The goal of this procedure is to prepare and remotely operate LED arrays to control neural activity in brain slices. This is accomplished using in vitro slices from animals with channel REDSIN two expressing neurons, and using an LED array in conjunction with a microscope to activate those neurons. This video covers how to calibrate an LED and microscope to deliver known intensities of light to the slice chamber.
With this setup, the activation thresholds of channel two expressing neurons can be accurately measured in response to light stimulation through widefield stimulation of channel opsin two expressing neurons. This technique allows temporarily precise optical control of a large neuronal population. The main advantage of this technique over existing optogenetic methods, including those that use lasers or shutter based methods, is that it's very inexpensive and can be easily fitted to an existing patch clamp scope.
This method can help answer key questions in the field of adult neurogenesis, including how does activity shape the integration of new neurons into the adult brain. The implications of this technique extend toward therapies or diagnoses based on adult neural stem cells as it offers precise temporal control over the activity of adult born neurons integrating into existing neural circuitry. Though this method can provide insight into the functional consequences of adult neurogenesis, it can also be performed on any in vitro preparation.
Containing channel adoption expressing neurons Begin by selecting an LED array that produces its peak power at the desired wavelength. In addition, choose the array with a maximum current rating. Integrate the LED array to the microscope.
Start by attaching the LED array to a fan called heat sink using thermal paste to increase the effectiveness of the heat sink. Then remove the low power stock LED from its lens assembly and fix the higher power LED to the lens. Now replace the Brightfield lamp with the assembled LED apparatus, and make sure that the fan is securely grounded to the system's common ground.
Careful positioning of the LED arrays needed to ensure that the call made light is in line with the objective.Objective. Achieve Kohler illumination by focusing the condenser so that the image of the field diaphragm is focused on the slice chamber and is centered under the objectives optical path. A thin tissue of lens paper can help focus the image of the back aperture at other depths.
Now, drill pinholes of known diameters in an opaque material that can act as an aperture for the power meter. Place one of these small pinholes over the sensor of the optical power meter. Then fix the power meter to the microscope to focus the condenser on the pinhole.
Manually position the power meter until it gives a maximum reading. Affix the power meter in this position. If the power meter maximum is above the plane of the slice, reestablish Kohler illumination at that plane.
Next, fully open all the apertures. Systematically move the power meter relative to the optical light path using the stage manipulators. Once finished, measure the uniformity of optical power within the illuminated area by systematically taking power readings in a grid around the maximum power, which should lie under the focus of the objective, The microscope is properly set up with cooler illumination centered at the objectives focus, the maximum power of the array should be at this focus regions outside of the focus should now receive a known amount of power according to the uniformity plot.
Now, measure the maximum power for each size pinhole with knowledge of the drill bit diameter used to make each pinhole plot the optical power as a function of the pinhole's area. Because the drilled pinholes are not perfect, averaging these values will produce a good estimation of optical power density. The LED array is gonna be used for patching optics.
Make sure you take into account all the optical elements needed for patching when you build the power plot and the uniformity plot. For instance, many microscopes have a flippable diffuser screen that you can use that will sacrifice power for the sake of uniformity. Prepare brain tissue slices using standard methods, and while allowing the slices to recover for 30 to 45 minutes in warmed A CSF, go and make the patch pipettes.
Once the tissues have recovered, gently place a slice into the recording chamber of the microscope under constant perfusion of oxygenated A CSF. Confirm the presence of EYFP channel redsin two channels by epi fluorescence under fluorescent illumination. Locate in the slice a healthy channel two EYFP neuron with mature morphology.
Then locate this neuron soma under patching optics and produce a giga ohm seal between the plasma membrane and the walls of the patch electrode. If the electrode is sufficiently closed to a spiking neuron, spontaneous activity should produce a measurable local field potential even without a giga ohm seal. Now activate channel adopts two in this neuron by flashing different doses of light because light doses a function of both LED power and duration calculate how much light is necessary to evoke an action potential at multiple powers and durations.
An Olympus BX 51 wi microscope was configured with an LED inline with two apertures and a condenser lens. Sufficient brightfield contrast for patch clamp recording was obtained by closing both the field diaphragm and the aperture diaphragm. A uniformity plot of LED light intensity was constructed in a region around the objective field of view indicated by the dotted circle.
With all diaphragms fully opened, slicers were exposed to maximum light power for channel adoptin activation. This configuration produces light three orders of magnitude more powerful than the configuration used for patch clamp visualization. Widespread labeling of adult born olfactory bulb granule and per glomerular neurons were observed four weeks after lentiviral infection of migrating neuroblast in the rostral migratory stream.
Loose patch recordings from a single born channel Adoptin two EYFP expressing granule cell indicated that for this neuron, a five millisecond stimulation at maximum power was sufficient to evoke spiking. Once mastered, this technique can be done in an afternoon. It's important to remember after this procedure to always monitor your equipment and your settings for drift over time Since their development, such optogenetic techniques have paved the way for researchers in the field of neuroscience to explore synaptic transmission and integration in a number of model organisms such as worm, fly mouse, and even primate.
