small plastic Petri recording dishes by filling it with ~1mm of snail Ringer's.
12) Use the fine Vanna scissors to carefully detach the buccal ganglion by cutting through each connective, as far from the ganglion as possible. Leaving long connectives attached will make the ganglion easier to handle, make it easier to pin out, and minimize damage to the neurons within the ganglion. Using the finest Dumont forceps, transfer the ganglion to the recording dish, grasping only the connectives and not the ganglion itself.
13) Relocate the brain and cut through the dorsal commissure. This will "unfold" the brain and make it easier to see the multiple lobes in each hemisphere. Carefully detach the brain and transfer it to the recording dish, again cutting each connective as long as possible and handling only the connectives.
14) Use the tiny Minutin pins in the recording dish to pin out both the brain and the buccal ganglion dorsal side up. Try to pin both out near the center of the dish, so that they will be more accessible when it comes time to record from them with microelectrodes.
B. Desheathing the Ganglia with Protease
1) Use a plastic pipet to remove the Ringer's from the two ganglia and immediately cover each with a droplet of protease solution. Leave the protease on the buccal ganglion for ~1 minute and on the brain for ~2 minutes. When you remove the protease, immediately recover each ganglion with fresh Ringer's. Do not leave either ganglion exposed to the air for more than 20 seconds. If you leave the protease on two long the ganglia will simply turn to mush and be unusable.
2) When you are finished with the protease treatment, carefully rinse both ganglia by replacing the Ringer's at least twice more, then add enough Ringer's to refill the dish, so that the ganglia are covered.
3) If necessary, re-pin each ganglia so that it is stretched and relatively immobile. If the ganglia can move, then impaling cells with a microelectrode will be much more difficult.
C. Recording Setup
1) Pull at least four microelectrodes on the Sutter Instruments puller, using the program specified by the instructor. Fill and mount one in the wire-core half-cell holder and at least one in a pellet half-cell holder.
Note: Use 0.6 M K2SO4 to fill the electrodes.
2) Turn on the fiber-optic microscope lights, the PC, the function generator, the audio amplifier, and the PowerLab.
3) Set the Audio Selector Switch to VCO and the audio amplifier to TUNER with the MONO switch out. Adjust the function generator and the audio amplifier to produce a pleasant tone at a low volume.
4) Turn on the Model 1600 Neuroprobe amplifier with the switch on the back. Confirm that the OUTPUTS are connected as follows: X1 to Powerlab Ch1, X10 to the function generator VCO input (on the back), and Current to PowerLab Ch 2. Confirm that the CURRENT INJECTION TRANSIENT inner knob is turned all the way counter-clockwise (off), both CAP COMP knobs are turned all the way counter-clockwise (minimal), and all of the black push-buttons (except METER OFF) are in the out (off) position.
5) Start Scope. Set Input A to Ch 1 and Input B to Ch 2. Set both input channels for single-ended recording with no filters. Set Ch A to 200mV range and Ch B to 100mV range. Set the time base to 200mesc and maximal sampling rate. Set Sampling to Repetitive. Adjust the screen display so that Ch A occupies about 2/3 of the vertical display area. At this point, you may want to save a Scope Settings file named "Snail Brain Settings" to the desktop, to make setup faster for future sessions.
Note: Input A (Ch1) and the audio tone will monitor the voltage at the microelectrode, exactly as in the previous muscle RP lab. Input B (Ch2) will monitor the current injected through the microelectrode by the Neuroprobe amplifier. The display scale for CH2 is 10mV per 1nA injected current.
6) Check that all of the ground and shield cables within the cage and between the cage and the PowerLab and amplifiers are appropriately connected.
D. Electrode Testing and Compensating
1) Position either a Petri dish or a ganglion chamber filled with Ringer's in the center of the microscope stage. If necessary, secure it in place with a few small pieces of clay.
2) Mount an electrode and holder to the headstage in the micromanipulator. Position the electrode tip in the Ringer's. Make sure that either the headstage ground pellet, or the cage shield ground pellet is in the Ringer's bath.
3) To zero, impedance test, capacitively compensate, and current compensate your microelectrode, follow the entire Set-Up Procedure on pages 9 and 10 of the Neuroprobe Amplifier Manual, up to step #20. Only use electrodes with a tip impedance between 10 and 60 Mohms.
4) Congratulations! You are now ready to record from your snail brains for the remaining five minutes of the lab session (hopefully more). Save sufficient traces to complete Data Sheet Item # 1 first to demonstrate this.
II. INITIAL NEURON RECORDING
A. General Guidelines
1) In general, keep Scope activated and on Repetitive Sampling mode while you try to stick and initially evaluate cells. Switch to Multiple Sampling mode to save data traces. Alternatively, you can switch over to Chart, keep the screen scrolling and toggle the Save Data mode (at the lower right) on and off as necessary.
2) Change your Scope (or Chart) settings as often as you need to to match and capture what you are trying to record. A good chunk the entire first half of this semester has been practical training in how to do this.
3) Save your data as frequently as you can, so that you don't lose anything interesting. Keep good notes so that you can find relevant traces later on.
4) BE PATIENT AND PERSISTENT. Do not throw away any snail brain preps unless you are absolutely certain that they are irrevocably damaged.
5) Always keep a mounted electrode or two as a backup, so that you can switch them out rapidly. If you are sitting around with nothing to do - go pull, fill, and mount some electrodes.
6) If everything seems to be fine, except that you just can't seem to successfully stick any cells, then you may need another protease treatment. Carefully remove the electrode (always turn off the Neuroprobe amplifier first), remove the recording chamber, and repeat the sequence in IB above. Be sure to rinse out the protease with several careful Ringer's bath replacements.
B. Stick a Cell
Assuming that you have successfully extracted, pinned out, and desheathed one or more ganglia, that the recording chamber is mounted on the microscope stage, and that the electrode/amplifier have been correctly zeroed, capacitively compensated, and current compensated:
1) Carefully reposition the recording chamber so that the ganglion and spot from which you want to record is near the exact center of the stage. Secure the chamber with a few small pieces of clay.
2) Carefully lower your electrode until the tip is in the immediate vicinity of your candidate cell. Maximally zoom in the microscope and refocus.
3) Advance your electrode tip using the fine advance knob on the micromanipulator, watching and/or listening for evidence that you have penetrated the cell. As with the muscle, successful penetration of the cell will be accompanied by a drop in voltage (the resting potential) and a rise in audio monitor tone pitch.
4) Remember your tricks for sneaking into cells. When the electrode tip is up against the cell, the signal will get noisy, due to the high impedance of the covered tip. You can often penetrate the cell by "buzzing in" with the Cap Comp Override, or by GENTLY tapping the micromanipulator knob.
III. POSSIBLE EXPERIMENTS
There are a multitude of possible experimental protocols and recording regimes that you can try once you have successfully stuck a cell. Which approach is most likely to yield interesting results will depend on the nature of the "native" activity pattern of your cell. The instructor and TA will help you choose a procedure which seems most appropriate for your particular cell. Some of the possibilities are sketched out below, along with possible Data Sheet Items showcasing your findings.
A) My Cell is Quiescent with a Stable Resting Potential
1) Try depolarizing the cell by injecting continuous positive current. Set the CURRENT INJECTION switches to + POLARITY and CONT (continuous). Slowly turn the CURRENT know clockwise to increase injected current and deplolarize the cell.
2) Look closely for transient negative voltage events. These may be shunting IPSPs carried by chloride currents. Such IPSPs will reveal themselves as voltageevents only when the membrane is driven away from rest by injected current. If you see such events, then record several traces at different + and - injected current levels. Assuming that chloride is passively distributed (not pumped or exchanged), then ECl should be about the same as VRest. The reversal potential for these events will also equal the resting potential and the polarity of the PSP will flip over or reverse (- to +) as Vm goes from above VRest to below VRest.
3) If you don't see any IPSPs, continue to slowly increase the injected positive current and see if you can induce action potentials in the cell. The most likely patterns will be a) sustained repetitive firing, b) a stable pattern of repetitive "bursts" of APs, or c) one or more APs or bursts which space out and then stop. How best to proceed will depend on which pattern you see:
a) Tonic (Sustained) Periodic Firing - Incrementally increase the injected current and record one or more Scope traces at each level. Keep good written notes or Scope trace internal labels, recording the level of applied current for each trace. You can read the level of applied current from display Channel B or by setting the Neuroprobe LED display to CURRENT. You will ultimately want a data set which will allow you to plot and illustrate the relationship between injected current level and AP firing rate.
b) Tonic (Sustained) Periodic Bursting - You will probably want to either increase the time base, or switch over to continuous Chart recording at a high sampling rate for this. Incrementally increase the injected current and record one or more Scope traces at each level. Keep good written notes or Scope trace internal labels, recording the level of applied current for each trace. You can read the level of applied current from display Channel B or by setting the Neuroprobe LED display to CURRENT. You will ultimately want a data set which will allow you to plot and illustrate the relationship between injected current level and such variables as burst frequency, # APs per burst, or intraburst AP frequency.
c) Phasic (Diminishing Rate) Repetitive Firing or Bursting - You will definitely want to switch over to continuous Chart recoding at a high sampling rate for this. Start with the CURRENT switch turned OFF. Preselect a level of current to apply, using the CURRENT knob and POLARITY switch, monitoring this on the Neuroprobe LED METER set to CURRENT. To apply the current, depress the CURRENT switch to MOMEN (momentary) for as long as required for the cell to begin firing and then accommodate back to quiescence. Test and record several current levels, keeping notes so that you can find and interpret your data.
4) Use high sampling rate recording and the display ZOOM function to closely examine several APs. Are the APs smooth and relatively short (<10msec) in duration. If so, they are "Na APs" mediated mostly by inward sodium currents. Are the APs longer (>10msec) or do they show a "shoulder" during the repolarization phase? If so, they are, at least partially, "Ca APs", mediated in part by inward calcium currents.
B) My Cell Has a Resting Potential with Superimposed PSPs
1) Save multiple traces of the native activity to answer the following questions: Are all of the PSPs identical in polarity and amplitiude? Do the PSPs ever overlap and summate?
2) Again, try injecting both negative and positive current in the CONT (continuous) mode. Can you discover the reversal potential for any of the PSPs? What does this tell you about most likely ions mediating these PSPs? PSPs might be expected to reverse at about:
-90mV for K+ conductances
-70mV for Cl- conductances
-20mV for non-selective Na+/K+ conductances
+60mV for Na+ conductances
+100mV for Ca++ conductances (unlikely)
3) As you depolarize the cell with + injected current, do depolarizing EPSPs lead into APs? Alternatively, can you see negative-going IPSPs interspersed with the APs?
4) Can you induce the cell to fire upon the release of a sustained negative applied current? This is the anode-break phenomenon.
C) My Cell Has a Native Activity Pattern of Spontaneous Firing of Individual APs
1) Save several traces to document this spontaneous activity pattern. Is it periodic or irregular?
2) Apply continuous positive current to depolarize the cell and negative current to hyperpolarize the cell. There are two likely consequences:
a) Firing is Periodic and Rate Depends Directly on Applied Current - Your cell is probably intrinsically active, suggesting a steady leak conductance which continuously depolarizes the cell, initiating each AP. Save enough traces to document the relationship between applied current and firing rate.
b) Applied Current Does Not Directly Affect Firing Rate - Your cell is probably being synaptically driven by other cells. Applying a negative current can suppress APs and reveal the underlying PSPs which are driving them. Applying a sufficient level of positive currentcan also suppress APs by preventing the cell from recovering from H-H sodium channel inactivation and potassium channel activation.
3) Again, do your APs seem to be pure Na, pure Ca, or hybrid Na/Ca APs (see section IIA4 above)? What are the apparent reversal potentials for the underlying EPSPs.
D) My Cell Has a Native Activity Pattern of Spontaneous AP Bursts
1) Save several traces to document this spontaneous activity pattern. Are the bursts periodic or irregular?
2) Apply continuous positive current to depolarize the cell or negative current to hyperpolarize the cell. There are two likely consequences:
a) Bursting is Periodic and Rate Depends Directly on Applied Current - Cool!! Your cell is probably an intrinsic burster, capable of acting as a central pattern generator all by itself! Save enough traces to document the relationship between applied current and the rate of bursting. Record several high sampling rate records of individual bursts. Does the firing within each burst seem to first accelerate, then decelerate, with a little positive bump right at the end of the burst? Way cool!! Your cell is a parabolic burster. Ask the instructor to help you do a high-speed spike-triggered recording for a single burst to document how AP spacing and duration change as the burst progresses.
b) Applied Current Does Not Directly Affect Bursting Rate, but May Affect Timing and AP #s Within the Burst - Your cell is probably a driven burster, being synaptically driven into a bursting "regime" by input from other cells. Experiment with applying positive and negative currents at different levels to determine whether the cell is capable of displaying other activity patterns. Experiment with applying transient negative currents within a burst to see what happens.
IV. OTHER POSSIBLE EXPERIMENTS
If time permits we can explore other aspects and dependencies of spontaneous activity in snail brain neurons. These may include:
1) Ionic dependence of APs using ion substituted Ringer's solutions.
2) Temperature dependence of spontaneous activity patterns
3) Pharmacological dependence of activity patterns using neurotransmitters, neuromdulators, channel agonists (mimics), and/or channel antagonists (blockers or uncouplers).
V. SHUTTING DOWN AND CLEANING UP
1) Make sure that you have saved all of your data to the hard drive, then quit Scope. Turn off the PowerLab box. Turn off the Neuroprobe amplifier. Turn off all of teh other electronic hardware.
2) Under the microscope, remove your snail ganglia and dispose of them. Replace the Minutin pins in the Sylgard - they are NOT disposable! Carefully flush out the chamber with RO water, then recap it. Flush out your larger glass Petri dish and dispose of all extraneous snail parts
3) Carefully and completely clean and completely dry ALL of your surgical instruments, especially the fine scissors and forceps.
4) Make sure that both the microscope and fiber-optic lights are turned off. Make sure that both micromanipulators are magnetically secured to the steel plate.
5) Make sure that the Microelectrode R/C Meter and the Sutter Instruments electrode puller in the back of the room are turned off.
6) Account for and rinse out ALL of the electrode half-cell holders that you used. Dispose of ALL of the used pipet microelectrodes in the Sharps container. DO NOT throw out the half-cell holders!
7) Return all Ringer's solutions to the refrigerator and all protease tubes to the freezer in 103.
VI. PREPARATION OF THE LAB DATA SHEET
Your data sheet should include all THREE of the items described in the boxes above (remember that DS#2 requires TWO data sets. 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