This protocol provides accurate spatial temporal information on neuroactivity. The main advantage of this technique is ability to manipulate and evaluate neuroactivity with high spatial temporal resolution. It may be useful for illustrating the pathogens of neuropsychiatric disorders that lead to abnormalities in neural activity.
This method can be applied to the study of neurons as well as health in the other organs. Demonstrating the procedure will be Zhongtian Guo, PhD graduate student from our laboratory. One day after head plate implantation administer 1.32 milligrams per kilogram dexamethasone sodium phosphate intraperitoneally one hour before surgery to avoid cerebral edema.
Under a stereoscope, perform a circular craniotomy approximately two millimeter in diameter using a dental drill. To reduce brain damage, operate the dental drill carefully with constant slight movement and light downward pressure. Remove the bone fragments several times using a suction system.
After removing the bone fragment, use an artificial spinal fluid or ACSF solution to remove and wash any debris remaining on the brain's surface. Repeat this cleaning procedure several times to suppress inflammatory reactions. Using a pressure injection system, set the appropriate pressure to inject 500 nanoliters of AAV solution through a glass capillary with a tip diameter of 10 to 20 micrometers for 10 minutes.
If the level of the AAV solution in the glass capillary decreases gradually, the AAV solution is being administered to the brain. Leave the glass capillary in place for an additional 10 minutes to prevent backflow. Repeat this three times to administer a total of 1.5 microliters of AAV solution into the brain.
Apply 2%low melting agarose to the brain surface of S1 using a micropipette, and then place a glass window over the craniotomy with two cover glasses. Press the cover glass against the agarose while it is still liquid. This prevents the formation of air bubbles in the agarose.
Seal the edges of the cranial window with dental adhesive resin cement. To calibrate the holographic stimulation system, place the surface of a red fluorescent slide at the sample plane and set the microscope in live imaging mode with a weak excitation light. Run the calibration GUI.
mfile, check the parameters pane and click on the Save button. Click the Z Scan button in the step one pane if it'll automatically generate three random spots in 21 different axial planes two micrometers apart from each plane. Move the slide bar and check the live image.
Find the perfect plane where the spots appear the smallest and the brightest, and then click the Save button. This will automatically generate an offset spherical wavefront for the digital hologram. Click the Go button in the step two pane and then click six spots on the left square.
Check the live image. If there are six distinguishable fluorescent spots, then type their x and Y axis to the edit boxes and click the Save button. This will automatically generate a fine transform coefficient to coordinate calibration between the holographic stimulation and imaging system.
Then, click the Scan button in the step three pane. It will generate 441 digital holograms to perform single spot scanning across the field of view in 21 by 21 steps. Check the images while changing patterns in the list box.
Adjust the laser power to obtain spot images within the dynamic range of the imaging device. Put the imaging device in recording mode and click the Play button. After the play completes, click Generate Weight Map in the step four pane and choose a stacked image.
Then close the calibration GUI window. It will automatically generate a weight map to compensate for the unbalanced intensity in each spot. Place the AAV injected mouse with a head plate under the microscope and perform two-photon imaging using a holographic microscope and a mode locked titanium sapphire laser tuned to 920 nanometers with a 25x objective.
Open the commercial imaging software in the live imaging mode. Adjust the voltage of the image detector and the power of the imaging laser to optimize the brightness of the neurons expressing GCaMP8f. Capture images of the neurons expressing this protein.
To illuminate the specific neurons with holographic illumination, run the SLMcontrol. m script file. Click the reference image and choose the acquired image.
Then, click the Spot button to choose specific pixels on the neurons in the image and press the Enter button on the keyboard to finalize it. To detect neural activity with high temporal resolution using an image sensor, set the exposure time, imaging area and binning, then perform the image acquisition. After placing the mouse under the microscope and performing two-photon imaging, open the commercial imaging software.
In the live imaging mode, adjust the voltage of the image detector and the power of the imaging laser to optimize the brightness of the neurons expressing GCaMP-6m-p2A-ChRmine. Capture images of the neurons expressing these proteins and then illuminate the specific neurons following the procedure shown earlier. To investigate the functional connectivity within layer two and three neurons, use a spatial light modulator to generate holographic patterns of optogenetic stimulation and combine it with two-photon calcium imaging.
Set the intensity of the imaging laser to 920 nanometers at 10 to 20 milliwatts and the field of view to 256 micrometers by 256 micrometers measured at a depth of 100 to 150 micrometers from the cortical surface. Set the pixel dwell time at 1.5 microseconds for two hertz or 100 nanoseconds for 30 hertz. Use both two hertz and 30 hertz as the imaging frame rate to see if a single holographic stimulus caused a calcium response in the neurons.
Set the intensity of the holographic stimulation laser that stimulates a single neuron at 10 milliwatts which is sufficient to induce neural activity. Simultaneously, image the calcium iron response at 920 nanometers with 10 holographic stimuli at 1040 nanometers and eight seconds intervals for a duration of 50 milliseconds after a baseline period of 10 seconds. After that, return the mouse to its home cage.
The representative image and traces of neurons expressing GCaMP8f in 100 hertz imaging with holographic stimulation and an image sensor are shown here. This graph shows the neural response to holographic stimulation at each laser power. The representative Calcium 2+traces during holographic stimulation of 10 different neurons at two hertz and 30 hertz imaging frame rates are presented here.
The same color indicates the same neuron. A schematic diagram evaluating functional connections between neurons is depicted here. When the orange neuron is stimulated, the red neurons respond at the same time indicating that there is functional connectivity between these neurons.
A typical image of the primary somatosensory cortex neurons expressing GCaMP6m in wild type is shown here. These graphical images represent the typical Calcium 2+traces during holographic stimulation at two hertz and 30 hertz imaging frame rates. Neuro responsiveness to holographic stimulation can be detected at both two hertz and 30 hertz imaging speeds.
These steps are important to reduce brain motion and two, obtain stable results. We believe that this microscope can induce behavior in in writing neurons with specific patterns. With this technique, we are now able to assess and manipulate the activity of individual neuron and characterize neuropsychiatry in detail.
