Spinal Cord Preparation from Adult Red-eared Turtles for Electrophysiological Recordings during Motor Activity

[Abstract] Although it is known that the generation of movements is performed to a large extent in neuronal circuits located in the spinal cord, the involved mechanisms are still unclear. The turtle as a model system for investigating spinal motor activity has advantages, which far exceeds those of model systems using other animals. The high resistance to anoxia allows for investigation of the fully developed and adult spinal circuitry, as opposed to mammals, which are sensitive to anoxia and where using neonates are often required to remedy the problems. The turtle is mechanically stable and natural sensory inputs can induce multiple complex motor behaviors, without the need for application of neurochemicals. Here, we provide a detailed protocol of how to make the adult turtle preparation, also known as the integrated preparation for electrophysiological investigation. Here, the hind-limb scratch reflex can be induced by mechanical sensory activation, while recording single cells, and the network activity, via intracellular-, extracellularand electroneurogram recordings. The preparation was developed for the studies by Petersen et al. (2014) and Petersen and Berg (2016), and other ongoing studies.

Here, we provide a detailed protocol for setting up the integrated adult turtle preparation, sometimes called the ex vivo preparation (Guzulaitis et al., 2014), with intact spinal motor network. The preparation provides the opportunity for measurements of the central pattern generator in the lumbar spinal segments (Figure 1), which is similar to the lumbar spinal cord of mammals and other animals (Walloe et al., 2011). This preparation includes the spinal segments D3-S2 en bloc. Measurements of the scratch reflex can be performed entirely in the absence of chemical anesthesia. Intracellular, as well as high-density extracellular recordings, can be acquired in the spinal cord concurrent with both ipsilateral and contralateral electroneurogram recordings of muscle nerves (ENG). The scratch reflex is induced by mechanically touching the ventral side of the carapace and therefore identical or close to a natural behavior. A smaller version of the integrated turtle preparation was introduced by Keifer  The preparation steps can be split into two parts, typically performed over two days. First part can be performed without a microscope. All procedures of the first part are completed over 3 h. The first 2 h to induce anesthesia and the last hour for dissection. The procedures of the second part can be performed at a setup using a microscope and will take about an hour to complete.   c. Take the turtle from the ice bath. Two conditions must be met to ensure that the turtle is fully anesthetized: 1) Its eyes must be closed and 2) No pedal withdrawal reflex response.
Proceed, when the anesthesia is confirmed.  f. Crush the head with the large pliers and dispose it in the plastic bag.
Note: Now that the turtle is decapitated the dissection can begin. In the next steps, you will perfuse the cardiovascular system with cold Ringer's solution, by injecting Ringer's solution through the heart of the turtle. This removes blood and cools the nervous tissue.
g. Place the turtle on its back, and use the cast saw cutter to make a square opening in the plastron. The location of the four cuts is shown in Figure 3. The two cuts, orthogonal to the spinal cord, are oriented along the edges of the two central scutes (smaller plates). Keep the minimum distance between the cuts, parallel to the midline, above 5 cm. Make sure that the square cutout plastron is completely released with the cast saw cutter before continuing. Verify this by gently pushing on the inner corners of the cut-out plastron, if the square piece moves freely it is sufficient. 6 www.bio-protocol.org/e2381   the heart, the heart rate should increase dramatically at first, but stabilize at a pace around 20-40 bpm. If the heart rate is not in this range adjust the flow accordingly: increase the flow if a lower heart rate is observed, and decrease the flow if the heart is pumping too fast.       The preparation is now left in a Petri dish in cold Ringer's solution ( Figure 11). The muscles and connective tissue covering the vertebra has to be removed carefully with a scalpel and forceps. The muscles tissue is carefully scraped off until the vertebra is fully exposed while leaving the nerves intact ( Figure 12A). 11 www.bio-protocol.org/e2381   c. Dry the cut rostral carapace edge with a paper towel. d. Apply a thin layer of cyanoacrylate adhesive along the cut edge that will be the area of contact with the rostral Plexiglas plate (Figure 13). Be careful not to get adhesive into the spinal column, which will obstruct the flow of Ringer's solution.    a. Identify the motor nerves originating from D8-S2 and dissect them free for ENG-recordings ( Figures 17A and 18). Figures 18 and 19 show the location of the nerves and corresponding muscles respectively: Hip-flexor, Hip Extensor, three Knee-extensors (FT-KE, IT-KE and AM-KE), dD8 and HR-KF (Mortin and Stein, 1990). Muscle tissue along nerves should be dissected free to minimize noise in the ENG recordings. The nerves are robust but can easily be damaged during the dissection without obvious visible signs.
b. Gently cut out the muscle tissue and free the nerves. A good technique to free the nerves from surrounding tissue is to place the tip of the fine scissors in the tissue close to the nerve and pull the sharp edge distally along the nerve. 15 www.bio-protocol.org/e2381     integrated preparation. Figure 17 shows example electrophysiological recordings and histology (Petersen and Berg, 2016 The solution is saturated with 95% O2/5% CO2, by aeration for 30 min to obtain pH level of 7.6

Preparation for extracellular and intracellular recordings
Note: Either Ringer's solutions can be used in this protocol.