In Vivo Chronic Two-Photon Imaging of Microglia in the Mouse Hippocampus

The roles of microglia in the hippocampus are not yet fully understood. This protocol can directly image how microglia contact and regulate neurons. In vivo imaging of resting microglia and neurons in the hippocampus has been very difficult.

This method can solve this problem. To implant the observation window, make a three millimeter circular groove on the skull using a dermal punch, ensuring the alignment of the groove with the previously marked area. When the dermal punch reaches the innermost layer of the cranium before touching the underlying dura, gently lift the central bone island using a 30 gauge needle.

Next, carefully remove the dura using a dura picker. Aspirate the pia, pial vessels, and cortical tissue from the surface. Keep the depth of exposed tissue surface homogeneous over the entire cranial window and gradually deepen the aspiration until fibers of the external capsule are exposed.

After positioning the suction tip to the external capsule near the lateral ventricle at the rostrolateral end of the cranial window, remove the surface layer of the external capsule running in the caudomedial to the rostrolateral direction. Then remove the inner layer running in the mediolateral direction. Confirm the removal of the entire external capsule by checking the full exposure of the surface of the alveus and the dorsal hippocampal commissure.

After ensuring that the bleeding has completely stopped, fill the hole with sterile saline to the level of the skull surface. Next, insert the glass bottom metal tube vertically into the hole while aspirating the excess water overflowing from the sides of the tube until the glass bottom lightly presses against the alveus and partially flattens the underlying CA1. Using the dental resin cement, fix the wall of the inserted tube to the surrounding skull.

Set the mouse in the head holding device under the objective lens of the two-photon microscope and on the motorized XY scanning stage. Then fill the space between the glass bottom and the objective lens with water without creating air bubbles. Adjust the focus to CA1 with the guidance of fluorescence emitted from the brain parenchyma and the reflected light from the edge of the metal tube under continuous illumination by the pulsed laser.

Adjust the angle of the mouse head by tilting the head holding device so that the glass bottom is parallel to the imaging plane. Then adjust the correction collar of the objective to achieve the highest resolution at a depth of the target structure in the CA1. Next, confirm that the fluorescent cells in the molecular layer of the dentate gyrus or DG at a depth of 500 micrometers from the glass bottom are imaged in the entire field of view.

Only in case of a successful surgery, the DG signals can be detected immediately. Raise the postoperative mouse in individual housing for several weeks. Set the anesthetized mouse under the two-photon microscope.

Perform the imaging of the hippocampus. Confirm if the quality and intensity of the images captured after the reduction of postoperative edema are better or comparable to those captured on the surgery day. Ensure that microglia have recovered their ramified morphology.

Then perform in vivo imaging on the layer of interest in the CA1. The microglia in CX3CR1-GFP mice captured immediately after the surgery showed no prominent activation. After successful surgery without induction of edema, the two-photon imaging depths exceeded the entire CA1 layers and reached 500 micrometers from the surgical surface.

The microglia in the chronic phase of more than three weeks after surgery exhibited higher fluorescent intensity and resolution and more homogenous distribution due to reduced edema and inflammation. Microglia in all layers already restored their ramified morphology and exhibited immobile cell bodies and highly modal processes. The high resolution images over the entire CA1 were provided for several months.

The signals from fluorescent microglia help identify hippocampal layers. Microglia in the alveus had elongated cell bodies and processes extending along the axonal tracts. Microglia in the stratum pyramidale could be distinguished by the lower density of their processes.

The border between the CA1 and the DG could be recognized by thick blood vessels running along the boundary which was better recognized by labeling vessels with sulforhodamine 101. As an application example, GFP positive microglia and tdTomato labeled pyramidal neurons were simultaneously imaged. Neuronal labeling helped to understand the exact layer structures of the CA1.

The most important point is to reduce surgery damage. Avoid tissue damage by aspiration. Apply gentle pressure when inserting the glass bottom and avoid dental cement from touching the brain surface.

This approach will help identify the role of microglia in the hippocampus. For example, how microglia interact with neurons, how they are involved in learning, and how they control brain diseases.