Ophthalmic Drug Delivery for Noninvasive Neuromodulation in a Mouse Model

Start with a genetically modified mouse whose hippocampal neurons express designer receptors exclusively activated by designer drugs or DREADDs.

These engineered receptors selectively respond to synthetic test drugs while remaining unresponsive to endogenous molecules.

Scruff the mouse regularly to minimize stress and acclimate it to the procedure.

Next, weigh the mouse to calculate the correct drug dosage.

During mouse handling, use a dim red light to prevent circadian disruption.

Gently scruff the mouse to immobilize it.

Using a pipette, take the test drug solution and form a droplet at the tip. Now, approach the eye

and release the droplet onto its surface.

Return the mouse to its home cage. Repeat the drug delivery several times.

The drug absorbs through the eye and enters the brain.

It then crosses the blood-brain barrier and reaches the hippocampal region. Later, it binds to DREADD receptors and activates neurons, enabling precise, noninvasive neuromodulation.

For repetitive CNO delivery, beginning four weeks post injection, acclimate the animals to handling by scruffing each mouse three minutes daily for three to four days prior to the administration of the eyedrops. When the mice have become acclimated to handling, weigh each mouse to determine the appropriate amount of CNO to be delivered to achieve a one milligram of CNO per kilogram of body weight concentration.

Two hours before lights off during the inactive phase, load one to three microliters of the prepared CNO solution into a P10 micropipette and immobilize the mouse via scruffing. Slowly expel the solution until a stable droplet forms on the pipette tip and carefully bring the droplet close to the cornea until the solution is delivered without touching the pipette tip to the mouse's eye. Then return the mouse to its home cage before applying the CNO eyedrop to the eye of the next animal.

In cases where CNO needs to be delivered during the mouse active phase, ensure the presence of a dim red light for proper animal handling and CNO delivery.