Browser Warning

The web browser you are using does not have features required to display Crawdad correctly.
Please use the most recent version of one of the following:

Snail Ganglia: Control of Feeding (DRAFT)

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). 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.

Video 11.1, Feeding Behavior, shows one such rhythmic behavior. In this lab exercise, you will record from neurons in the buccal ganglia, which control feeding. For background, see Murphy (2001) and Ramakrishnan et al. (2014) for Helisoma or Benjamin (2008) for Lymnaea.

Dissection

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 [switch to Lymnaea]. Lymnaea [switch to Helisoma].

The dissection has five parts. The first three are the same as in Lab 11, Snail Nerves, and are diagrammed in Figure 11.2, Dissection Steps. (1) Remove much of the shell. (2) Open the body cavity to expose the buccal mass and circumesophageal ganglia. (3) Cut the esophagus and tilt the buccal mass forward to expose the buccal ganglia and nerves. (4) Grasp one of the buccal nerves or connective tissue attached to a nerve and cut all nerves. (5) Place the ganglia in a small dish and pin them out flat by the nerves.

Recording

The ganglia have a sheath covering the cell bodies. Before recording, this sheath must be softened. Remove saline from the dish and blot it away from the ganglia with a tissue. Place a drop of protease solution on the ganglia, let it sit for 2-3 minutes, then flood the dish with normal saline and begin recording. [NOTE: make a video of this]

By now, you should have some experience with intracellular recording. The intracellular recording procedure used here is the same as that used in Lab 4, Resting Potential (Figure 4.1). Here, you need an electrode with a resistance of 10-20 MΩ. Once the electrode is lowered into the saline, and again after recording from each neuron, check the resistance and balance the bridge. Video 12.2, Ganglion Recording, shows checking resistance and balancing the bridge, followed by aiming the electrode for a recording. Your instructor will show how to do this with your equipment. See Appendix C, Recording Tips, for general information about recording and troubleshooting procedures.

When recording, aim the electrode at a visible cell body. If you cannot see cell bodies, adjust the lighting, magnification, and focus; ask your instructor for help if necessary. See the examples below for recordings from several neurons in Helisoma.

Be sure to keep notes about where you are recording. If your data-acquisition software permits, add these notes directly into your data file. Use Figure 11.3, Buccal Ganglia (Diagram), as a guide. You may want to print this diagram and mark recording locations on it.

Example recordings

These are short samples, to illustrate the diversity of neurons found in buccal ganglia. When doing your own recordings, try to hold the recording as long as possible; do stimulation series and neuromodulation experiments as appropriate.

Experiments

Feeding stimulants -- crushed lettuce, sugar (ask why this shouldn't work...)

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.

Further Exploration

Place a suction electrode on a nerve as in Lab 11, Snail Nerves, then record intracellularly from neurons. Look for coordinated activity (spikes in the nerve that correspond to APs in the neuron). What happens if you stimulate the nerve? What if you depolarize the neuron to fire more APs?

Lab Cleanup

Discard glass electrodes and clean out KCl beakers and syringes. Clean up any spilled saline and rinse the ground electrode with deionized water. Put the snail parts in the freezer or trash (ask your instructor) and rinse the dissection dish and tools with fresh water.

Questions

  1. For each neuron you record from, identify some candidate cell types based on the location (refer to Figure 11.3, Buccal Ganglia). Then look up those neurons in Murphy (2001) and Ramakrishnan et al. (2014). Is the activity pattern you saw consistent with the published behavior of a candidate neuron? If it is a motor neuron, which nerve contains its axon? Does its activity resemble any spiking pattern you saw while recording from that nerve in Lab 11, Snail Nerves?
  2. If you recorded rhythmic bursts, determine their frequency (inverse of average time between burst onsets) and duty cycle (proportion of the burst interval during which the nerve is active). Because burst interval can be highly variable, you may need to do this for 2-3 different sections of a recording.
  3. Describe changes that occurred after administration of feeding stimulants or neuromodulators. Be quantitative if possible (changes in burst frequency and/or duty cycle). How long do the effects last?
  4. Propose mechanisms by which feeding stimulants or neuromodulators could initiate or change the feeding motor program (Quinlan et al., 1997; Trimble and Barker, 1984).
  5. If you found that two different neuromodulators had the same effect (e.g. increasing burst rate), how can you account for that?
  6. If you found that one neuromodulator had different effects in two different neurons (e.g. increasing activity in one and decreasing it in another), how can you account for that?
  7. [NOTE: Add questions about mechanisms of rhythmic behavior, intrinsic and exogenous.]

References