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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). A pair of small buccal ganglia connected to these controls rhythmic feeding movements.
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. Cell bodies are arranged in a single layer in the surface of the ganglion, surrounding a central neuropil of dendrites and axons. This is similar to the organization of our brains, where gray matter surrounds white matter.
Pond snail buccal ganglia have been used as models of rhythmic control circuitry. The ganglia are smaller and more accessible than the vertebrate spinal cord circuitry that control rhythmic behaviors such as locomotion, while their neurons are relatively large and easy to record from. Video 11.1, Feeding Behavior, shows one such rhythmic behavior. In this lab exercise, you will record from motor nerves that extend from the buccal ganglia to muscles of the buccal mass, which are involved in scraping and swallowing food. For background, see Murphy (2001) and Ramakrishnan et al. (2014) for Helisoma or Benjamin (2008) for Lymnaea.
This exercise can be done with any pond snail, but Helisoma trivolvis and Lymanea stagnalis are the most studied. The description and videos below are for Helisoma. Lymnaea.
The dissection has three parts. First, you will remove much of the shell. Second, you will open the body cavity to expose the buccal mass and circumesophageal ganglia. Third, you will cut the esophagus and tilt the buccal mass forward to expose the buccal ganglia and nerves. These are diagrammed in Figure 11.2, Dissection Steps.
By now, you have experience recording nerves with suction electroes. The setup here is the same as that used in Lab 2, Nerve Recording (Figure 2.1). However, you may need to increase amplifier gain to 10,000× and may need to adjust filters. Video 11.4, Nerve Recording, shows recording of two nerves. Before starting to record from the nerve, empty the saline out of the dissecting dish and replace it with fresh cold saline.
Use a suction electrode just slightly wider than the nerves. If the electrode is curved, turn it so that it curves downward; a curved electrode may give easier access to some nerves. Bring the suction electrode into the saline and pull gently on the syringe until the tip of the electrode holds enough saline to reach the wire inside the electrode. Place the electrode onto a nerve and apply suction. It is not necessary to suck a loop of nerve into the electrode tip. Unless there is a lot of electrical noise, spontaneously firing spikes should be obvious on the oscilloscope and speaker.
All nerves should have some spontaneous activity. While bursts of spikes may be found in any of the nerves, PBN, LBN, and VBN are particularly active. Be patient — it may take a minute or two before rhythmic activity starts, and the interval between bursts may be long. Slowing down the data acquisition display may make it easier to see activity patterns.
Be sure to keep notes about which nerve you are recording. If your data-acquisition software permits, add these notes directly into your data file as you record. Use Figure 11.3, Buccal Ganglia (Diagram), as a guide. Note that LBN, VBN, and CBC may travel together for some distance from the ganglia before becoming distinct.
If your signal-to-noise ratio is always poor, you may be able to improve it by cutting the nerve and pulling the cut end (ganglion side) into the electrode. However, if you do this with more than 3-4 nerves, the ganglia may flap loosely, making further recording difficult.
See Appendix C, Recording Tips, for general information about recording and troubleshooting procedures.
Feeding stimulants — crushed lettuce, sugar
Neuromodulators — suggest types (dopamine, serotonin) and concentrations (and how to do it); refer to Pharmacopeia section. Suggest concentrations to use. Cholinergic agonists and antagonists (especially for Lymnaea). See Appendix D, Pharmacopeia, for other chemicals and concentrations to try.
Analyzing bursts — diagram of measurements.
Record from two or more nerves simultaneously. Are different nerves on the same side (e.g. PBN and LBN) synchronized? If they burst at the same rate, are they in phase? Are the same nerves on opposite sides (e.g. left LBN and right LBN) synchronized?
Stimulate nerves and observe the effect on buccal mass movements.
Stimulate one nerve and see if it affects activity in other nerves.
Cutting CBC might prevent the feeding rhythm from responding to feeding stimuli (but the response isn’t certain anyway)
Sucrose is reported to stimulate feeding (Benjamin, 2008). Do artificial sweeteners also stimulate feeding?
Eject saline from the suction electrode. Remove the glass electrode from its holder and save it or discard it (ask your instructor). Clean up any spilled saline and rinse the ground electrode with distilled water. Put the snail parts in the freezer or trash (ask your instructor) and rinse the dissecting dish and tools with fresh water.