Sound attenuated chamber

Pre-pulse inhibition (PPI) was performed in sound-attenuated chambers (Med Associates Inc., St. Albans, VT, USA) as previously described (16 (link)) with minor revision. In brief, a 65-dB background whiter noise was maintained throughout the test sessions. Mice were acclimated to the testing chamber for 5 min as the first test session. Then, mice were individually placed in a tube fixed on a plastic frame to monitor movement. After 12 initial startle pulses (20 ms, 120 dB) were presented, 12 prepulse/startle trials (prepulse, 20-ms white noise at 70, 80, or 90 dB at 100-ms intervals; 80-ms interval; startle pulse, 20 ms,120 dB) were performed immediately. The initial startle pulses provided a baseline of startle reactivity for the following test session. The different trial types (12 times for each trial type) were presented pseudo-randomly with a 15-s inter-trial interval (10-20 s), and two consecutive trials were always different. Mice movements were analyzed for 100 ms from startle stimulus onset. Each complete test session lasted about 25 min. For each pre-pulse intensity, PPI (%) was calculated as: [1 - (startle amplitude on “pre-pulse followed by pulse” trial/startle amplitude on “pulse-alone” trial)] × 100.

Locomotor activity was tested for 30 min under ambient light levels (190–210 lux) in sound attenuated chambers (43 × 43 cm) equipped with an infrared beam detection system (Med Associates Inc.). Data are presented as cumulative distance traveled in 5 min intervals.

Animals were first habituated to the sound-attenuated chambers for several days (Med Associates Inc.). Auditory stimulation was performed using an audio generator, and electrical signals were recorded with a Neurologger and analyzed offline. Within a recording session, the ERP protocol (300 repetitions of white noise click pairs, spaced 8 s apart) or ASSR protocol (300 repetitions of 2-s-long click trains consisting of 80 white noise clicks, spaced 10 s apart, at 85 dB) were presented. All mice received all treatments in a randomized cross-over design.

We used the (Quirk et al., 2000 (link)) method for auditory fear conditioning. Rats were placed into sound attenuated chambers (Med Associates Fairfax VT) with a 33×28×25 cm interior chamber within a 63×45×58 cm sound attenuating outer box and aluminum walls and aluminum rod floor. The fear conditioning paradigm was controlled by Ethovision software (Noldus Information Technology) and consisted of 3 days. On day one, (acquisition) rats were allowed to habituate to the chamber for 5 min followed by 5 x 30 s tones paired with shock, 0.5mA for 0.5 s, administered with 3 min inter-trial intervals (ITI). Rats were removed to their homecage and returned to the chamber 24 hr later. For day two (extinction), after another 5 min habituation, 20 x 30 s tones, with 3 min ITIs, were played with no shock at termination. Rats were again returned to the homecage for 24 hr. The last day, day three (extinction recall), consisted again of a 5 min habituation and 20 x 30 s tones, with 3 min ITIs. Freezing, the complete cessation of all movement other than respiration was measured by Freezescan software (CleverSys, Inc) during the 30 s tones.

To assess locomotor activity, mice were placed in sound-attenuated chambers equipped with photo beams (Med Associates, St. Albans, VT, USA). Mice were habituated for 30 min and then observed for an additional 90 min. Number of ambulations, measured by the number of beam breaks, was quantified in 5-min intervals after the habituation period.

Mice were placed in the center of a transparent acrylic arena with 42 cm by 42 cm floor dimensions, 30.5 cm high walls, and a removable, perforated lid (Figure 1A). The arena was housed within a sound-attenuated chamber (Med Associates; Fairfax, VT) with lights and a fan in the interior. Mice were allowed to explore the arena undisturbed for 15 min. Movement was monitored in the arena using infrared beam brakes (Versamax, AccuScan Instruments; Columbus, OH). Behavioral metrics analyzed included total distance traveled (cm) and time in center (center defined as > 7.875 cm from the walls of the arena). The arena was wiped down with 70% ethanol and allowed to dry before adding each animal. Mice were only tested once.