Well, in this protocol, we use a miniature microscope to investigate the neuroactivities in the dentate gyrus of the hippocampus. We aim to investigate neuron coding and spatial representations during the different disease conditions. I believe the key challenge of studying dentate gyrus in vivo involves obtaining high quality calcium imaging data.
Whether the various expression is satisfactory, whether the GRIN lens is implanted at the proper location, and the details like this during the entire surgery are critical importance for the success of the study. In the traditional calcium imaging surgery, the virus injection and the GRIN lens implantation are typically performed as separate operations. In our protocol, we combined these two steps into a single procedure, which not only reduces the waiting time, but also ensures the accurate placement of the GRIN lens.
Our lab uses various microscopy techniques to study the neurodynamics underlying cognitive functions. We hope to understand the neuroactivity patterns that support cognition, and combining theoretical modeling and causal manipulations, we hope to dissect the processes of information flow and neurocomputation in the brain. To begin, place the anesthetized mouse on a stereotaxic apparatus.
Make a continuous incision along the midline of the scalp using surgical scissors, and cut off part of the scalp to expose the skull surface. Make the craniotomy in the specified region with the micro drill, and set four points as the border. Stop drilling when brain tissue becomes visible.
Use the control panel to set the injector to inject 80 nanoliters of the virus. Click the Inject button on the control panel to inject the virus into the dentate gyrus at a flow rate of one nanoliter per second. To suction the brain tissue, revolve the tip of the blunt needle closely above the brain tissue, and press gently to facilitate aspiration.
Rinse continuously with cold saline during suctioning to maintain a clear view of the brain. Observe the tissue features closely to monitor the depth of aspiration. The cortical tissue is pale pink.
The corpus callosum underneath appears as white fibrous tissue, and the hippocampal CA1 layer is dark red and gray. Stop the suction when the surface of the tissue becomes smooth and a large area of CA1 is exposed. Move the holder attached to the GRIN lens above the drilled hole.
When the GRIN lens contacts the surface of the brain tissue, set the Z axis to zero. Insert the GRIN lens into the hole at the dorsal ventral axis minus 1.32 millimeters. Allow the holder to stand for five to 10 minutes, then raise the holder and rinse the hole with saline.
Using a needle, apply the ultraviolet resin around the GRIN lens. Illuminate the area with ultraviolet light for 15 seconds to ensure that the resin becomes solid. Carefully remove the holder from the GRIN lens.
Cover the entire exposed skull with ultraviolet resin. Place the head plate on the skull in the appropriate position. Illuminate the entire skull and head plate with ultraviolet light for 15 seconds.
Cover the exposed GRIN lens with a 3D printed protective cap. Fix the cap to the head plate using M1.6 screws. Turn on the mini scope control software and click Select Config File, then select VS code, followed by user configuration files.
Now click Run, then slide the LED power button to adjust the brightness. Slide the focus adjustment button to set the value near zero. Attach the base plate to the mini scope, and readjust the position of the mini scope to obtain a field of view with good imaging quality.
Carefully apply the first layer of denture base material around the mini scope base plate. Once the first layer of the cement becomes hardened, apply a second layer of cement from the base plate to the head plate. After all denture base material hardens, loosen the set screw and detach the mini scope from the base plate.
Put the base plate cap into the base plate to protect the exposed GRIN lens and tighten the set screw. After imaging, convert the imaging data from AVI to HDF5 format. Apply the non rigid motion correction, and down sample the motion corrected movie.
Apply extract on the down sample data to identify single cell signals, then apply cell check and manually select the potential cells. Click Save Data before closing the cell check window. Finally, save the data files.
In unsuccessful in vivo calcium imaging, no active cells were observed in the imaging field of view. Successful in vivo calcium imaging showed fewer than 10 active cells without obvious blood vessels, more than 50 active cells with visible blood vessels, and hundreds of active cells with clear blood vessels. Extracted individual cells from a successful in vivo calcium imaging recording were superimposed on the field of view, with representative calcium traces shown.
The position of GCaMP6f expression and the track of the GRIN lens were verified in a mouse brain slice.
