Many challenges can arise when using miniaturized microscopy for in vivo calcium imaging of the amygdala. This protocol provides time-saving guidelines for achieving successful in vivo calcium imaging. In vivo calcium imaging allows the continuous simultaneous imaging of genetically defined cell populations over a period up to several weeks, a process that was previously not possible.
For AAV injection, with the anesthetized mouse secured in a stereotaxic frame, align the height of the bregma and lambda with the pin and identify the specific location on the exposed and disinfected skull for the craniotomy. Use a drill equipped with a 0.6 millimeter bit to carefully make a 1.6 millimeter craniotomy with an 18, 000 revolutions per minute drill speed and wash the skull with sterile PBS. Subtract the dorsoventral difference between the bregma and the exposed brain surface from 4.2 millimeters, the dorsoventral coordinate of the lens implantation based on a bregma to calculate the value A distance, and load three microliters of mineral oil and 0.7 microliters of a greater than one times 10 to the 13th viral genomes per milliliter of the AAV solution into a glass pipette.
Remove the pin from the stereotaxic probe holder to allow the pipette to be fixed to the frame and position the pipette over the prepared injection site. When the bleeding has completely stopped, insert the glass pipette tip 4.3 millimeters into the brain tissue from the dorsoventral bregma coordinate and use an injection pump to deliver the entire volume of virus before slowly removing the pipette. When all of the virus has been injected, drill the skull two more times as demonstrated to allow the implantation of two skull screws and wash the skull fractions with PBS.
Next, implant the 70%ethanol sterilized screws as deeply as possible and install a motorized surgery arm on the stereotaxic frame. Connect the required hardware to a laptop computer on which the controlling software is installed and use a cannula holder to stably secure a 26 gauge needle onto the stereotactic frame. After calculating the coordinates for the needle insertion site, use the stereotaxic manipulator to position the needle tip at the exposed surface of the brain and hydrate the brain tissue with two to three drops of PBS.
Enter the appropriate dorsoventral and lateral amygdala coordinates into the software and click GoTo to lower the needle. When the needle has completely stopped, enter 45 millimeters to the start position box and click GoTo to elevate the needle. When the needle stops moving, remove the PBS from the brain surface and uninstall the needle and cannula holder from the stereotaxic frame.
Attach the lens holder to the stereotaxic rod and fix the GRIN lens to the lens holder. Sterilize the lens with 70%ethanol and attach the stereotaxic rod to the stereotaxic frame. Position the tip of the GRIN lens at the designated site on the brain surface and subtract the absolute value of A from the dorsoventral coordinate of the brain surface to determine the dorsoventral coordinate for lens implantation.
Hydrate the brain tissue with an additional two to three drops of PBS and use the motorized arm to set the lowering depth to 1, 000 micrometers and the raising height to 300 micrometers. When the GRIN lens reaches the target site, remove the PBS and carefully apply resin dental cement around the GRIN lens, screws, and screw sidewalls. When the dental cement has hardened, remove the lens holder from the lens and apply acrylic cement to the skull surface.
Next, replace the lens holder with a head plate taped to the tip of the rod in a horizontal orientation. Slowly lower the rod until the head plate is positioned at the top of the lens cuff before lowering the head plate 1, 000 micrometers until the lens is located at the right edge of the internal ring of the head plate. Use acrylic cement to attach the head plate to the cement layers and use paraffin film to protect the implanted GRIN lens surface from dust, then apply removable epoxy bond onto the paraffin film to secure the film to the lens surface and return the animal to its cage with monitoring until full recumbency.
To evaluate the quality of the GRIN lens implantation, place a carbon cage under the anesthetized mouse on the head bar of a mobile home cage system and turn on the air flow so that the cage moves freely without friction. Use a microscope gripper and a stereotaxic manipulator to vertically align a miniaturized microscope to the frame before lowering the microscope until the surface of the implanted GRIN lens appears in the image field of view within the software. Align the center of the implanted GRIN lens with the center of the field of view and capture the image of the implanted GRIN lens surface, then slowly raise the objective lens of the microscope while observing the image for the appearance of GCaMP expressing cells.
In animals with a successful lens implantation, both GCaMP expressing cells and blood vessels can be clearly observed within a single focal plane range. In contrast, in animals with off-target implantation, a clear image of GCaMP expressing cells is not observed within the focal plane range, although the blood vessels can be observed with some difficulty. In the case of an out-of-view lens, neither blood vessels nor GCaMP expressing cells can be visualized and a bright edge can be observed on the implanted GRIN lens.
In animals with a successful GRIN lens implantation, approximately 50 to 150 likely spontaneously active cells typically display a significant fluorescence change within the field of view in the lateral amygdala even without tone. Upon tone presentation, only a few cells display a tone-specific change of GCaMP signal as determined by delta F image analysis. When the same analysis is conducted on mice injected with GFP-expressing AAV as a control, GFP-expressing cells are detected within the focal plane range and no cells display a significant fluorescence change with or without tone.
Histological verification of GCaMP expression and GRIN lens targeting reveals no sign of tissue damage due to brain tissue inflammation around the GRIN lens. If the speed of the needle or the lens is not consistent, it can cause motion artifacts and poor image quality. Using a motorized surgery arm can help.
If the mobile home care system is not ready during the baseplate attachment step, a low concentration of isoflurane gas can be used to anesthetize the mouse during the baseplate attachment.
