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Zombie Snails: Mindless Methodical Movement

A study on central pattern generators (CPGs) that control eating behaviors in pond snails.

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Do you consistently think “breathe in, breathe out” or "left, right, left, right" when you're walking? Unless you're London Tipton (http://dai.ly/x31iwo0?start=346 to 6:02), you probably don't. How is this possible? All humans have neural networks called central pattern generators (CPGs) that control rhythmic movements like breathing and walking. Unfortunately, it is nearly impossible to study this in humans, so we use mushy invertebrates that can show us these CPGs in real time. In the pond snail, Lymnaea stagnalis, there is a buccal CPG that regulates mouth movements, including feeding and laying eggs.

By anesthetizing the snail, implanting an electrode on one of these buccal neurons, observing its behavior and aligning it with the electrical activity, I will be able to see CPGs in action.

This project mainly replicates a 1999 paper by Jansen et al. that studies pattern generators in the buccal ganglia of freely behaving snails with a few tweaks of my own. The buccal ganglia is a group of neurons (ganglia) that controls buccal movements - any behavior related to the mouth. In regards to the freely behaving part, it's very important in neuroscience for the subject to be freely able to move and behave of its own will in order to see the neurons acting in real time.

In theory, the project isn't that difficult; put an electrode around a neuron in the buccal ganglia and watch the spikes occur at the same time the snail is eating. However, what's most challenging about this project is the damn setup to get to the point of data collection. Jansen et al. used these 40 mm pond snails called Lymnaea stagnalis which are super cute but incredibly small for a first-time neuroscientist (think the size of your thumbnail).

he paper focused on data collection from three neurons in the buccal ganglia: the posterior jugalis nerve (PJN), the lateral buccal nerve (LBN), and the ventral buccal nerve (VBN) which can be seen in the picture shown. The LBN and VBN actually stem from the same neuronal branch and eventually split into their respective neurons, so for simplicity's sake, I'm implanting my electrode around the initial branch for both nerves. **This has been updated to put the electrode on one of the esophageal trunks that control the movements of the esophagus.**

In terms of the buccal movement, snails rasp or scrape their toothy tongue called a radula across a surface, like a tank with algae or a piece of spinach, in order to collect food or clean. They rasp when they eat or, interestingly enough, when they lay eggs. The substrate that they lay their eggs on needs to be clean so they rasp it clean. Both of these movements are controlled by the buccal ganglia. The buccal ganglia stimulates the idea of "start rasping," sends it down one of the neurons, like the LBN or VBN, which then stimulates a muscle that controls the radula to initiate rasping. Jansen et al. found that the electrical activity seen in the spikes changed in frequency and amplitude depending on the behavior at hand. More explicitly, when the snail was eating, the spikes occurred more often with large amplitude versus when the snail was cleaning to lay eggs, the spikes occurred less often and with a lower amplitude (see picture). Although this would be awesome to see for myself, due to time constraints, I will only be observing the electrical activity in accordance with feeding.

  • 1 × Pond snails Lymnaea stagnalis, number depends on how many trials you do
  • 1 × Tank with water, moss, and rocks For keeping the snails happy and healthy in a good ecosystem!
  • 1 × Magnesium chloride (MgCl2) 50 mmol/L or 0.7g in 147g H20
  • 1 × Syringe For anesthetization, 3 mL or more
  • 1 × Needle 1 needle for each snail; can use the same syringe

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  • Lights, please

    Nancy Sloan07/10/2016 at 22:58 0 comments

    After a long and difficult time discovering the buccal ganglia, I recommend using a microscope with at least 6-8 Watt LED bulbs or else you will struggle to find the right area. The buccal ganglia is almost bikini-shaped as shown in the previous log from Ramakrishnan et al. 2014 and is seated directly behind the mouth. Best of luck!

  • A New Target

    Nancy Sloan07/01/2016 at 02:07 0 comments

    Upon reading a new paper, I have determined a new location for the electrode (when I get that point in the experiment): the esophageal trunk! Ramakrishnan et al. in 2014 studied the buccal A cluster (BAC) cells that fill up the buccal ganglia, 40 in each. These cells vary in location, size, and the cluster that they're in but essentially are responsible for telling different muscles to move, like opening the mouth or bringing the radula to the surface. All of these BACs have axonal projections through different nerves branching from the ganglia that we've talked about before: the lateral buccal neuron (LBN), the posterior buccal neuron (PBN), the esophageal trunks (ETs), and a few through the cerebro-buccal connective (CBC) that all then connect to different muscles. However, every one of these BAC projections goes through the esophageal trunks and none go through the ventro-buccal nerve. My plan was to attach the electrode to the trunk of the lateral and ventral buccal nerves, which is technically still okay, but only one nerve will be receiving signal. In the picture below from Ramakrishnan's paper, you can see that there are connections in every neuron except for the VBN with the lightest grey view.


    HENCE I will be placing the electrode around one of the esophageal trunks for a *hopefully* stronger signal. Until I get to the point of electrode placement, I am continuing the search for the buccal ganglia.

  • On the Road to Postoperative Recovery

    Nancy Sloan06/27/2016 at 20:36 0 comments

    After going through surgery, usually humans can wake up from the anesthesia and function quite normally. Snails are not the same. After this procedure, they need to stay hydrated, but in something less anesthetic and more similar to their blood plasma. This is called a snail saline solution, or Ringer's solution. It's essentially 1 liter of water but with some very key components, like sodium chloride (NaCl) and a solution called HEPES. Below is the recommended concentration for each component which can be converted into grams of milliliters to add to the water. Sodium hydroxide (0.1 M NaOH) was added last in very small quantities to make sure the final pH of the solution was 7.9. This saline was provided by very kind and knowledgeable Lymnaea experts at Cornell. The snails can recover in this within 24 hours so place them in it for comfort and hopefully a speedy recovery!

  • Stop that snail!

    Nancy Sloan06/27/2016 at 18:54 0 comments

    More clearly explained, inject the snail before it hides behind its trap door anyway. For the past 2 weeks, I've had considerable trouble getting the needle into the snail to inject it with the anesthesia solution before the snail locked itself away in its trap door. It's actually a very fast and strong protection mechanism, considering it's a snail. I ordered needles off of Amazon that were wonderfully priced, however were not sharp enough to do the job. Even when trying to shave it down, it just didn't work. Your local CVS will have a needle for insulin injection so use that if you must. It has a beveled tip to the needle and a very nice 3 mL syringe for injection. Make sure you have these items before starting!!!

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  • 1
    Step 1

    Step 1 sets up a safe and healthy environment for the snails! These are pond snails so in nature, they reside in bodies of water with slow moving current. This can be reproduced with a tank filled with 2 gallons of water (rule of thumb is 2 gallons per 20 snails), a bubbler to keep the water moving, rocks on the bottom and moss that floats on top to encourage good bacteria to grow, and a constant temperature area around 70F. The rocks and moss aren't totally necessary but all my snails have been living very happily so I'd recommend it if you don't have a lot of time to let an ecosystem build naturally.

  • 2
    Step 2

    Step 2 involves preparing the electrode for implantation. Take about 2 feet of your 25 micron-diameter wire, fold it in half, and wrap it around itself. Determine which end is going into the snail and the other will go into the channel of the SpikerBox.

    On the side going into the snail, start by removing the insulation from the ends to reveal the stainless steel wire beneath (my insulation was Teflon PFA and could be carefully burned away). Curl one end into a little hook and let the other one hang next to it but NOT touch. The hook will go around the neuron and the other will act as a ground electrode inside the snail's medium.

    On the other end of the wire, make two more ends to attach onto the male RCA channel connection (if this is the side where the bend is, you can just cut that bend to make two wires). Again, remove the insulation from these ends as well. Using a voltage meter, figure out which end on this side is connected to the hook on the snail side. That one will attach to the smaller metal stand inside connector. The ground will attach to the taller metal stand. Finish the connections by soldering the wires to the connectors.

    **Note: I attached my electrode to two slightly larger wires that in turn connected to the RCA connector. It made things a little easier because the electrode wire was so small and could also be elongated using these wires.

    **Note: I also insulated my entire wire with silicone glue in case it was ever in touch with water; this is up to choice.

    **Reference: inspiration was taken from Cullins et al. 2010 paper.

  • 3
    Step 3

    Step 3 prepares the snail for surgery. Start by injecting the snail with 1.5-2 mL of magnesium chloride (50 mmol/liter) to anesthetize them. This should plump them up enough to have them hang out of their shell just a wee bit and allow for easier navigation of the neurons. From experience, make sure to use a really sharp needle or you will be frustrated for a long time!! A beveled needle used for injecting insulin should do the trick.

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Discussions

de∫hipu wrote 07/01/2016 at 15:35 point

I wonder, do snails even have any kind of equivalent of pain? Or is the anesthesia just to prevent them from moving?

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Nancy Sloan wrote 07/01/2016 at 17:59 point

Hi Radomir! Great question - so the anesthesia is to put them to sleep while the electrode is being implanted, similar to what humans do for surgery. Technically, the snail is an invertebrate and doesn't have a spinal cord or nociceptors to comprehend or feel pain, but it is very certain that they don't like being prodded with needles!

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