Non-invasive Strategies for Chronic Manipulation of DREADD-controlled Neuronal Activity

DREADDs are the most popular chemogenetic approach for remote control of neuronal activity. Here we offer new alternatives for repetitive or chronic DREADD control, focusing on less invasive CNO delivery methods. We describe two strategies for the prolonged CNO delivery in mice by repetitive eyedrops, or chronically through the animal's drinking water.

The protocols described here are examples of chronic and noninvasive options for CNO delivery that can be adapted to a variety of experimental designs including different DREADD variants, tissues, or even species. A key issue for a CNO delivery is a animal's stress and pain. These protocols minimize this problem and are easy to perform and adapt to different experimental settings.

Before performing the surgery, clean and sterilize the stereotaxic frame and all needed instruments. When the frame is ready, confirm a lack of response to toe pinch in an anesthetized mixed background wild type male mouse, shave the top of the head, and fix the head of the mouse to the frame. Then apply ocular protective lubricant on the eyes and finally, clean the exposed skin with sequential povidone iodine and 70%ethanol scrubs.

Use a sterile scalpel to expose the skull and calibrate the frame to the bregma point. Drill at a medialateral coordinate of 2.9 millimeters, and an anterior posterior coordinate of minus 2.7 millimeters to target the hippocampus. When the brain is exposed, use a micro injector and pulled microcapillary pipettes to unilaterally inject 90 nanoliters of the adenoassociated virus into the hippocampus at a dorsal ventral depth of minus three millimeters.

Then close the incision with nylon sutures, and remove the animal from the frame for analgesia administration. 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. For chronic CNO treatment, beginning four weeks post injection and three days before starting the treatment, replace the regular water bottles with small bottles stopped with rubber spouts and covered with aluminum foil containing 10 milliliters of regular water. Secure to the cages with tape to allow mice to acclimate to the bottles.

Measure the daily water consumption for each mouse, and weigh each mouse. Test a range of CNO concentrations to determine the dose that displays the maximum effectiveness with the minimal CNO concentration. Then fill each small bottle with eight milliliters of regular water plus the required amount of CNO.

For restricted CNO treatment, beginning four weeks post injection and three days before starting the treatment, place a small bottle containing 10 milliliters of water plus 1%sucrose on the cage away from the original water bottle during the last portion of the animal's active phase. At the end of the exposure, remove the water plus sucrose solutions from each cage and measure the sucrose water consumption for each animal. For CNO delivery, fill the bottles with five milliliters of water plus 1%sucrose and one milligram per kilogram of CNO.

Place the bottle in the same position as during the sugar water acclimation period during the last portion of the active phase, removing the bottles and measuring the amount of water, sucrose and CNO consumed as demonstrated. At the end of the experiment, harvest the treated animal brain into fresh fixative. After 9 to 12 hours, cryoprotect the brain tissue in a 30%sucrose solution.

When the brain sinks, section the sample on a cryostat. When all of the brain sections have been obtained and the nonspecific binding blocked, incubate the tissue samples with an anti-c-Fos antibody solution at four degrees Celsius overnight with constant agitation. The next morning, wash the samples with three five-minute washes in fresh PBS per wash before incubating the samples with an appropriate fluorescence conjugated secondary antibody.

After one hour at room temperature and constant agitation protected from light, obtain digital images of the labeled tissue sections by fluorescence confocal microcroscopy. After loading the images into ImageJ, outline at measure the areas of Adeno-associated infected mCherry positive cells and quantify the number of C false positive cells within this region to obtain the number of activated cells per area. Repetitive CNO delivery using eyedrops elicits a robust induction of cFos expression in most infected neurons demonstrating that the effectiveness of CNO delivery is sustained during the repetitive exposure.

Furthermore, a significant induction of cFos is observed in samples collected two hours after CNO treatment compared to samples obtained six hours after CNO exposure, indicating that changes induced by CNO are time dependent. The daily consumption of water plus CNO is not significantly different compared to the total volume of regular water consumed. Similarly, the amount of water plus 1%sucrose consumed during the night is not affected by the addition of CNO, nor are differences in the daily consumption of both water plus CNO, and water plus sucrose plus CNO observed over a five-day experimental period.

Similar to the application of CNO eyedrops, a robust induction of cFos is observed after two, but not six hours of CNO drinking water access. In addition, there is a clear threshold of effectiveness for CNO as a low CNO dose does not elicit cFos activation compared to a saline control, whereas higher doses induce a robust and similar cFos induction. We recommend performing a dose response analysis to define the lowest CNO dose that does not significantly reduce neuronal activation, and considering the use clozapine as a ligand.

Here we demonstrated using CNO-mediated cFos induction as a result of neuronal activation. However, these protocol can be easily adopted to electrophysiological analysis or behavioral tests. DREADD technology is a powerful tool for remote control of neuronal activity, and designing alternative strategies for CNO delivery.

We'll increase the spectrum of options for experimental settings and potential clinical interventions.