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The overall goal of this procedure is to allow the simultaneous visualization of neuronal activity across multiple layers of cortical neurons using calcium indicators in a freely behaving mouse and to understand neural circuit functioning using a miniaturized head mounted fluorescence microscope. This method can help answer key questions in systems neuroscience field such as, how is the information propagator across multiple layers of the cortex. The main advantage of this technique is that, it allows one to visualize large populations of genetically encoded neurons across multiple cortical layers in freely behaving animals, reliably over time.

In order to visualize genetically encoded neurons from multiple cortical layers in a freely behaving animal, the following procedures need to be performed, and this protocol highlights the prism probe implantation, baseplate installation and in vivo imaging in a freely behaving mouse. To begin this procedure, one to two weeks after virus injection, place the animal in the stereotaxic apparatus, and prepare for the prism probe implant surgery, by disinfecting it in 70%ethanol and cleaning it with lens paper. Next, open a round craniotomy, using a micro drill with a 0.5 millimeter burr.

Ensure that the craniotomy diameter is just larger than the prism diameter. Then, position the craniotomy such that when the prism is inserted into the cortex, its flat edge is facing the virus injection site and is within a 150 to 200 micron radius. After that, remove the dura with fine forceps.

Always keep the tissue moist with sterile saline, once the brain tissue is exposed. This will also maintain pressure on the tissue. To alleviate pressure in the brain tissue during the prism probe insertion, create an insertion tract by attaching a straight edged dissection knife to the electrode holder arm of the stereotaxic apparatus.

Mount it at an angle, such that the knife plate is perpendicular to the curvature of the skull and in a plane parallel to the virus injection column. Carefully position the knife above the craniotomy, along its interior media edge and about 200 microns lateral to the virus injection site with the cutting edge facing posteriorly. Zero out the Z axis when the knife tip touches the pia.

Then, lower it gradually to a depth that which the prism probe will be inserted. Subsequently, move the knife one millimeter posteriorly, to create a path for the prism's leading edge. Slowly retract the knife using the stereotaxic arm micromanipulator in 10 micron per second increments, and place a piece of gel foam sponge soaked with sterile saline over the incision.

Following that, attach the lens holder to the stereotaxic manipulator arm at the same angle as the knife in the previous step. Align the prism such that the flat side of the prism is above the incision and parallel to the virus injection column. Once the prism is at the correct angle, gradually lower it into the brain in 10 micron increments to a final Z level of 1.1 millimeter from the brain surface, for this probe.

Cover any exposed tissue around the prism in the craniotomy, with a very thin protective level of elastomer adhesive, using a 25 gauge needle. To prevent the lens from moving inside the craniotomy, after the elastomer adhesive is cured, use a 25 gauge needle to apply some cyanoacrylate adhesive to attach the glass of the prism lens, to the adjoining skull over the layer of elastomer adhesive. Once the cyanoacrylate adhesive is cured, unscrew the lens holder and carefully remove the microscope.

Then, slowly retract the stereotaxic manipulator arm, to leave the prism probe securely implanted. Apply a layer of dental acrylic or cyanoacrylate adhesive around the implant, to cover the exposed skull surface without touching the surrounding retracted muscle tissue. Covering a large area of the skull with this cranial cap, will later help in the base plate attachment.

Subsequently, mix the catalyst end base from a silicone adhesive syringe and put a drop of the elastomer inside the prism probe cuff, to cover the probe lens top, in order to prevent any damage and dust from settling. One week to 10 days after implanting the prism probe, check for virus expression in the tissue through the implanted prism probe. For this, remove the silicone adhesive cap over the surface of the implanted prism probe lens.

Examine the probe lens surface, and clean off any debris gently, with lens paper and 70%ethanol, to ensure the imaging surface is clean. Next, attach a baseplate to the microscope and fasten the baseplate set screw to hold it in position. Then, secure the microscope into the microscope gripper on the stereotaxic micromanipulator arm.

Attach the gripper to a rod which can be mounted on the stereotaxic micromanipulator arm. Subsequently, position the microscope above the prism probe lens, using the stereotaxic micromanipulator arm. Visually inspect the orientation by viewing the prism lens from the side and back of the animal stage.

The optical axis of both the microscope objective and prism probe, must be aligned. Following that, launch the acquisition software, select the project, and turn on the microscope LED through the software. Evaluate the quality of the microscope alignment by focusing on the top phase of the implanted prism probe lens in the acquisition software, by moving the manipulator arm of the stereotax.

When properly aligned, the edges of the prism probe lens should appear sharp. Adjust the microscope's physical distance above the implanted prism probe, using the stereotaxic manipulator arm to obtain the desired focal plane inside the tissue. Then, use dental acrylic or cyanoacrylic to permanently attach the base plate to the acrylic cap to bridge the gap.

It is critical not to apply adhesive on any part of the microscope's body or the set screw while gluing the base plate to the animal's head. Now, release the microscope from the gripper and retract the gripper from the microscope. If cyanoacrylic or another transparent adhesive was used, cover it with black nail polish or a layer of black dental cement to prevent ambient light leakage into the head cap, which can contaminate future images acquired during experiments.

Then, protect the implanted prism probe with the base plate cover. In this procedure, plug the microscope to its data acquisition box connected to the computer and launch the acquisition software. Then, remove the base plate cover, by turning the base plate set screw counter-clockwise and lifting out the base plate cover.

Seed the microscope in the base plate on the animal. The microscope should snap into place with the aid of the magnets on the base plate. Then, advance the base plate set screw until a slight resistance is felt.

After that, select the acquisition settings to be used to gather the data, which includes the frame rate for capturing data. Check the image histogram when selecting the settings to ensure good signal to noise ratio. Then, start acquiring the data.

When the recording is complete, loosen the base plate set screw and detach the microscope from the base plate, by gently pulling up the microscope. Subsequently, replace the base plate cover and gently tighten the base plate set screw. Here is a demonstration of an animal mounted with the microscope, freely interacting with the objects in the behavior arena.

During awake behaving imaging with the system, the activity of the somatosensory cortical neurons was recorded when the mouse was exposed to three different environments. Open field on day one, object familiarization on days two to four, and to novel object on day five. Following cell extraction using the calcium image data analysis software, spatial filters corresponding to cell locations, were overlaid on the mean fluorescence intensity projection of the microscope recording data.

A white dashed line separates layers two, three and five cells. Corresponding calcium traces from five cells from the each of the layers, show the farming pattern of the cells in two different behavioral contexts:Object familiarization and novel object exposure. Layer two, three cells were more active compared to layer five cells on the day when the mouse was exposed to a novel object.

Here are the rest of the plots of cell activities from the superficial entity players, when the animal was in open field, object familiarization and novel object exploration. Once mastered, each technique can be completed within an hour if performed properly. After its development, this technique paves the way for systems neuroscience researchers, to explore specific cell types in different brain areas, while leveraging different behavior paradigms in droning and other models of neurophysiology in disease.

From this protocol, one should have a good understanding of how to label multiple layers of somatosensory cortex, implant a prism probe and attach a head mounted miniaturized microscope.