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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.

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

Spontaneous Tonically Firing Cells

Bursting Cells

Use Figure 8.8 as a guide in making the following measurements.

Cells with Synaptic Potentials

All Cells

Examples

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.

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

  1. 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.
  2. 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.
  3. Present graphs showing neuronal responses to different levels and polarities of current injection and any other interesting quantitative observations you made.
  4. 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)?
  5. 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).
  6. Suggest two possible mechanisms for postinhibitory rebound.

References