The overall goal of this procedure is to apply modern optogenetic techniques to photo stimulate targeted neuronal pathways in in vitro slice preparations. This is accomplished by first expressing channel rod Dossin in the neuronal cell lines of interest. The second step of the procedure is to make acute slice preparations of the relevant brain structures to be studied.
The next step is to record responses from neurons in the slice preparation in response to photo stimulation of the channel, rod Dossin expressing fibers. The final step is to image the distribution of channel opsin expressing neurons and fibers. Ultimately, this approach enables an assessment of the functional topography and synaptic properties of neural circuits using laser scanning photo stimulation of optogenetically targeted neural components.
The main advantages of this technique over traditional electrical stimulation methods are the ability to specifically target and stimulate particular neuronal cell types and examine their long range projections in vitro. This method can help answer key questions in the field of systems neuroscience, such as the functional organization of complex war sockets. Prepare a syringe and needle to inject the viral solution, which contains four micrograms per milliliter of poly to enhance transfection.
Then pressure inject 200 nanoliters of the solution using the syringe pump, two to three weeks later, there will be adequate expression of channel rod Dossin slice. Preparation should be performed as quickly as possible After removing the injected brain, make a block of the brain in the desired orientation using a clean razor blade. Next, affix the blocked surface of the brain to the cutting stage using instant adhesive glue.
Then transfer the stage to a freezer chilled vibram cutting chamber and pour ice cold A CSF into the cutting chamber. Slice the brain sections using a viome into four to 500 micron thick sections. Transfer the brain slices to an oxygenated holding chamber and allow them to incubate at 30 degrees Celsius for at least an hour before proceeding with the experiment.
When ready to begin the experiment, transfer the brain slice to the stage of the electrophysiological recording rig. Record a neuron using standard whole cell patch clamp techniques. Briefly position the recording pipette by the neuron while applying positive pressure to the pipette.
After contact with the cell is made, release the pressure and apply suction to form the giga seal. Then apply brief suction to break the membrane for whole cell configuration and take recordings in voltage or current clamp modes. Operate the laser scanner with either tidal wave or EFAs data acquisition software.
The software must be calibrated to the photo diode before use to control the power of the laser. A variable neutral density wheel is placed in its path. Rotating the wheel adjusts the laser's power by measuring at the back focal plane.
The power is typically set to between 25 and D milliwatts. The pulse parameter of the shutter controls the length and timing of stimulation in the software. An image of the region to be stimulated can be made from the grab video function.
Next, set the stimulation grid such as a 16 by 16 grid with 80 micron spacing. Then overlay the grid onto the acquired image and adjust the XY coordinates and degree of offset to optimize the orientation to start the stimulation. Activate the map function.
The data is retained and can be analyzed using the offline analysis program in tidal wave, or it can be analyzed using the map analysis program in the EFAs software. After completing the physiological recordings on the slice, the expression pattern of YFP tagged channel opsin can be documented. First, fix the slice overnight in 4%PFA in 0.01 molar PBS at four degrees Celsius.
The next day, transfer the slice to 30%sucrose 4%PFA solution for overnight cryo protection. The following day, cut 50 micron sections of the slice on a cryostat. Rinse the slices in PBS and mount them for imaging with an anti fade medium at the confocal microscope.
After finding the region of interest in white light, use a 514 nanometer excitatory wavelength and a 527 nanometer emission wavelength. To image YFPA viral construct that drives expression of channel rod Dobson, EYFP. In excitatory glutamatergic neurons was injected into the primary visual cortex of a BSY mouse.
Viral expression was found in tracks between the primary and secondary visual cortices, and in the cortico thalamic fibers of the LGN whole cell patch clamp recording from a neuron in the secondary visual cortex marked by the star revealed e PSCs. Following laser scanning photo stimulation of the channel, rod Dossin expressing fibers in the lower layers of V two cortico thalamic projections from layer six of the primary somatosensory cortex. The ventral posterior nucleus were studied using a CRE LAX approach to express channel rod dossin in layer six cortico thalamic neurons, and in their projections as expected transfection by a flocked a a V construct into a CRE transgenic mouse resulted in channel rod dosin expression in the targeted layer six neurons and their fibers extending to layer four.
Channel rod Dobson, EYFP expression was also noted in fibers projecting to VP physiological recordings from a VP neuron. Using whole cell patch clamp revealed that e PSCs could be elicited following photo stimulation near the recorded neuron, which is marked by the star. Offline analysis depicts the mean response amplitude to photo stimulation in VP Resectioned 50 micron slices of layer six cortico thalamic neurons expressing channel Rodin EYFP revealed a fine pattern of labeled fibers traversing from layer six to layer four where they terminated and arbor extensively.
In addition, cortico thalamic fibers were observed entering the white matter and traversing towards the thalamus. After watching this video, you should have a good understanding of how to apply modern optogenetic techniques to photo stimulate targeted neuronal pathways in in vitro slice preparations. Don't forget, working with lasers can be extremely dangerous when you perform calibration steps associated with this procedure.
Wear protective eyewear.
