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).
The 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.
Below are the complete instructions for this experiment, if you want to see the whole process (every step and attempt to achieve this project) you can check out the following logs:
- Log 1: Stop that Snail (June 27)
- Log 2: On the Road to Postoperative RecoveryInstructions (June 27)
- Log 3: A new target (July 1)
- Log 4: Lights, Please (July 10)
For this experiment you will need:
|1||×||Pond snailsLymnaea stagnalis, number depends on how many trials you do|
|1||×||Tank with water, moss, and rocksFor keeping the snails happy and healthy in a good ecosystem!|
|1||×||Magnesium chloride (MgCl2)50 mmol/L or 0.7g in 147g H20|
|1||×||SyringeFor anesthetization, 3 mL or more|
|1||×||Needle1 needle for each snail; can use the same syringe|
|1||×||Microscope,40x-80x, 8 Watt LED lightsFor locating the neurons|
|1||×||Dissection Minuten PinsFor pinning and locating the neurons, 0.1 mm in diameter|
|1||×||WireStainless steel or wire, 25 micrometers in diameter, better if insulated|
|1||×||Backyard Brains SpikerBoxAmplifies spikes so they can be heard|
|1||×||Backyard Brains SpikeRecorder softwareShows a visualization of the spikes on a computer or mobile phone|
|1||×||Snail salineSee Log 2 for the components|
- 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.
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.
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.
Step 4 involves the surgical process of cutting them open and locating the buccal ganglia below the radula. While the paper put electrodes around 3 neurons, I’m working with just 1 neuron, the lateral and ventral buccal nerve, which is a solid trunk at the ganglia but branches off into the respective nerves. Use lots of pins to specifically locate the ganglia and make sure it doesn’t disappear into the snail!
Step 5 is the implanting of the electrode onto the neuron. Place the hook electrode around either the ipsilateral or contralateral ET, glue it with spray super glue for easy drying, surround it in a Vaseline and mineral oil mixture, and let the connection ease back into the snail. Place the snail in a ringer solution or water with special minerals in it to help the snail heal (I used SmartWater that has a good amount of electrolytes for the snail). Leave the snail alone to heal for a day or 18-24 hours.
Step 6 is hooking up the electrode to the Backyard Brains SpikerBox and the SpikeRecorder software on the computer and watching the neuron spike away! These spikes should align with when the snail opens its mouth and rasps for food or cleaning.
By Nancy Sloan