The overall goal of this procedure is to learn how to prepare leach ganglion for intracellular recording from individual neurons, how to prepare a patch of skin to map a sensory receptive field and how to culture leach neurons. This is accomplished by first using ice to anesthetize the lech and pin it in a ventral side up position. The next step is to dissect out about three separate ganglia and stretch them out for recording.
The final step is to impale the individual neurons and record action potentials. Overall, the behavior of the neural network can be characterized by making physiological characterizations of the various cell types in conjunction with experimentation. The main advantage of using leches over other simple invertebrate preparations like S slugs or crustacean, is that the ganglia have just over 400 easily accessible neurons, and these neurons can stay active in culture for several days.
Visual demonstration of this method is critical as the skin preparation and pulling out single neurons are difficult to learn. One might easily damage a ganglion that invasives a patch of skin or damage neurons when culturing them. A question that you can naturally ask on hearing that you're gonna see a film and read a text about the nervous system of the leach is, why on earth would anybody work on an invertebrate brain when the human brain is so much more interesting?
And if you were gonna work on an invertebrate, why would you pick the leach a horrible blood sudden mucus secreting disgusting animal? Well, there are several good reasons for it. The first is that the leach has an extremely simple nervous system compared to our own.
And in it you can study individual nerve cells, see the way they're connected, and see how those connections result in the behavior of an animal in the way that you can't possibly do in a more complicated animal. Another reason is that the Leach, which was first introduced into neuroscience in 1960 by Stephen Koffler, offers opportunities for studying basic problems, which actually are relevant to the way in which our own brain works. And there are many examples which I could point to.
One is that the majority of cells in the brain, our brains are not nerve cells, but so-called glial cells, satellite cells, they were first studied in the leach, and then once one knew how they worked there, you could see if they were similar in our own brains, and they are, and that was important for many clinical things too. But the principle reason for working on this animal, you will see in the film, it has a beautiful nervous system. The nervous system consists of a chain of ganglia, groups of nerve cells, a limited number.
You can see all the cells you can record from them individually. You can find out how they're connected, rather like studying a map of the Paris metro. And it's this beauty that makes the preparation so fascinating to work with, coupled with the ability to discover certain fundamental principles Prior to beginning the dissection.
Be sure to first set up the recording software and recording electrode. Prepare to dissect a leach in a large silicone elastomer lined dish filled with ringer solution using pins. Stretch the animal out ventral side up with a number 10 or number 11 scalpel.
Make a longitudinal incision with very shallow cuts in the ventral midline. Try not to cut through the ventral blood sinus until the animal is fully opened and the blood sinus is stretched without kinks. The VNC is contained within now.
Use fine iris scissors to nick the blood sinus slip one blade of the scissors under the sinus and slightly raise the blade to separate the sinus from the nerve tissue. Then cut the sinus the length of the VNC while avoiding the nerve roots, segmental nerves or the connectives between ganglia. Next, remove part of the ventral nerve cord by cutting the roots distal to where the blood vessel joins the roots.
Now transfer the ganglion in a clean dish filled with saline and pin it down by the nerve roots and connectives. Use this preparation to obtain intracellular recordings from the cell bodies of the sensory neurons. Pin the ganglia ventral side up in a clean dish containing L 15.
With fetal FCS, the roots should be stretched slightly with fine scissors. Nick, the glial capsule at one end slip, one scissor blade under the capsule and across the capsule. Next, add 100 microliters of collagenases dys displays to the dish and place the dish on a shaker for 15 minutes later, examine the cells by moving the culture media around the exposed cell bodies.
Return the cells to the shaker for another 15 or more minutes until the cells can be extracted from the ganglion using minimal suction from a fire polished pipette with a bore size of about 50 microns, slightly larger than the body diameter. To release the cells ECM, add a few drops of L 15 with FCS medium to the cells and incubate them for a few hours to 24 hours at room temperature. When they are ready, plate the cells next to each other in a substrate coated microwell dish.
With L 15 medium. Do not use FCS. After 24 hours, they will be ready for electrophysiology and will remain viable for several days.
Initially, the electrical properties are not exactly the same as an in vivo prep. However, after a few days, the impulses are nearly indistinguishable. Pin the leach dorsal side down in an elastomer lined dish as previously described.
Next, pin the skin to one side with roots on the other side of the severed ganglion. Alternatively, make a window on the ventral aspect of the leach over a ganglion with all the roots to the body wall intact. After obtaining a stable intracellular recording in one of the sensory cells, use a small fire polished rod to lightly touch the skin in the same segment with t and p cells.
Lightly touching the skin will generate an electrical response. With end cells, more pressure from the rod will be required, and in most cases, an end cell will only respond if the skin is pinched with tweezers. Therefore, to minimize potential tissue damage record from the N cells last to determine if the examined neurons have processes in certain locations.
The connectives between ganglia can be cut and the receptive fields can be reexamined for any losses. Intracellular recordings from sensory neurons in the leach ganglion are characterized by their distinct time traces. Their resting potential is between negative 45 and 60 millivolts.
If measured any lower, a new glass probe or new preparation should be used. Spontaneous action potentials with a small amplitude indicate that the cell may be a motor neuron. In the ganglion body wall preparation, a current injection can be used to determine if the neuron innervated any still attached muscles.
For instance, the annulus erector motor neuron on the ventral side of the ganglion will make the ann eye rise. If after current injection a uncharacteristic waveform is seen, it could be due to an unbalanced bridge or offset capacitance compensation. When the bridge is balanced, stimulus artifacts are seen at the beginning and end of the current pulses after seven hours.
In primary culture, measurements were taken from a pair of retia cells. The amplitude of the synaptic potentials was around one to two millivolts. After 56 hours in culture, the synaptic potentials were larger than 10 millivolts.
The shape of the action potential also changed. In the same preparation, the nerve roots or connectives were stimulated. The black trace was evoked by a sub-threshold stimulus applied with an extracellular electrode to the connective.
The red trace shows how increasing the stimulus voltage caused the cell to fire and action potential. An injection of Lucifer yellow into a retia cell was made by passing a negative current through the electrode. Shortly after the injection, the dye remained localized to the soma.
30 minutes later, the dye had already diffused into the nerve roots After its development. This technique help the researchers in the field of neurobiology to explore the underlying mechanism behind shapes of the action potentials and to further investigate the neuro circuitry. Also to promote an understanding of regeneration and repair in identifiable neurons.
This video demonstrates a series of experimental techniques. Using the leach could be applied to synaptogenesis, neuromodulation, or fundamental aspects of neurophysiology.
