Snail Brain: Diversity of Neuronal Excitability
Introduction
The nervous systems of gastropod molluscs have been models to explore fundamental processes of brain function, including neural network plasticity underlying learning and memory (Elliott and Susswein 2002; Byrne et al., 2009). A snail brain consists of several ganglia, the circumesophageal ganglia, fused to form a ring around the esophagus (Figure 8.1). Each ganglion contains a relatively small number of neurons. Compared to vertebrate neurons, these neurons are very large, with cell body diameters up to hundreds of microns. Many of these cells are consistently identifiable between animals. The small number, large size, and identifiability of individual cells have made invertebrate preparations, such as the snail, good models for study of many basic system, cellular, and molecular properties of neurons. The cell bodies are arranged in a single layer around the central neuropil of a ganglion. An invertebrate ganglion is similar in structure to an orange, with the rind consisting of cell bodies and the inside being the neuropil in which synaptic connections are made. This is the arrangement in crayfish as well as snails.
In this lab exercise, you will record from and characterize a variety of neurons from the subesophageal and buccal ganglia of snails. This exercise was originally designed using the land snail Helix aspersa, but pond snails of the genera Lymnaea and Helisoma can be used instead (for an overview of Lymnaea, see Benjamin, 2008). You will see neurons with a variety of activity patterns, including silent cells, tonically firing cells, rhythmically bursting cells (that fire regularly repeated groups of action potentials), cells with different responses to current injection, and cells with different ionic mechanisms of action potential generation. Like the different classes of mammalian brain neurons, each type of snail brain neuron has a unique “electrical personality” suited to its function in a neural network (McCormick, 2009; Toledo-Rodiguez et al., 2005). This characteristic pattern of spontaneous and evoked activity depends on the neuron’s population and distribution of ion channels.
Dissection
The snail brain dissection is similar for all species of snails. The description and videos below are for the land snail Helix aspersa but will give you a good general understanding applicable to any species. Your instructor may also direct you to different videos for other species.
The Helix dissection has four main parts. First, you will open the snail and remove a mass of gut with the ring of circumesophageal ganglia attached. Second, you will pare this down to the point where only the ganglia and their associated nerves remain and pin the ganglia out ventral side up. Third, you will cut through the aorta, a tube running through the subesophageal ganglia, remove the pedal ganglia, and pin the ganglia dorsal side up. Finally, you or your instructor will remove a sheath of fat and connective tissue that covers the ganglia.
- Before starting the dissection, anesthetize a snail by injecting it with 2 ml of 50 mM MgCl2. This is not shown in the videos; ask your instructor for help. Anesthetize pond snails by placing them in a 5% ethanol solution mixed in pond water and wait until they stop moving.
- Video 8.1, Entire Snail Dissection, shows the entire dissection up to desheathing. You may wish to watch this for an overview or for review after reading the descriptions and watching the videos below.
- Figure 8.2 and Video 8.2, Removing the Shell. Remove the anterior portion of the snail’s shell by splitting it with scissors and peeling it away. Place the snail on a piece of foam. When it has stretched out, put a pin through the visceral mass where the shell was. If the snail retracts, wait for it to stretch out fully again and put another pin through the head between the four tentacles.
- Figure 8.2 and Video 8.3, Removing Ganglia. Place the foam with the pinned snail under the microscope at its lowest power. Use medium scissors to cut posterior to anterior along the white line that runs in the center of the dorsal surface of the snail. Cut all the way to the anterior tip of the head, keeping the scissor tips shallow so that no underlying tissue is damaged. Pin out the skin to each side of this cut. You should now see the gut, a thick orange tube, with the light-colored cerebral ganglia on top of it.
- If you are unable to find the gut and brain at this point, try moving the shiny white structures (part of the reproductive system) and pinning them out of the way. If you still cannot find the brain, proceed with the next step and ask your instructor for help finding the brain later.
- Grasp the gut near the posterior pin and lift it. Cut down vertically through the gut behind your forceps and into the floor (thick foot tissue) of the snail. Gently lift the gut and the attached tissue under it and cut beneath it to free the tissue you are holding. Keep cutting anteriorly as close to the floor of the snail as possible. It is important to cut as close to the floor as possible to avoid damaging the subesophageal ganglia. Finally, cut through the gut at its most anterior attachment to the body wall. You are now holding a mass of tissue that includes the gut and brain. If you take more than 5 min to do the above steps, rinse the snail occasionally with saline to keep it from drying out.
- Figure 8.3a and Video 8.4, Pinning. Place the tissue mass in a dissecting dish filled with snail saline and hold it down with minutien pins in the outside edges. Find the brain and esophagus (use the dark-pigmented eyespots mentioned in the video as a landmark). Free the ganglion ring from the other tissue by cutting the nerves going to the tissue. Keep the nerves as long as possible. Pin the ring ventral side up very tightly but do not pin into the ring of ganglia itself. (The ventral side of the subesophageal ganglia has many nerves leaving it, while the dorsal side is relatively smooth.)
- Figure 8.3b and Video 8.5, Cutting the Aorta. Find the aorta, a hollow tube running through the subesophageal ganglia. It is easiest to find the aorta on the inside of the ring. It should be near the surface of the subesophageal ganglia if the ganglia are ventral side up. Insert a blade of fine (Vannas) scissors into the aorta opening inside the ring. Follow this tube and slit the aorta toward the outside of the ring along its whole length, cutting through the pedal ganglia. The ventral subesophageal ganglia should now fall open, leaving the smooth walls of the aorta separating the cut from the parietal and visceral ganglia underneath. Do not cut below the smooth walls of the aorta. Carefully cut away the pedal ganglia.
- Figure 8.3c and end of Video 8.5, Cutting the Aorta. Unpin the nerve ring and turn it over so the dorsal surface is up. Pin it again tightly stretched out.
- Video 8.6, Desheathing (not necessary for pond snails). The subesophageal ganglia are now ready for desheathing. Your instructor will probably do this for you. To desheathe the ganglia, find a nerve entering the ganglia, especially one of the larger nerves near the midline, and grasp the sheath above the nerve with fine forceps. Pull up on the sheath and cut between the raised sheath and the nerve. Slide a scissor blade under the sheath, lift, and snip the edges of the sheath until it can be pulled off completely. It is especially important to use fine sharp scissors and to keep their blades raised above the ganglia under the sheath. You should now be able to see cell bodies in the subesophageal ganglia (Figure 8.4).
- At this point, there is still a thin sheath covering the neurons. It is like a tight cellophane wrapper and will break electrode tips if you try to record from cells now. Apply a solution of protease enzyme mixed in snail saline to the preparation to disrupt this sheath. Leave this on for 20 to 30 min at room temperature and then replace it with fresh cold saline. You can begin recording while the brain is in protease solution, but don’t forget to replace it with saline.
- After the dissection, clean your dissecting tools thoroughly. Do not let snail slime dry and harden on them. If that happens, soak the tools in warm water with a little dishwashing detergent. Rinse and wipe the foam sheet. Put leftover snail parts in a bag in the freezer.
Recording
Figure 8.5 shows the setup for intracellular recording. Snail brain cell recordings require a more delicate approach than the crayfish muscle recordings did. First of all, you need sharper electrodes (15 to 30 MΩ). Second, instead of driving the electrode into the cell with the manipulator, you will need to bring the electrode tip gently to the cell surface and then use the Buzz button on the amplifier to enter the cell. This sequence is shown in Video 8.7, Recording, for one cell. In this recording, good lighting is crucial. It is best to have some light coming through the ganglia from below (to help you see cells) and some from the side (to help you see the electrode). The brain cells are also easier to see if there is only a small volume of saline covering the ganglia.
Pull a pair of glass microelectrodes and place them, sharp end up, in a beaker with a small amount of 3 M KCl. After a few minutes, the tip of the electrode 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), then put it in the electrode holder. With the micromanipulator, place the tip of the electrode in the saline over the exposed brain and measure the electrode resistance. (The procedure for measuring resistance varies with different amplifiers; ask your instructor for help.) A resistance of 15 to 30 MΩ is good. If the resistance is below 10 MΩ, the electrode is probably broken. If the resistance is much over 40 MΩ it is probably clogged; an extended press of the amplifier’s Buzz button may clear it. Be sure to dispose of broken or used electrodes safely in a closed canister. For more information on intracellular recording, see Appendix C, Recording Tips.
At high magnification, focus the microscope on the tip of the electrode. (The actual tip is too small to be seen and is somewhat lower than what you are focused on.) Adjust lighting for best visibility of the electrode; your instructor can suggest ways to make the electrode more visible. Now bring the electrode closer to the brain, refocusing along the way, until the visible part of the electrode appears to be a short distance from the ganglia. If you have trouble locating the electrode tip, try using the manipulator to move it slightly back and forth horizontally; this movement will make the tip easier to find than if it is stationary. Choose a cell body and position the electrode tip just over it, compensating for the fact that the tip is a little beyond what you can see. After this, looking under the microscope is no longer useful because the electrode tip is invisible. Instead, watch the oscilloscope and use the fine-adjust knob of the manipulator to advance the electrode. When the oscilloscope trace starts to wobble slightly, the electrode is just against the cell membrane. Push the Buzz button on the amplifier to get into the cell. When the electrode is in a cell, the oscilloscope should indicate a negative voltage, the resting potential, and will often show spontaneous activity of the cell. This sequence is shown in Video 8.7, Snail Brain Recording, for one cell. If you do not get into the cell at first, move the electrode down very slightly and buzz again. Repeat this process until you get into the cell or it is obvious that the electrode is deep into the ganglion.
Most cells will fire action potentials after the initial penetration because of membrane damage; give them some time to recover. The resting membrane potential can vary greatly, from a silent cell at −70 mV to an actively firing cell at −30 mV. If a cell does not fire when you inject depolarizing current, it may be a glial cell or a damaged neuron.
Experiments
Record from and describe the activity of at least five different neurons of the visceral and right parietal ganglia. If time is limited, concentrate on recording the characteristics of one or two cells and share data with other lab groups. It is easier to record from large cells than small ones, but it is not necessary to aim the electrode at a specific cell. Draw the ganglia and note the locations of the cells from which you record. Try to identify the cells on previously published maps of the ganglia (Figure 8.6 for Helix; see Murphy, 2001, for pond snail buccal ganglia). Characterize the physiological properties below as applicable.
First, classify the cell by its activity level. Is it silent? Does it fire spontaneous action potentials intermittently, in a regular rhythmic pattern, or in regular bursts? Are postsynaptic potentials (PSPs) visible? These could be excitatory (EPSPs), inhibitory (IPSPs), or EPSPs leading to spikes. Depending on the activity level, measure the following properties. Use Figure 8.7 as a guide when making the following measurements.
Silent Cells
- Record the resting potential.
- Stimulate the cell by injecting depolarizing current. Record the response (number of APs vs. current amplitude and duration) at different levels and durations of current. Begin with 0.5 nA and 1000 ms. If you are unable to elicit spikes at any level of depolarization, abandon this cell and record from another one.
- Measure the amplitude of the first spike (baseline to peak and baseline to trough separately). Does spike amplitude change over the course of the stimulus?
- Measure the duration of the first spike at half-amplitude (half of the way from baseline to peak). Spikes that rise and fall quickly with durations under 10 ms are based on Na+. Spikes with durations of 20 ms or greater that fall slowly in two phases are based on Ca2+. Intermediate spikes based on both ions can also be seen (Kerkut and Gardner, 1967).
- Determine the relative refractory period by noting the longest time interval at which the second spike begins to be smaller than the first.
- Determine the absolute refractory period. First determine the level of current injection at which the number of APs no longer increases with increased current levels. At this level of current injection, measure the interval between the first two spikes.
- Describe any changes in the duration of APs in the train of elicited spikes.
Spontaneous Tonically Firing Cells
- Record the firing frequency (APs per second) without current injection.
- Estimate the resting potential by measuring the membrane potential immediately after a spike.
- Estimate the threshold for AP generation. The depolarization phase of the AP starts with a shallow slope and then depolarizes rapidly. The transition between these phases is the threshold.
- Measure the spike amplitude and duration as described for silent cells.
- Inject varying amounts of depolarizing and hyperpolarizing current and record the effect on activity level, resulting in plots of AP frequency vs. stimulus amplitude or duration. Also note any changes in spike amplitude or duration at different potentials.
Bursting Cells
Use Figure 8.8 as a guide in making the following measurements.
- Measure the burst frequency (bursts per second) and number of spikes per burst.
- Record the amplitude of the oscillating potential by measuring from the bottom of the trough between bursts to the level at which spikes begin to fire.
- Determine the threshold for AP generation. This is the potential at which spikes begin to fire.
- Measure the spike amplitude and duration as described for silent cells.
- Look at the spikes during a burst. Does the spike amplitude, interspike interval, or depth of afterhyperpolarization after a spike change during a burst?
- Inject varying amounts of depolarizing and hyperpolarizing current and record the effect on burst frequency and duration and on the number of spikes within a burst.
- Does excitatory synaptic input contribute to initiation of a burst? Look for excitatory postsynaptic potentials leading up to AP firing.
Cells with Synaptic Potentials
- Inject depolarizing and hyperpolarizing current and determine the effect that each has on the amplitude of the PSP and the probability that the cell will fire an action potential.
All Cells
- Check for the presence of postinhibitory rebound (Figure 8.9). Inject a 3 to 5 nA, 1 to 5 s hyperpolarizing current. At the end of the current pulse, note whether (1) a silent cell returns briefly to a more depolarized RP than before and may even fire an action potential, (2) a spontaneously active cell briefly fires at a higher rate than before, or (3) a bursting cell fires a burst immediately at the end of the pulse.
Examples
- Video 8.8, Na+ Potentials, shows a cell that fired one action potential when the electrode penetrated it and was then silent. It fired action potentials only when depolarizing current was injected. Note the short duration of the action potential. The action potentials are probably based on Na+.
- Video 8.9, Ca2+ Potentials, shows a spontaneously active cell. Note the long duration of the action potentials and their characteristic shapes. This action potential is probably based on Ca2+. (Human heart muscle fires calcium-based action potentials that look very similar to these.)
- Video 8.10, Bursting, shows a cell that spontaneously fires rhythmic bursts of action potentials.
- Video 8.11, Stimulation, shows another spontaneously active cell with action potentials based on calcium. Note the decrease in frequency after the end of the depolarizing pulse and the slight increase in frequency after the end of the hyperpolarizing pulse.
- Video 8.12, Synaptic Input, shows a cell that fires an action potential after receiving a postsynaptic potential from another cell.
- Figure 8.9 shows an example of postinhibitory rebound in a normally silent cell.
Further Exploration
Try to explore as many cells as possible with the above protocol. The more cells you record from, the more you will appreciate the diversity of neuronal “personalities” present even in a small snail brain. You may want to design your own experiments to examine the effects of electrical stimuli other than those covered above, keeping in mind what these perturbations might tell you about a cell’s intrinsic currents. In addition, you can carry out the following experiments.
- Examine the effect of brief positive and negative current injections on the behavior of a bursting cell. For example, when in the cycle can a depolarizing current pulse advance the onset of the next a burst? When in the cycle can a hyperpolarizing current pulse delay the next burst?
- Examine the effects of neuromodulators such as serotonin and dopamine on the burst characteristics of bursting cells (for example, Quinlan et al., 1977).
- Nerves leading into the ganglia can be stimulated with a suction electrode, altering activity of the neurons (Kerkut et al., 1975).
- Determine the input resistance (Rinput) and membrane time constant (τ) of a neuron using small hyperpolarizing current pulses. Rinput is a measured resistance of the whole cell; it includes membrane, internal, and external resistance (Equation 5, Rinput = ½(Rm(Ri + Ro))½). See Lab 1, Membrane Properties, for more information. Ask your instructor for help making an accurate measurement of the voltage across the membrane using a single electrode.
Lab Cleanup
During the lab, be sure to immediately discard used glass electrodes in the appropriate container. After the lab, remove your last electrode from its holder and discard it as well. Clean up any spilled saline and rinse the ground electrode with distilled water. Put the snail brain in the trash and rinse the dissecting dish with fresh water.
Questions
- Present a map showing the locations of the recorded cells. Compare this with previously published maps of these ganglia (Figure 8.6; Kerkut et al., 1975; Murphy, 2001) and try to identify the cells from which you recorded.
- Present a table of characteristics of the cells from which you recorded, including resting potential, type of activity, presence of synaptic input or postinhibitory rebound, spike size and duration, and any other features you observed. Illustrate the different types of cells with example traces from your recordings.
- Present graphs showing neuronal responses to different levels and polarities of current injection and any other interesting quantitative observations you made.
- Did you observe any silent cells that fired only a few action potentials during a continued depolarizing stimulus? This type of response is called accommodation or spike rate adaptation. How would accommodation affect the response of a sensory neuron to the continued presence of a stimulus? What is accommodation called when it happens in a sensory system? How is it important in perception (see Lab 7, Stretch Receptor)?
- This exercise should suggest to you that neurons may have many different types of currents to produce such varied electrical activity. Suggest some specific types of currents that might account for the activity patterns you observed (McCormick, 1999; Toledo-Rodriguez, 2005). In particular, suggest ionic mechanisms for (a) spike-frequency accommodation and (b) rhythmic bursting activity (initial depolarization, depolarization maintenance, AP firing, and burst termination). Finally, some mammalian brain neurons show very different activity in wake and sleep states; consider the ionic currents that cause a thalamic neuron to fire tonically during wakefulness and rhythmically during sleep (McCormick, 2009).
- Suggest two possible mechanisms for postinhibitory rebound.
References
- Benjamin PR (2008). Lymnaea. Scholarpedia 3:4124. [doi]
- Byrne JH, LaBar KS, LeDoux JE, Lindquist DH, Thompson RH, Teyler TJ (2009). Learning and memory: Basic mechanisms. In: Byrne JH, Roberts JL (eds.), From Molecules to Networks: An Introduction to Cellular and Molecular Neuroscience (Academic Press, San Diego), pp. 539-608.
- Elliott CJ, Susswein AJ (2002). Comparative neuroethology of feeding control in molluscs. J Exp Biol 205:877-896. [pdf]
- Kerkut GA, Gardner DR (1967). The role of calcium ions in the action potentials of Helix aspersa neurones. Comp Biochem Physiol 20:147-162. [doi]
- Kerkut GA, Lambert JDC, Gayton RJ, Loker JE, Walker RJ (1975). Mapping of nerve cells in the suboesophageal ganglia of Helix aspersa. Comp Biochem Physiol A 50:147-162. [doi]
- McCormick DA (2009). Membrane potential and action potential. In: Byrne JH, Roberts JL (eds.), From Molecules to Networks: An Introduction to Cellular and Molecular Neuroscience (Academic Press, San Diego), ch. 5.
- Murphy AD (2001). The neuronal basis of feeding in the snail, Helisoma, with comparisons to selected gastropods. Prog Neurobiol 63:383-408. [doi]
- Quinlan EM, Arnett BC, Murphy AD (1997). Feeding stimulants activate an identified dopaminergic interneuron that induces the feeding motor program in Helisoma. J Neurophysiol 78:812-824. [pdf]
- Toledo-Rodriguez M, El Manira A, Wallen P, Svirskis G, Hounsgaard J (2005). Cellular signalling properties in microcircuits. Trends Neurosci 28:534-540. [doi]