The overall goal of this procedure is to analyze leg muscle physiology. Its timing and kinematics in a tethered insect during different walking situations. The stick insect raus miosis is used for this study because for an insect, it is relatively large and has clearly separated segmental ganglia.
First, the animal is tethered and labeled for fluorescent video tracking. Next EMG electrodes are placed in the protractor and retractor muscles. Then the preparation of the tethered insect for tarsal contact recording on a slippery surface follows.
The final step of the procedure is the simultaneous recording of leg muscle activity and tarsal contact during different walking situations. Ultimately, results can be obtained that show alterations in muscle timing. Depending on the behavioral context, a leg is used in The main advantage of this method over existing techniques like studying.
Walking on trek balls is that mechanical coupling through the ground is removed. This method can help answer key questions in motor control, such as how much a single leg contributes to walking in a motor leg animal. The implications of this technique open the field of adaptive locomotor behavior because we can now study changes in walking speed, in walking direction on the single leg level, demonstrating the setup will be hunts.
Peter Boha, a technician in my laboratory, Two firmly joined nickel coated brass plates insulated by each other through a two millimeter wide strip of two components. Epoxy are used to prepare the slippery walking surface for the animal, lubricate each half of the plate separately with an electrically conductive glycerine saline mix using a piece of soft tissue. When correctly applied, the solution should be between 0.1 and 0.2 millimeters thick and create a surface kinematic viscosity of about 430 centi stokes.
Two round ground glass screens are positioned in front of the plate such that during an experiment, they are 70 millimeters from the animal's eyes. When a moving striped pattern is projected on these screens, the animal will begin to walk forward. Walking is induced by a progressive pattern on both screens with stripes moving outward or by a single black beam on white background placed between the screens, which can elicit longer straight walking sequences.
If both progressive stripe patterns moving the same direction turning is induced. Once the experimental setup is assembled, the animal can be prepared for an experiment using dental cement tether, a female adult stick insect ventral side down onto a thin bolsa stick, which is attached to a brass tube with a socket. The head and legs protrude from the front and side of the dell to allow their free movement.
Next fluorescent pigments mixed with dental cement are used to mark the head thorax and the distal femur and tibia of each leg for later. Video tracking. The animal's muscle activity is recorded by means of implanted EMG electrodes into the thorax.
The signal is amplified 100 times in a pre amplifier and then amplified 10 times and filtered from 100 hertz to 2000 hertz before conversion to a digital signal. Electrodes are made from two coated copper wire smear with glue, then twisted around each other, sold a one end of the twisted wire to a mini plug and cut the other end using a minute pin punch small holes into the cuticle through which the electrode will pass. To contact the muscles placed the mini plug on one end of the wire to the socket attached to the brass tube to which the animal is tethered.
Insert the cut end of an electrode through the hole just beneath the cuticle into a muscle such that the two wires of each electrode are placed in separate holes about one millimeter apart. To hold the wires in place, we use a small drop of histo acrylic glue. One more wire is inserted into the abdomen, which will act as the reference electrode in order to understand the relationship between the kinematics of insect walking and muscle physiology.
Tarsal contact during walking is measured during EMG recording burn off the insulation at the end of a 15 centimeter long piece of copper wire with alcohol to be able to measure current flow through the tarsus. Next, a loop is formed on one end of the wire slid over the tass of the animal and tightened at the distal end of the tibia. The wire is then fixed on the thorax of the animal with a drop of dental cement, and the bolsa stick in the brass tube to which the animal is glued is connected to a micro manipulator.
In order to allow height adjustment of the animal between seven and 12 millimeters above the slippery surface, this corresponds to the height of the insect during free walking through an alligator crimp. The other end of the wire is then connected to the differential lock-in amplifier. We apply a drop of electrode cream on the areas of contact to improve conductivity of the wire At its ends, the circuit is completed by connecting the plate to the amplifier with plugs at the base of the plate.
A pulse generator creates a signal which is applied to both halves of the plate via these plugs. One of the leads from the plate is connected to the amplifier and used as a reference signal. Now with the animal in place and the wires attached, its leg becomes a switch and a current will flow through the tarsus to close a circuit between the plate and the amplifier.
This produces a signal with a lead time of less than one millisecond at contact and a delayed signal that lift off. The signal strength is tested by moving the tarsus up and down. The EMG wires are then connected to the amplifiers.
The trigger signal for the camera is recorded simultaneously with the tarsal contact and the EMG trace to allow frame by frame correlation of the traces. Now, a walking sequence can be recorded with the animal in place and everything attached. The visual pattern and the video recording are started simultaneously in order to create a visual recording of the movement as a control.
Using a 100 frames per second high speed video camera record the leg movement from above and through a mirror placed at a 45 degree angle behind the animal. If the animal does not start locomotion spontaneously, it can be stimulated at the abdomen with a brush or with a puffer there to the antennae. Any tracking of the fluorescent markers on the animal can be improved by illuminating the animal with an array of blue LED lights and attaching a yellow filter to the camera lens.
This suppresses the short wavelength of the activation light and adds contrast to the video. First record the walking sequence of the intact animal as a control. Changing the visual stimuli changes the direction of locomotion.
If desired, amputate the legs by pinching the femur with a pair of forceps and allow the animal 30 minutes to recover. Now the physiological role of the missing legs can be assessed. The dots in this image show the positions of electrodes in the protractor and retractor muscles of the middle leg.
In this EMG preparation, the front and hind legs have been removed to study the effect of the neighboring legs on the kinematics and muscle activity of the middle legs. Now the data in this trace of a turning animal, the top trace shows the tarsal contact trace. While the bottom trace shows the EMG from the retractor muscle.
During forward steps at the beginning of the trace, the retractor is active during stance while the leg has ground contact. After a short switch, the leg engages in backward steps and the retractor is active during swing, while the leg is in the air. Here is a complete view of all the data collected while the animal walks on top is the EMG trace from the protractor muscle.
At center is the EMG trace from the retractor muscles, and below is the tarsal contact signal. In the inset, you can see the animal's actual movements captured by the high speed camera. The alternating activity in the EMG traces corresponds to the activity of the two muscles in the steps cycle.
The protractor muscle is active just before and during swing phase, and the retractor is active after touchdown and during the stance phase. Once mastered, the preparation can be done in less than 90 minutes, and data can be acquired for several hours following this procedure. Other methods like kinematic analysis or single cell recordings can be performed in an almost intact behaving animal After its development.
This technique has helped researchers in the field of motor control to address questions on how single appendages generate flexible and adaptive locomotive patterns.
