Behavioral tests

The sucrose preference test utilizes a two-bottle choice paradigm to measure anhedonia. For this test, an experimental cage resembling the animal’s home cage was set up with two bottles: one filled with plain drinking water and the other with a 3% sucrose solution (Sucrose, Sigma-Aldrich). No food was present during the test. One day before the actual test, animals were acclimated to the cage equipped with two water bottles. Additionally, they were familiarized with the sucrose solution in their home cage. Before the test commenced, animals underwent mild food and water deprivation. As a baseline reference, the SPT was conducted without DBS on day 0. On subsequent days (days 1–3), the two original bottles were replaced: one with water and the other with the 3% sucrose solution. The 120 Hz DBS was initiated at the beginning of the test. To track liquid consumption during the 1-h test, both bottles were weighed before and after placement in the cage. 24 h after the final SPT, animals were retested using the SPT setup, but without the DBS intervention. To mitigate potential side bias, the positions of the bottles were alternated daily. The sucrose preference index was determined by dividing the volume of sucrose consumed during the test by the total volume of liquid consumed.

The forced swim test was used to evaluate “depressive-like” states and behavioral despair. The test was conducted in a plastic cylinder measuring 25 cm in height and 20 cm in diameter. Prior to the test, the cylinder was filled to a depth of 15 cm with water maintained at 23 °C. Animals were then introduced into the cylinder, allowing them to swim for a duration of 5 min. As a baseline reference, the FST was conducted without DBS on day 0. On day 1, DBS was administered for 5 min within the animal’s home cage. Immediately after the stimulation concluded, the animal was placed into the FST cylinder. The entire process was captured on video and subsequently analyzed using the Ethovision software. Two primary behaviors were recorded: immobility, indicative of passive coping, and swimming, reflecting active coping. Immobility was characterized as minimal movement, primarily small efforts to keep the head above water. In contrast, swimming was detected when the animal moved around the cylinder with all four paws underwater.

For the analysis the cumulative duration of the last four minutes was taken, and the results for the two behavioral states were reported in seconds.

To evaluate potential changes in general appetite and spontaneous locomotor activity post-DBS administration, we designed an experimental setup to monitor both standard chow consumption and movement within the home cage. On day 0, the baseline was established for chow intake over a 3-hour span and distance moved was captured using video recording. From days 1–3, following a 1-h DBS session, chow consumption was monitored for the subsequent 3 h. The total distance covered during the initial 40 min of the DBS session was recorded using Ethovision software.

To assess the impact of DBS administration on reward-related behavior, we employed a motivation test. Over the initial two days, mice were familiarized with both the experimental chamber, measuring 60 cm in length and 40 cm in width, and chocolate-flavored pellets (Bio-Serv) which were provided in their home cage for a duration of 30 minutes. The testing environment was illuminated with white light at an intensity of 40 lux. On day 2, animals were permitted to explore the experimental chamber for a 10-min duration. During this time, they had unfettered access to two different trays: one containing standard chow and the other stocked with chocolate pellets. The trays were placed a specified distance (15 cm) apart. On day 3, the test was repeated, but with the addition of DBS administration throughout the test’s duration. All test sessions were video-recorded and subsequently analyzed using the Ethovision software. To estimate motivation, we measured the time in seconds taken to first approach and first consume the food items, along with the total consumption of both food types (standard chow vs. chocolate pellets).