In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

We present an in vivo two-photon imaging protocol for imaging the cerebral cortex of neonatal mice. This method is suitable for analyzing the developmental dynamics of cortical neurons, the molecular mechanisms that control the neuronal dynamics, and the changes in neuronal dynamics in disease models.

This method can help to answer key questions in neuroscience, especially those related to neuron socket formation. The main advantage of this technique is that researchers can analyze the morphological changes of individual neurons in living neonatal mice. Begin with an anesthetized post-natal day five pup and proceed only in the absence of a reaction to the tail pinch test.

First, sterilize the scalp by wiping it with 70%ethanol. Then, use scissors sterilized with 70%ethanol to remove approximately 20 square millimeters of the skin covering the skull. Next, use sterile forceps and a clean cotton swab soaked in cortex buffer, to remove the fascia of the skull.

Use a loading tip to apply tissue adhesive to the incised skin surface to stop the bleeding while avoiding the area to be imaged. Place the pup on another heating pad, set to 37 degrees Celsius, and allow it to recover from anesthesia. Wait approximately 30 minutes until the tissue adhesive has dried and solidified.

Once the tissue adhesive has dried, begin the cranial window preparation on the re-anesthetized mouse. Apply one drop of cortex buffer onto the skull, then use a sterile razor blade to carefully open a one millimeter diameter area of the skull, leaving the dura intact. Apply cortex buffer to keep the brain surface moist.

Use a small piece of gelatin sponge soaked in cortex buffer to stop any bleeding. Use a fresh piece to compress one side of the craniotomy and drain any buffer and blood from the dural surface without touching the dura. This is the most critical step to prepare clear window.

The dura must be undamaged and the brush should be removed from the exposed dura. Next, use a pipette tip to apply a thin layer of one percent low melting agarose, dissolved in cortex buffer. Apply a round, three millimeter glass cover slip onto the agarose gel layer.

Remove all bubbles between the cover slip and the agarose gel layer by pouring in excess of agarose gel between them. Use tweezers to remove the excess gel, protruding from under the cover slip. Mix cement powder and the cement liquid.

Use a pipette tip to apply the mixture to the skull before it becomes solidified. Then, attach a custom made titanium bar to the cranial bone using dental cement. Align the titanium bar, and the cover slip in parallel to easily capture images.

Cover the exposed skull with dental cement. Then, subcutaneously inject an analgesic. Place the pup in a recovery cage on a 37 degrees celsius heat pad for an hour until the dental cement has solidified.

To begin imaging, first set the two-photon laser wavelength. For RFP excitation, use a wavelength of 1000 nanometers. Wipe the surface of the cover slip with 70%ethanol.

Attach the anesthetized pup to the titanium plate on the imaging stage, using the titanium bar. Use the goniometer stage, to adjust the head, such that the cover slip is parallel to the objective lens. Maintain the body temperature of the pup during imaging using a heating pad set to 37 degrees Celsius and reduce the isoflurane concentration to 0.7%to 1%Place the imaging stage under the 20x objective lens of the two photon microscope.

Apply one drop of water onto the cover slip. Set the software to acquire Z-stack images at 1.4 micron intervals. For layer four neuron imaging, set the Z width to between 150 and 300 microns to image the entire dendritic morphology.

Use slow scanning and averaging to get clear images showing the neuronal morphology. It usually takes more than 20 minutes to acquire the entire dendritic morphology. This image shows a representative Z-stack image of the layer four cortical neurons of P5 pup.

The arrow indicates the neuron to be analyzed. Neurons with blurred dendrites should be removed from the analysis. This image shows a higher magnification image of the indicated neuron in the prior image.

Blue arrowheads indicate dendritic tips that will be retracted at the next time point, and the small white arrowheads indicate the axon of a neighboring cell. After 4.5 hours, the dendritic tips with blue arrowheads are retracted, and the dendritic tips with yellow arrowheads are elongated. A representative dendritic morphology reconstruction is shown here, neurons showing disconnected dendrites as seen here, should be excluded from analyses.

While attempting this procedure, it's important to keep the dura intact, during the process. Using this procedure, other methods like in the propulsion imaging can be performed to answer additional questions related to the neuron activity pattern in neonatal brain.