We mounted speakers to standard mouse cages and positioned colored light sources at all four walls. (Figure 1). To mimic television viewing we piped audio from the “cartoon channel” into the mouse cage at 70 decibels, which is well below the levels of 100–115 db that are typically used for acoustic stress models and consistent with National Institutes of Health acceptable noise levels26,27,28,29,30,31. A photorhythmic modulator was used to change colors and intensities in concordance with the audio thereby simulating television that cannot be avoided (e.g. flashing lights on all four sides of the cage).

Figure 1 Mouse overstimulation chamber and experimental procedure. Full size image

Beginning at postnatal day 10 (P10), mice were randomly divided into two groups: (a) a control group which was reared according to approved and established protocols at the Seattle Children's Research Institute Vivarium; and (b) an overstimulated group which was treated identically to the control group except that they were exposed for 6 hours every night to auditory and visual stimulation intended to generate “non-normative sensory stimulation.” Exposure lasted for 42 days, which is comparable to the length commonly used in enriched environment studies8. Mice in both groups remained with their mother throughout this period to avoid any additional handling. Following the exposure period, lights and speakers were removed, but the mice remained in their familiar, regular mouse cages. Beginning 10 days later, we performed the following behavioral tests: the Open Field test32, Light Dark Latency test33, the Elevated Plus Maze34, the Novel Object Recognition test35 and the Barnes Maze36. For all tests, we made use of the VideoTrack (ViewPoint LS) tracking software to track mouse movements. The tracking software records where the mouse was, how long it was in certain areas, as well as the distance the mouse traveled in each area. Experiments were videotaped and in all cases, technicians blinded to research group made assessments.

We first assessed anxiety and activity. The Elevated Plus Maze measures the anxiety induced by open spaces, as well as height. The apparatus was an elevated maze consisting of four arms making the shape of a plus sign (Figure 2). Two of the arms had walls around them, whereas the other two arms project out of the center without walls. All mice were placed in the center of the maze and were allowed to run freely around the maze for 5 minutes. We calculated the amount of time the mouse spent in each arm, the distance traveled and the number of entries the mouse made into each arm. Overstimulated mice spent significantly more time in the open arm (Fig. 2E), had more entries into the open arm (Fig. 2F) and traveled greater distances in the open arm (Fig. 2G) compared to controls.

Figure 2 Results of Elevated Plus Maze (EPM) and Light Dark Latency (LDL) Tests. 2A & 2B demonstrate an illustrative example of a control and an overstimulated mouse's travel pathway on the EPM. Overstimulated mice spent more time in the open arm (mean±SEM; Controls: 9.93±2.11s, n = 48 and Overstimulated: 31.03±2.78s, n = 61, p < 0.001). (2E); had more entries in the open arms (mean±SEM; Controls: 7.97±1.22, n = 48 and Overstimulated: 11.92±1.06, n = 61, p < 0.001). (2F); and traveled greater distances in the open arms (mean±SEM; Controls: 73.55±23.61cm, n = 48 and Overstimulated: 176.23±14.62cm, n = 61, p < 0.001). (2G). 2C & 2D demonstrate an illustrative example of a control and an overstimulated mouse's travel pathway on the LDL. Overstimulated mice spent more time in the light chamber (mean±SEM; Controls: 53.79±4.17s, n = 48 and Overstimulated: 82.39±6.41s, n = 61, p < 0.001) (2H); took less time to fully enter the light chamber (mean±SEM; Controls: 223.00±17.49s, n = 48 and Overstimulated: 98.00±15.33, n = 61, p < 0.001) (2I) and traveled greater distances in the light chamber (mean±SEM; Controls: 276.42±42.03cm, n = 48 and Overstimulated: 436.89±40.06cm, n = 61, p <0.05) (2J). Full size image

The Light Dark Latency test measures risk-taking and anxiety in a related but different way. It is based on the rodents' innate aversion to brightly illuminated areas as well as their competing exploratory instincts37,38. The apparatus consisted of two compartments. One compartment is made of translucent fiberglass the other is painted black. The two compartments are separated by a black dividing wall with a small doorway for the mouse to travel between them. A 100 W light was directed at the translucent (light) chamber from approximately a 15 cm distance. The light is blocked out of the black chamber with a lid making it dark. Each mouse was placed in the dark chamber and was allowed to run freely between the two chambers for 5 minutes. Latency to enter (defined by all four paws entered), time spent, entries and distance traveled in the light chamber were recorded. Overstimulated mice were significantly different from control mice for all domains (Figure 2).

The Open Field Test measures hyperactivity and anxiety in yet a third way. The open field box consisted of a square black box made out of plexiglass with an outlined center area. Each animal was placed in the box for ten minutes. Overall activity in the box (measured with videotrack) was measured as well as the amount of time and distance traveled in the center area of the maze. Mice naturally prefer to be near a protective wall rather than exposed to danger out in the open, but a competing foraging instinct will motivate them to explore. (Figure 3A). Overstimulated mice spent significantly more time in the center (Fig. 3A,B), entered significantly more times into the center and traveled more distance within the center than control mice (Figure 3B). Moreover, the overall distance traveled by the overstimulated mice was significant higher than the distance traveled by the control mice, suggesting that overstimulated mice are hyperactive. (Figure 3C).

Figure 3 Results of Open Field Test: Tests. 3A & 3B demonstrate an illustrative example of a control and an overstimulated mouse's travel pathway on the Open Field Test. Overstimulated mice spent more time in the center of the open field (mean±SEM; Controls: 24.97±2.04, n = 64 and Overstimulated: 44.25±3.76, n = 72, p < 0.001) (3C), had more entries into the center of the open field (mean±SEM; Controls: 20.69±1.72, n = 72 and Overstimulated: 34.94±2.72, n = 72, p < 0.001) (3D) and they were more active as measured in distance traveled in the center of the open field field (mean±SEM; Controls: 505.79±47.45, n = 72 and Overstimulated: 883.03±71.82, n = 72, p <0.001), (3E) They also traveled greater distance overall (mean±SEM; Controls: 5836.66±194.26, n = 72 and Overstimulated: 6612.43±200.5, n = 72, p< 0.001) (3F). Full size image

We next assessed memory and learning. The Novel Object Recognition Test assesses short term memory by testing a mouse's ability to remember if it had previously encountered an object or not39. It is based on the idea that a mouse will spend more time investigating and exploring an object which it has never seen (novel object), compared to an object it has encountered before (familiar object). The same box, which was used in the Open Field Test, was used for the Novel Object Recognition test. Mice were habituated to the box a day before the test, during the Open Field Test. On the test day, mice were placed in the box for the acquisition period with two identical objects and were allowed to explore and familiarize themselves with the objects for 5 minutes. The mice were given a one hour inter-trial interval (ITI) and were then placed back in the test box. Everything was the same as during the acquisition period except that one of the two identical objects was replaced with a new, novel object. During the testing period, mice were allowed to explore both of the objects for 5 minutes. This trial was recorded with a video camera and scored by a trained and blinded experimenter to determine the time the mouse spent investigating each object. During the testing period, overstimulated mice spent less time on the novel object compared to controls. (Figure 4E).

Figure 4 Results of Barnes Maze Test (BM) and Novel Object Recognition Test (NORT). 4A & B demonstrate an illustrative example of a control and an overstimulated mouse's travel pathway on the BM. 4C summarizes the results of the BM over 4 days of successive testing. Overstimulated mice trended towards finding the escape hole faster than controls during the first day of the experiments (mean±SEM; Controls: 30.60±4.64, n = 12 and Overstimulated: 20.08±2.35, n = 72, p = 0.09) which is consistent with our findings that exposed animals travel greater distances and thus are more likely to encounter the escape hole. However, on day 4, control mice took significantly less time to find the target hole compared to overstimulated mice (mean±SEM; Controls: 3.44±0.39, n = 12 and Overstimulated: 6.33±0.67, n = 10, p < 0.001) Overstimulated mice continued to make more errors before finding the hole suggesting impaired memory and learning. Figure 4D summarizes the mean times mice spent on both objects during the acquisition (mean±SEM; Controls: 44.35±4.61, n = 39 and Overstimulated: 57.49±4.01, n = 42) and the test trial (mean±SEM; Controls: 44.87±5.64, n = 39 and Overstimulated: 53.81±3.77, n = 42). Figure 4E shows the discrimination ratio for the controls and the overstimulated mice. This shows how well the mice were able to discriminate between the novel and the familiar object. Overstimulated mice spent less time with a novel object compared to controls. Accordingly the discrimination ratio calculated as ((Time Spent on the Novel Object – Time Spent On the Familiar Object)/Total Time) is significantly reduced compared to controls (mean±SEM; Controls: 0.32±0.07, n = 39 and Overstimulated: 0.16±0.05, n = 42, p < 0.05). Full size image

Finally, we tested the mice with the Barnes Maze which is a circular platform with an escape hole that leads to a small chamber attached underneath the platform. There are 19 other imitation holes circled around the maze in order to distract from the real hole. The imitation holes look like the escape hole, but do not lead to an escape chamber. The Maze is lit up with a 100 w light bulb. On day one, the mouse was placed in an opaque box in the center of the maze. After ten seconds had elapsed, the opaque box was removed and the mouse was guided by hand to the escape hole. The first trial began after 15 minutes. On trial one, the mouse was once again placed in the start box for 10 seconds. Once released, the mouse had three minutes to find the escape hole. As soon as the mouse found the hole the light was turned off. The mouse spent 1 minute in the escape chamber and was then returned to its original housing cage. This procedure was recorded with the video camera and tracking software and latency to find the escape hole, as well as distance traveled was recorded. Mice were given four learning trials a day with 15-minute ITI's. On each day, the mean time to find the target hole was calculated. Overstimulated mice performed poorly compared to control mice on day 4 of testing. (Figure 4C).