In this protocol, we begin with feeding the larva of appropriate genotype with E based food containing all trans A TR.Each lab is pinned down and dissected in a normal saline for the replace the normal saline with two milli calcium ringer solution and set the LABA preparation on the microscope stage. Motion of the dissected LABA is videotaped with CCD camera. By illuminating the nervous system locally, we can perturb neuro activity of a subset of neurons in a specific timing while monitoring the motion of the same inact preparation.
Hi, I am Tega in Dr.A lab. Hi, I'm Hi Kosaka, also in dr. A lab Oph level.
Locomotion is a useful model system for studying motor circuit. Because of highly developed genetic techniques in this protocol, we shall optogenetic manipulation of motor neurons while monitoring level motion. By using lasers in the confocal microscope, a subset of neurons can be stimulated and motor responses to the spatial temporal perturbation of the neurons can be analyzed.
So let's get started. Maintain fly lines of K six G four US CHR two, or K six G four US NPHR in plastic bios containing standard live food. Add a TL solution to yeast based at the appropriate concentrations and mixed well Spread the yeast based on an apple juice aate.
Pick up second or third in SLA from the vials. Put the lobby on the aate with yeast space containing a TR.Cover the plate with aluminum foil to keep a TR in dark. Grow the lobby at 25 degrees C for an appropriate period of time.
This is a CCD camera for visualizing of same intact lab. Attach the CCD camera to a conventional confocal microscope with a SEMA detachment. Pick up the A TRA lobby and rinse them with water.
To remove residual food from the body, put the lava on the C guard cold dish dorsal side off. Pin down with insect pins of the head and tail. Add calcium free normal saline.
Make a small incision near the tail with micro scissors from the incision. Make a longitudinal cut along the dorsal midline. Be careful to avoid damaging the ventral nerve core and the peripheral nerves.
Make a small lateral incision of the head pin down each corner of the dissected body wall. Remove the internal organs except for the brain and the ventral nerve cord. Replace the normal saline with 2 million model calcium ringer solution.
Attach a full power dry objective lens in the confocal microscope and set the lab preparation on the microscope stage. Obtain a transmission image of the dissected preparation with a red laser to locate the ventral nerve code by confocal microscope. Define the region of interest on the transmission image zoom mold is useful for determining the specific region of interest.
Change the filter set of the microscope to illuminate with 488 nanometer or 559 nanometer light. Visualize the body wall by illuminating the preparation with the halogen lamp. Monitor the preparation with a CCD camera attached to the confocal microscope.
Recall the motion of the body wall and switch the laser beam only all to temporarily manipulate neural activities while monitoring the motion peristaltic motion in the same intact preparation is observed and recorded by the CCD camera. This lab expresses CHL two in modern neurons. Illuminating one or two segments of the ventral nerve core activates modern neurons in the segments, which in turn induces muscle contraction in the corresponding body wall segments stimulating other segments in the ventral nerve.
Cold induces contraction of other body segments. NPHL was expressed in motor neurons when a few segments of the ventral nerve call were stimulated with a yellow laser. The propagating wave was arrested at the corresponding segment of the body wall.
The wave then restarted from the arrested segment when the laser elimination was switched off. This protocol elucidates the procedure for performing optogenetic perturbation of neural activity with lasers in same inact circular level in motion. The ventral nerve code and the peripheral nerve must not be damaged during this dissection to achieve sufficient stimulation.
Illumination power should be adjusted by changing laser power scanning speed or number of repetitions in confocal microscope. This is the entire protocol. Thank you for watching and good luck in your experiments.
