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

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18-Oct 2016



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-, extracellular- and electroneurogram recordings. The preparation was developed for the studies by Petersen et al. (2014) and Petersen and Berg (2016), and other ongoing studies.

Keywords: Adult turtle (成年海龟), Integrated preparation (综合制备), ex vivo (离体), Spinal cord (脊髓), Electrophysiology (电生理学), Intracellular and extracellular recordings (细胞内和细胞外记录), Single units (单一单位), Electroneurogram (神经电图), Scratch reflex (搔反射), Central pattern generator (中枢模式发生器)


The investigation of spinal electrophysiology has traditionally been associated with mechanical complications due to the many moving parts and the flexibility of the spine. To circumvent this issue, the spinal cord has often been dissected out of the column and moved to a chamber where stable electrophysiological recordings can be performed. Nevertheless, this procedure has shortcomings, for instance, it is complicated to properly activate the motor circuitry especially if multiple motor behaviors are to be investigated. Furthermore, the absence of blood supply and lack of oxygen has serious ramifications on the health and integrity of the circuitry. An experimental model, which can circumvent all these issues, is the turtle preparation (Keifer and Stein, 1983). Here, one can study not only the fully developed vertebrate spinal cord, but also several different types of complex motor behaviors without the need of neuro-active substances such as glutamate agonists, 5HT, and dopamine. Since the neurons in the turtle central nervous system are able to perform anaerobic metabolism, the integrity of the circuit remains for much longer than in the mammalian experiments. Last, the turtle carapace organization allows stabile multi-electrode recordings of unprecedented quality. 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 and Stein (1983) and subsequently adapted and modified (Currie and Lee, 1997; Alaburda and Hounsgaard, 2003; Alaburda et al., 2005; Berg et al., 2007 and 2008; Kolind et al., 2012; Vestergaard and Berg, 2015). The present preparation was developed for the study by Petersen et al. (2014) and Petersen and Berg (2016) where electrode arrays are inserted perpendicularly into the lumbar spinal cord (Berg et al., 2009).

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.

Figure 1. The integrated adult-turtle preparation with implanted electrodes. A. Schematic of the placement of the silicon probes in the spinal cord; B. The preparation with three silicon probes and intracellular glass electrode. Suction electrodes for electroneurogram recordings are attached (pointing from top and bottom and right and left). C-D. Close up of the spinal cord with silicon probes and intracellular glass electrode (only inserted in the spinal cord in D). The tips of suction electrodes are also visible. Modified from Petersen and Berg (2016) with permission. E. The spinal cord after the silicon probes have been retracted. Blue DiD markings are visible from the first and third shank (8 markings for each of the probes, highlighted with arrows).

Materials and Reagents

  1. Scalpel handle #3 (Fine Science Tools, catalog number: 10003-12 ) with scalpel blade #15 (Fine Science Tools, catalog number: 10015-00 )
  2. Cast cutter blade: BSN 2.5” stainless steel sectioned blade (BSN medical, catalog number: 480-4183-145 )
  3. Plastic bag, 5 L size
  4. Gloves
  5. 45 mm Rotary cutter blades (WorldKitchen, OLFA, catalog number: RB45-5 )
  6. Cyanoacrylate adhesive (Panacol, catalog number: Cyanolit® 202 )
  7. Plexiglas plate: 80 x 15 x 2 mm
  8. Paper towel
  9. 100 mm or 120 mm Petri dishes (VWR, catalog number: HECH41042024 or HECH41042030 )
  10. Earmuff (PeltorTM OptimeTM 98 Earmuff) (3M, catalog number: 10093045080912 )
  11. Syringe needle 27 G, 31 mm (BD, catalog number: 305136 )
  12. Red eared turtles (Trachemys scripta elegans, Nasco, Fort Atkinson, WI, USA) of weight 300-500 g and of both sexes were used in this procedure
  13. Crushed ice
  14. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S3014 )
  15. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P5405 )
  16. Sodium bicarbonate (NaHCO3) (Sigma-Aldrich)
  17. Magnesium chloride (MgCl2) (Sigma-Aldrich, catalog number: 208337 )
  18. Calcium chloride (CaCl2) (Sigma-Aldrich, catalog number: 21115 )
  19. Glucose (Sigma-Aldrich, catalog number: G7021 )
  20. Ringer’s solution (98% O2/2% CO2) (see Recipes)
  21. Ringer’s solution (95% O2/5% CO2) (see Recipes)


  1. Scissors
    Large scissors (Fine Science Tools, catalog number: 14014-17 )
    Fine scissors (Fine Science Tools, catalog number: 14090-09 )
    Fine serrated scissors (Fine Science Tools, catalog number: 14058-11 )
  2. Forceps:
    Dumont #5 forceps (Fine Science Tools, catalog number: 11251-10 )
    Dumont #5 fine forceps (Fine Science Tools, catalog number: 11254-20 )
    Dumont #7 curved forceps (Fine Science Tools, catalog number: 11271-30 )
    Toothed tissue forceps (Fine Science Tools, catalog number: 11022-15 )
    Serrated Graefe forceps (Fine Science Tools, catalog number: 11051-10 )
  3. Pliers: Liston Gross Anatomy Bone Cutters (Fine Science Tools, catalog number: 16104-19 )
  4. Course and fine Rongeurs: Lempert Rongeurs (Fine Science Tools, catalog number: 16004-16 ) and Friedman-Pearson Rongeurs (Fine Science Tools, catalog number: 16221-14 )
  5. Perfusion system: Custom-designed air-pressure system/outlet connected to the inlet of a 1 L Pyrex glass bottle with lid. The outlet of the bottle is connected to a flow meter and to a 21 G 5 mm syringe needle (BD, catalog number: 305129 )
  6. Cast saw cutter (Atlas International, American Orthopaedic, catalog number: T-CC-100 )
  7. Pyrex glass griffin beaker 1 L (Corning, PYREX®, catalog number: 1000-1L )
  8. Open Plexiglas container, Dimensions: 200 x 150 x 25 mm (D x W x H), Plexiglas thickness: 3 mm. Custom built
  9. Extracellular recordings: 64 channel Berg64-probe from Neuronexus. 8 shanks, each with 8 staggered recording-sites distanced 30 µm vertically, designed for ventral horn recordings from the turtle (Neuronexus, BERG A8X8-5MM-200-160 PROBE)
  10. Intracellular recordings: Glass pipettes pulled with a P-1000 Sutter Instruments and filled with a mixture of 0.9 M potassium acetate and 0.1 M KCl. For histological verification 4% w/v biocytin can be added to the mixture (Biocytin, Sigma-Aldrich, catalog number: B4261 )
  11. Nerve recordings: Electroneurogram (ENG) recordings can be performed with suction electrodes


Note: The surgical procedures comply with Danish legislation and were approved by the controlling body under the Ministry of Justice.

  1. First part of the preparation
    1. Anesthetization
      To induce hypothermic analgesia, the turtle is submerged in crushed ice in a bucket. Full induction of anesthesia takes about two hours.
    2. Ringer’s solution
      Prepare 2 L of cold Ringer’s solution (see Recipes) (5 °C) for the first day. Cold Ringer’s solution is used to keep the turtle anesthetized during the preparation.
    3. Initial surgery and perfusion
      Tools: Toothed Tissue Forceps, large pliers, large scissors, small scissors, cast saw cutter, cast cutter blade, perfusion system, plastic bag, gloves.
      1. Prepare perfusion-system: Fill the glass bottle with 500 ml Ringer’s solution and close the lid. Connect the bottle to a compressed air system/outlet.
      2. Turn on the compressed air and let the perfusion-system run for 30 sec with a flow above 120 ml/h as measured with the flow meter. Stop the flow.
      3. 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.
      4. Pull out the head of the turtle with the toothed Tissue Forceps to allow for decapitation.
      5. Hold the head in place with the large pliers (Figure 2), and cut the neck with the large scissors.

        Figure 2. Decapitation of anaesthetized turtle. Head is held in place with the toothed tissue forceps and large pliers. The large pair of scissors is used for the decapitation.

      6. Crush the head with the large pliers and dispose of 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.
      7. 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.

        Figure 3. The square cut in the plastron has been performed. Gently push in the inner corners of the square cut-out plastron to verify that it is released.

      8. Lift up the cutout plastron with the Graefe forceps and use the scalpel to carve it free from the soft tissue.
      9. Gently lift up the pericardium, the fine ‘pellicle’ containing the heart, with the Graefe forceps and cut it open with the fine scissors, to get access to the heart (Figure 4).

        Figure 4. The internal view of the window hole in the plastron of the turtle. The heart is marked by the circle and the right atrium by the arrow.

      10. Locate the ventricle and the atriums of the heart (the heart and the right atrium is marked by a yellow circle and an arrow in Figure 4). Cut a hole in the right atrium (located on the left and highlighted in Figures 4 and 5) with the fine scissors, and insert the perfusion needle into the center of the left part of the ventricle (Figure 5). Adjust the flow to a range around 75-130 ml/h, accordingly to the heart rate: When inserting the perfusion needle into 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.
        Note: Perfuse the turtle for about 10 min. Monitor the perfusion by checking outflowing liquid from the right atrium; as this becomes colorless the perfusion of the turtle is complete.

        Figure 5. The perfusion needle is inserted into the left part of the ventricle of the heart. The right atrium and ventricle are marked by the arrow and the circle respectively.

    4. Rostral and the caudal cuts of the carapace
      Tools: Cast saw cutter.
      1. Perform a rostral and a caudal transverse cut in the carapace orthogonal to the spinal cord. Place the turtle with the carapace facing upwards (dorsal side up). Figures 18 and 19 show the spinal segments and associated nerves. The central pattern generator is located in the spinal segments D8-D10 (Mortin and Stein, 1989; Mui et al., 2012; Hao et al., 2014), and the sensory input for the scratch reflex comes from every segment from D3 to S2 (Figure 18, excluding caudal scratching). In order to keep the sensory input to the network intact, the rostral cut is made between D2 and D3. The caudal cut is done between S2 and Ca1. The two cuts are seen in Figure 6. D2-D3 is located halfway along the 2nd central scutes and S2-Ca1 halfway along the 5th central scute (Mortin and Stein, 1990). It is important that the cuts are a) orthogonal to the spinal cord, b) performed in one cut all the way from the midline of the carapace to the cut edge of the plastron, since the perfusion through the spinal column must be sealed tight. The caudal cut should be angled perpendicular to the curvature of the carapace.

        Figure 6. Performing the rostral cut. The caudal cut has already been done. The rostral cut is done along the center of the first central scute, and the caudal cut along the center of the fifth central scute.

      2. When the two cuts have been made, the carapace is separated from the remains of the caudal part of the plastron. This separation is obtained with two diagonal cuts at the corners of the squared window in the plastron (the two yellow lines in Figure 7).

        Figure 7. The rostral cut at the ventral side (through the plastron). The two diagonal cuts are highlighted in yellow.

    5. Removing the internal organs, the plastron and the hind legs
      Tools: Large scissors, 1 L beaker with cold Ringer’s solution.
      1. Hold up the turtle with the caudal part upwards.
      2. Take the large scissors and cut the internal organs free of the carapace by cutting the thin membrane holding the organs in place along the inner side of the carapace from below and up (Figure 8). It is easier to cut along the carapace when the internal organs hang loose. Try to maintain the organ block in one piece when removing it.

        Figure 8. The internal organs are cut free along the inside of the carapace

      3. Cut the large head retraction muscle that inserts along the spine, and cut the remaining organs free along the carapace as far up as possible.
      4. Pull back one of the hind legs. Make an incision with the large scissors in the soft skin along the plastron (Figure 9). Begin from the diagonal cut made previously in the plastron, and aim towards the leg. Cut the thigh bone (femur) about 1 cm from the carapace (Figure 10).

        Figure 9. Cutting the soft skin along the plastron

        Figure 10. Cutting the thigh bone

      5. Repeat step A5d for the other hind leg.
      6. Cut the plastron and the caudal end of the carapace free without damaging the spinal cord. The preparation is now left in a Petri dish in cold Ringer’s solution (Figure 11).

        Figure 11. The preparation lying in a Petri dish without plastron and legs

      7. Clean off excessive blood from the preparation by quickly rinsing it in cold Ringer’s solution (about 50 ml) and place it in a 1 L beaker with cold Ringer’s solution when done.
    6. Clear the spinal column
      Tools: Graefe forceps, curved forceps, scalpel.
      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).

      Figure 12. The exposed spinal segments S1-Ca1 and the rostral transection. A. Ca1 and more caudal segments are all flexible segments, while D10 and more rostral segments are joined with the carapace. The yellow square highlights the segments. B. Rostral transection of the spinal cord with the rotary cutter blade to make the vertebra smoother, such that the tubing makes a tight seal. Area indicated by the yellow square, and location of second cut compared with the initial cut is indicated by the parallel lines.

    7. Transection the spinal cord
      Tools: Sharpened rotary cutter blade mounted in the cast saw cutter.
      To minimize damage to the spinal tissue, a special sharpened rotary cutter blade (Olfa, rotary cutter blade 45 mm) has been produced to transect the spinal cord with the cast saw cutter. At the spinal transection, the spinal cord retracts slightly into the spinal column. Therefore, perform the transection of the spinal cord in a firm and quick movement to get a smooth cut surface.
      1. Mount the rotary cutter blade in the cast saw cutter.
      2. Insert the edge of the blade into the cold Ringer’s solution in the Petri dish for ten seconds to cool it down. This will improve the quality of the spinal transection.
      3. Perform the rostral transection of the spinal cord about 1-1.5 mm from the previous cut. The transection must be perpendicular to the spinal cord (Figure 12B).
      4. Rotate the preparation 180° in the coronal plane and mount it in the clamp.
      5. Perform the caudal transection of the spinal cord. The second cut provides an improved perfusion.
      6. Put the preparation back in the glass beaker in Ringer’s solution.
    8. Attaching the Plexiglas plate to the preparation
      Tools: Cyanoacrylate adhesive, Plexiglas plate, Petri dish with lid, ice, scalpel, perfusion system, paper towel.
      1. Put a layer of ice in the largest Petri dish and place the smaller Petri dish on top of the ice, facing upwards, and fill it with cold Ringer’s solution.
      2. Place the preparation in the cold Ringer’s solution with the rostral end facing upwards.
      3. Dry the cut rostral carapace edge with a paper towel.
      4. 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.

        Figure 13. Applying adhesive to the rostral cut of the preparation. Put a fine line of adhesive along the cut corresponding to the contact area of the Plexiglas plate.

      5. Gently place the rostral Plexiglas plate on the preparation centered with the small hole over the cut spinal cord (Figure 14). Keep it firmly in place for about 30 sec. If the adhesive does not harden, apply some drops of Ringer liquid on the glue, which will help harden, while holding the plate in place.

        Figure 14. Attaching the rostral Plexiglas plate to the preparation

      6. Lift up the preparation and place an extra line of glue along the line of contact between the dorsal carapace and the Plexiglas. Leave it to dry for one minute before continuing.
    9. Mounting the preparation to the Plexiglas container
      Tools: Cyanoacrylate adhesive, Plexiglas container, paper towel.
      1. Take the Plexiglas container and place glue at the four contact points of the caudal carapace.
      2. Gently place the turtle preparation in the Plexiglas container (Figure 15). Keep it in place for about 30 sec.

        Figure 15. Preparation mounted with adhesive upside down in the custom Plexiglas container

      3. Gently fill the Plexiglas container with Ringer’s solution. The liquid will help harden the adhesive.
      4. Place the preparation in a larger plastic container and immerse it completely in Ringer’s solution.
      5. The procedures of the first part are now complete. Leave the preparation overnight in a refrigerator.

  2. Second part of the preparation
    1. Setup the spinal vertebral foramen perfusion
      1. Mount the steel tube and a silicone gasket to the hole in the Plexiglas plate. Press the gasket against the D4 vertebra, and push in the steel tube to obtain a tight seal (Figure 16). Connect the tube to a raised container with Ringer’s solution. Maintain a perfusion flow above 300 ml/h by adjusting the relative vertical position of the container with Ringer’s solution.

        Figure 16. Steel tube and silicone gasket pressing against the rostral end of the spinal column allowing Ringer’s solution to flow in the spinal column

    2. Dissecting out the nerves for electroneurogram recordings
      Tools: Finely serrated scissors, fine scissors, Graefe forceps, Dumont #5 forceps and Dumont #7 curved forceps. Other needs: 2 L of Ringer’s solution.
      1. 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.
      2. 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.
        Note: Figures 1B-1E show the finished preparation, mounted in the Plexiglas container and immersed in Ringer’s solution. The nerves dissected free are clearly visible as white branches originating at the spinal cord and going towards the hind limb muscles. Glass electrodes are used for the ENG recordings. Apply a slight negative pressure, and prepare the glass opening to fit the respective nerves you want to record.

        Figure 17. Electrophysiological recordings and histological verification. A. Intracellular and ENG recordings; B. Rastergram of ~200 extracellular units recorded with high density silicon probes; C. Histological verification of the location of shanks of the silicon probe; D. DiD is painted on the electrode before implant. ChAT and Nissl stains are applied as markers for motoneurons and neurons respectively. Sagittal and coronal slices in respectively C and D. Scale bars = 500 µm. Adapted from Petersen and Berg (2016).

        Figure 18. The sensory and motor nerves along the spinal cord. Adapted from Petersen et al. (2014) with permission.

        Figure 19. Major muscle groups of the hind-limb. Hip flexor, Hip Extensor, Knee Extensors (FT-KE, IT-KE, AM-KE) and Knee flexor (HR-KF, extend across both the hip- and the knee-joint). Reproduced from Bakker and Crowe (1982) with permission.

    3. Preparation for extracellular and intracellular recordings
      Tools: Fine forceps, fine Rongeur, fine scalpel, syringe needle tip (size: 27 G).
      Using the fine Rongeur, open the spinal column on the ventral side along the segments D8-D10. Gently remove the dura mater with scalpel and forceps. For each insertion site for the silicon probes, open the pia mater with longitudinal cuts along the spinal cord with the tip of a bent syringe needle tip (size 27 G). Perform the cuts parallel to the ventral horn between the ventral roots, as superficial as possible (Figure 1E). This completes the procedures to make the integrated preparation. Figure 17 shows example electrophysiological recordings and histology (Petersen and Berg, 2016).


  1. Ringer’s solution (98% O2/2% CO2)
    120 mM NaCl
    5 mM KCl
    15 mM NaHCO3
    2 mM MgCl2
    3 mM CaCl2
    20 mM glucose
    Demineralized water
    The solution is saturated with 98% O2/2% CO2, by aeration for 30 min to obtain pH level of 7.6
  2. Ringer’s solution (95% O2/5% CO2)
    100 mM NaCl
    5 mM KCl
    30 mM NaHCO3
    2 mM MgCl2
    3 mM CaCl2
    10 mM glucose
    Demineralized water
    The solution is saturated with 95% O2/5% CO2, by aeration for 30 min to obtain pH level of 7.6

    Note: Either Ringer’s solutions can be used in this protocol.


Funded by the Novo Nordisk Foundation (RB), the Danish Council for Independent Research Medical Sciences (RB and PP) and the Dynamical Systems Interdisciplinary Network, University of Copenhagen. Thanks to J. K. Dreyer and J. Hounsgaard for reading and commenting an earlier version of the manuscript.


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  17. Vestergaard, M. and Berg, R. W. (2015). Divisive gain modulation of motoneurons by inhibition optimizes muscular control. J Neurosci 35(8): 3711-3723.
  18. Walloe, S., Nissen, U. V., Berg, R. W., Hounsgaard, J. and Pakkenberg, B. (2011). Stereological estimate of the total number of neurons in spinal segment D9 in the red-eared turtle. J Neurosci 31(7): 2431-2435.


【背景】脊髓电生理学的研究传统上与机械并发症有关,因为许多运动部件和脊柱的灵活性。为了规避这个问题,脊髓经常从柱中解剖出来并移动到可以进行稳定的电生理记录的室。然而,该过程具有缺点,例如,如果要研究多个电动机行为,则适当激活电动机电路是复杂的。此外,缺乏供血和缺乏氧气对电路的健康和完整性有严重影响。一个可以规避所有这些问题的实验模型是龟制剂(Keifer and Stein,1983)。在这里,不仅可以研究完全发达的脊椎动物脊髓,还可以研究几种不同类型的复杂运动行为,而不需要神经活性物质如谷氨酸激动剂,5HT和多巴胺。由于乌龟中枢神经系统的神经元能够进行厌氧代谢,因此电路的完整性比哺乳动物实验中要长得多。最后,乌龟甲壳组织允许稳定的多电极录制前所未有的质量。在这里,我们提供了一个详细的协议,用于设置整合的成年龟制剂,有时称为“离体准备(Guzulaitis et al。,2014)),其中完整的脊髓运动网络。该准备提供了测量腰椎脊髓段(图1)中的中心型发生器的机会,其类似于哺乳动物和其他动物的腰脊髓(Walloe等人,2011) 。该制剂包括整个脊髓段D3-S2 。刮擦反射的测量可以完全在没有化学麻醉的情况下进行。细胞外以及高密度细胞外记录可以与肌肉神经(ENG)的同侧和对侧电图记录同时在脊髓中获得。通过机械地接触甲壳的腹侧并因此相同或接近于自然的行为而引起擦伤反射。 Keifer和Stein(1983)引入了一个较小版本的综合龟制剂,随后进行了修改和修改(Currie and Lee,1997; Alaburda和Hounsgaard,2003; Alaburda等人,2005; Berg 2007年和2008年; Kolind等人,2012; Vestergaard和Berg,2015)。本发明的准备工作是由Petersen等人(2014)和Petersen和Berg(2016)进行的研究开发的,其中电极阵列垂直插入腰椎脊髓(Berg& et al。 ,2009)。

图1.具有植入电极的整合成体 - 龟制剂。 A.硅探针在脊髓中的放置示意图; B.用三个硅探针和细胞内玻璃电极制备。吸附用于电子记录的吸引电极(从顶部和底部以及左右指向)。光盘。用硅探针和细胞内玻璃电极关闭脊髓(仅在D中插入脊髓)。吸电极的尖端也是可见的。经Petersen和Berg(2016)经许可修改。 E.硅探针之后的脊髓已经缩回。蓝色DiD标记从第一和第三柄可见(每个探针的8个标记,用箭头突出显示)。

关键字:成年海龟, 综合制备, 离体, 脊髓, 电生理学, 细胞内和细胞外记录, 单一单位, 神经电图, 搔反射, 中枢模式发生器


  1. 手术刀#15(精细科学工具,目录号:10015-00)的Scalpel手柄#3(精细科学工具,目录号:10003-12)
  2. 切割刀片:BSN 2.5“不锈钢切片(BSN医用,目录号:480-4183-145)
  3. 塑料袋,5 L尺寸
  4. 手套
  5. 45 mm旋转刀片(WorldKitchen,OLFA,目录号:RB45-5)
  6. 氰基丙烯酸酯粘合剂(Panacol,目录号:Cyanolit 202)
  7. 有机玻璃板:80 x 15 x 2 mm
  8. 纸巾
  9. 100毫米或120毫米培养皿(VWR,目录号:HECH41042024或HECH41042030)
  10. 耳罩(Peltor TM Optime TM 98耳罩)(3M,目录号:10093045080912)
  11. 注射针头27 G,31 mm(BD,目录号:305136)
  12. 在这个程序中使用重量为300-500克的红耳龟( Trachemys scripta elegans ,Nasco,Fort Atkinson,WI,USA)
  13. 碎冰
  14. 氯化钠(NaCl)(Sigma-Aldrich,目录号:S3014)
  15. 氯化钾(KCl)(Sigma-Aldrich,目录号:P5405)
  16. 碳酸氢钠(NaHCO 3)(Sigma-Aldrich)
  17. 氯化镁(MgCl 2)(Sigma-Aldrich,目录号:208337)
  18. 氯化钙(CaCl 2)(Sigma-Aldrich,目录号:21115)
  19. 葡萄糖(Sigma-Aldrich,目录号:G7021)
  20. 林格氏溶液(98%O 2/2%CO 2)(参见食谱)
  21. 林格氏溶液(95%O 2/5%CO 2)(参见食谱)


  1. 剪刀
    精细剪刀(Fine Science Tools,目录号:14090-09)
    精细锯齿剪刀(Fine Science Tools,目录号:14058-11)
  2. 镊子:
    Dumont#5镊子(Fine Science Tools,目录号:11251-10)
  3. 钳子:Liston Gross Anatomy Bone Cutters(Fine Science Tools,目录号:16104-19)
  4. 课程和精细Rongeurs:Lempert Rongeurs(精细科学工具,目录号:16004-16)和Friedman-Pearson Rongeurs(Fine Science Tools,目录号:16221-14)
  5. 灌注系统:定制设计的空气压力系统/出口连接到1 L Pyrex玻璃瓶的入口与盖子。瓶子的出口连接到流量计和21 G 5 mm注射器针头(BD,目录号:305129)
  6. 铸造锯(Atlas International,American Orthopedic,目录号:T-CC-100)
  7. Pyrex玻璃格里芬烧杯1升(康宁,PYREX ®,目录号:1000-1L)
  8. 开放式有机玻璃容器,尺寸:200 x 150 x 25 mm(D x W x H),有机玻璃厚度:3 mm。自定义
  9. 细胞外记录:来自Neuronexus的64通道Berg64探针。 8个轴,每个8个交错记录位置垂直距离30μm,设计用于从乌龟(Neuronexus,BERG A8X8-5MM-200-160 PROBE)的腹角记录
  10. 细胞内记录:用P-1000 Sutter Instruments拉出玻璃移液管,并填充0.9M乙酸钾和0.1M KCl的混合物。对于组织学验证,可以将4%w / v生物胞素加入到混合物中(Biocytin,Sigma-Aldrich,目录号:B4261)
  11. 神经记录:电子记录(ENG)记录可以用吸引电极执行



  1. 第一部分准备
    1. 麻醉
    2. 林格的解决方案
    3. 初始手术和灌注
      1. 准备灌注系统:向玻璃瓶中加入500毫升林格氏溶液并关闭盖子。将瓶子连接到压缩空气系统/出口。
      2. 打开压缩空气,让灌注系统运行30秒,流量超过120毫升/小时。停止流动。
      3. 从冰浴拿起乌龟。必须满足两个条件才能确保乌龟完全麻醉:1)眼睛必须闭合,2)没有脚蹬退回反射。进行麻醉时确认。
      4. 用带齿的组织镊子拉出乌龟的头部,以便斩首。
      5. 用大钳子(图2)握住头部,用大剪刀剪开颈部。


      6. 用大钳子粉碎头部,并将其放在塑料袋中。
      7. 将乌龟放在其背面,并使用铸造锯切机在plastron中形成方形开口。四个切口的位置如图3所示。与脊髓正交的两个切口沿着两个中心扫描线(较小的平板)的边缘定向。保持切割之间的最小距离,平行于中线,高于5厘米。在继续操作之前,请确保使用铸造锯切割器完全松开正方形切口。如果方块自由移动就可以轻轻地推开切口孔的内角来验证这一点。


      8. 使用Graefe镊子提起切口长条,并使用手术刀将其从软组织中清除。
      9. 轻轻地抬起心包膜,用心脏夹住精细的“防护薄膜”,用Graefe镊子,用精细的剪刀将其打开,以进入心脏(图4)。

        图4.乌龟的窗口中的窗孔的内部视图。 心脏被箭头标记为圆圈和右心房。

      10. 找到心室和心房的心房(心脏和右心房以图4中的黄色圆圈和箭头标记)。用精细剪刀在右心房(位于左侧并突出显示)中切开一个孔,并将灌注针插入心室左侧的中心(图5)。将流量调整到75-130 ml / h范围内,相应的心率:当将灌注针插入心脏时,心率首先应急剧增加,但稳定在20-40 bpm左右。如果心率不在此范围内,则相应地调整流量:如果观察到较低的心率,则增加流量,如果心脏抽速太快则减少流量。

        图5.灌注针插入心脏心室的左侧部分。 右心房和心室分别用箭头和圆圈标记。

    4. 腹部和甲壳尾切割
      1. 在与脊髓正交的甲壳中进行一个朝向和尾部的横切。放置乌龟与甲壳朝上(背侧向上)。图18和19显示了脊柱段和相关的神经。中心型发生器位于脊髓段D8-D10(Mortin和Stein,1989; Mui et al。,2012; Hao等人,2014),和用于擦伤反射的感觉输入来自D3至S2的每个部分(图18,不包括尾部划痕)。为了保持对网络的感官输入不变,在D2和D3之间进行射线切割。尾部切割在S2和Ca1之间完成。两个切割见图6.D2-D3位于沿着5号中央火山口(Mortin和Stein)的中部2号和S2-Ca1中途,1990)。重要的是,切割是a)与脊髓正交,b)在从甲壳的中线到plastron的切割边缘的一次切割中进行,因为通过脊柱的灌注必须密封。尾部切割应垂直于甲壳曲率成角度。


      2. 当两次切割已经完成时,甲壳与plastron尾部的遗体分开。这种分离是通过在平面窗口(图7中的两条黄线)的角落处的两个对角线切割获得的。

        图7.腹侧切口(穿过plastron)。 两个对角线切割以黄色突出显示。

    5. 去除内脏,腹腔和后腿
      1. 将尾部的乌龟向上拉。
      2. 拿起大剪刀,将甲壳内部的内脏从下面和上面切开,将夹带器官的薄膜沿着甲壳内侧切开(图8)。当内脏松动时,沿着甲壳切割更容易。在删除它时,尝试将器官块保持在一块。


      3. 切割沿脊柱插入的大头缩回肌肉,尽可能远地沿着甲壳切除剩余的器官。
      4. 拉回一条后腿。用柔软的皮肤沿着大型剪刀做一个切口(图9)。从先前在plastron中制作的对角线切割开始,瞄准腿部。从甲壳上切开约1厘米的大腿骨(股骨)(图10)。



      5. 对另一个后腿重复步骤A5d。
      6. 切开甲板和尾甲尾端,不损伤脊髓。准备工作现在留在冷林格尔解决方案中的培养皿中(图11)


      7. 通过在冷的林格氏溶液(约50ml)中快速冲洗,清除制剂中的过量血液,并在完成后将其放入1升烧瓶中,冷的林格氏溶液。
    6. 清除脊柱

      图12.暴露的脊髓节段S1-Ca1和切缘横切 A.Ca1和更多的尾段都是柔性段,而D10和更多的分泌段与甲壳连接。黄色方块突出显示细分。 B.用旋转刀片切割脊髓,使椎骨平滑,使得管道密封。平行线表示由黄色正方形指示的区域和与初始切割相比的第二切割位置。

    7. 横断脊髓
      1. 将旋转刀片安装在铸造锯切刀中。
      2. 将叶片的边缘插入培养皿中的冷林格氏溶液中十秒钟以将其冷却下来。这将提高脊柱横断的质量。
      3. 从前一次切割开始脊髓横切约1-1.5毫米。横断面必须垂直于脊髓(图12B)。
      4. 将准备物旋转在冠状平面内180°并将其安装在夹具中。
      5. 执行脊髓的尾部横断。第二次切口提供了改善的灌注。
      6. 将准备工作放回林格氏溶液中的玻璃烧杯中。
    8. 将有机玻璃板固定在准备中 工具:氰基丙烯酸酯粘合剂,有机玻璃板,带盖的培养皿,冰,手术刀,灌注系统,纸巾。
      1. 将一层冰块放在最大的培养皿中,将较小的培养皿放在冰面上,面朝上,并用冷的林格氏溶液填满。
      2. 将准备物置于寒冷的林格氏溶液中,将前端朝上。
      3. 用纸巾干燥切割的口头甲壳边缘。
      4. 沿着切割边缘涂一层薄薄的氰基丙烯酸酯粘合剂,该边缘将是与有机玻璃板接触的区域(图13)。注意不要将粘合剂插入脊柱,这会阻碍林格氏溶液的流动。

        图13.将粘合剂涂在制剂的切片上。 沿对应于有机玻璃板接触区域的切口放一条细线粘合剂。

      5. 轻轻地将传统的有机玻璃板放在以切开脊髓的小孔为中心的制剂上(图14)。将其牢固地保持在原位约30秒。如果粘合剂不会变硬,请在胶水上涂上一些林格液体,这将有助于硬化,同时将板固定到位。


      6. 抬起准备物,沿着背甲甲和有机玻璃之间的接触线放置一条额外的胶水线。保持干燥一分钟,然后继续。
    9. 将准备安装到有机玻璃容器上
      1. 取出有机玻璃容器,并将胶水放在尾甲甲四个接触点处。
      2. 轻轻将龟制剂放在有机玻璃容器中(图15)。保持约30秒。


      3. 用林格氏溶液轻轻地填充有机玻璃容器。液体将有助于硬化粘合剂。
      4. 将准备物置于更大的塑料容器中,并将其完全浸入林格氏溶液中
      5. 第一部分的程序现已完成。在冰箱里过夜一夜。

  2. 第二部分准备
    1. 设置脊柱椎孔灌注
      1. 将钢管和硅胶垫圈安装到有机玻璃板的孔中。将垫圈压在D4椎骨上,并推入钢管以获得紧密的密封(图16)。使用林格氏溶液将管连接到一个凸起的容器。通过用林格氏溶液调节容器的相对垂直位置,保持300ml / h以上的灌注流量。


    2. 解剖电图记录的神经
      1. 识别源自D8-S2的运动神经,并将其免费解剖为ENG录音(图17A和18)。图18和19分别显示了神经和相应肌肉的位置:髋关节屈肌,髋关节伸展器,三个膝关节伸肌(FT-KE,IT-KE和AM-KE),dD8和HR-KF(Mortin和Stein, 1990)。应该解剖神经中的肌肉组织,以尽量减少ENG记录中的噪音。神经很健壮,但在剥离过程中容易受损,无明显的迹象
      2. 轻轻切开肌肉组织并释放神经。将周围组织释放神经的好方法是将精剪刀的尖端放置在靠近神经的组织中,沿着神经向远端拉尖锐边缘。

        图17.电生理记录和组织学验证。 A.细胞内和ENG记录; B.用高密度硅探针记录的约200个细胞外单位的光栅图; C.硅探针柄位置的组织学验证D.DDD在植入前涂在电极上。 ChAT和Nissl染色分别用作运动神经元和神经元的标记。分别为C和D的矢状和冠状切片。比例尺=500μm。改编自Petersen和Berg(2016)。


        图19.后肢主要肌肉组。 髋关节屈肌,髋关节伸展器,膝关节伸肌(FT-KE,IT-KE,AM-KE)和膝关节屈肌(HR-KF,延伸穿过髋关节和膝关节)。转载自Bakker和Crowe(1982),获得许可。

    3. 细胞外和细胞内记录的准备
      工具:精镊子,精细的Rongeur,精细的手术刀,注射器针尖(尺寸:27 G)。
      使用精细的Rongeur,沿段D8-D10打开腹侧的脊柱。用手术刀和镊子轻轻取出硬脑膜。对于硅探针的每个插入位置,用弯曲注射器针尖(尺寸27 G)的脊部沿脊髓纵向切割打开皮肤。执行平行于腹侧根部之间的腹角的切口,尽可能肤浅(图1E)。这完成了进行综合准备的程序。图17显示了电生理记录和组织学示例(Petersen和Berg,2016)


  1. 林格氏溶液(98%O 2/2%CO 2)
    120 mM NaCl
    5 mM KCl
    15mM NaHCO 3
    2mM MgCl 2
    3mM CaCl 2
    20 mM葡萄糖
    通过曝气30分钟使溶液饱和98%O 2/2%CO 2,以获得7.6-7 /
  2. 林格氏溶液(95%O 2/5%CO 2)
    100 mM NaCl
    5 mM KCl
    30mM NaHCO 3
    2mM MgCl 2
    3mM CaCl 2
    10 mM葡萄糖
    通过曝气30分钟使溶液饱和95%O 2/5%CO 2,以获得7.6-7 /



由诺和诺德基金会(RB),丹麦自治医学科学理事会(RB和PP)和哥本哈根大学动力系统跨学科网络资助。感谢J. K. Dreyer和J. Hounsgaard阅读和评论手稿的早期版本。


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引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Petersen, P. C. and Berg, R. W. (2017). Spinal Cord Preparation from Adult Red-eared Turtles for Electrophysiological Recordings during Motor Activity. Bio-protocol 7(13): e2381. DOI: 10.21769/BioProtoc.2381.
  2. Petersen, P. C. and Berg. R. W. (2016). Lognormal firing rate distribution reveals prominent fluctuation-driven regime in spinal motor networks. eLife 18805.